JP5951990B2

Microorganisms for the production of adipic acid and other compounds

Abstract

This record has no abstract on file.

JP5951990B2, drawing sheet 1 — Sheet 1 of 47

Term

2.5 yearsleft in the term

Expires 27 March 2029.

5 claims: 4 independent, 1 dependent

1
天然に存在しない微生物であって、前記微生物は、アジペートを生産するための酵素を有し、アジペートを生産するのに十分な量で発現されるアジペートを生産する酵素(複数)をコードする外因性核酸(複数)を含み、前記アジペートを生産する酵素(複数)が、スクシニル-CoAおよびアセチル-CoAを3-オキソアジピル-CoAに変換するスクシニル-CoA:アセチル-CoAアシルトランスフェラーゼ;3-オキソアジピル-CoAを3-ヒドロキシアジピル-CoAに変換する3-ヒドロキシアシル-CoAデヒドロゲナーゼ;3-ヒドロキシアジピル-CoAを5-カルボキシ―2-ペンテノイル-CoAに変換する3-ヒドロキシアジピル-CoAデヒドラターゼ;5-カルボキシ―2-ペンテノイル-CoAをアジピル-CoAに変換する5-カルボキシ-2-ペンテノイル-CoAレダクターゼ;およびアジピル-CoAをアジペートに変換する、ホスホトランスアジピラーゼおよびアジピン酸キナーゼを含む、天然に存在しない微生物。
2
前記天然に存在しない微生物は細菌または酵母である、請求項1 に記載の天然に存在しない微生物。
3
前記天然に存在しない微生物は細菌である、請求項 2 に記載の微生物。
4
前記細菌はEscherichia coliである、請求項 3 に記載の微生物。
5
アジペートを生産する方法であって、請求項1に記載の天然に存在しない微生物を、アジペートを生産する条件下でおよび十分な期間にわたって培養することを含む、方法。

Independent claims5

96 paragraphs, as filed

0001This application claims the priority benefit of US Provisional No. 61 / 040,059 filed March 27, 2008, the entire contents of which are incorporated herein by reference.

0002The present invention generally relates to biosynthetic processes, and more specifically to organisms capable of biosynthesizing adipic acid, 6-aminocaproic acid and caprolactam.

0003Adipic acid (a dicarboxylic acid with a molecular weight of 146.14) is a commercially important compound. Its main use is the production of nylon 6,6 (a linear polyamide produced by condensing adipic acid and hexamethylenediamine, which is mainly used to produce different types of fibers). Other uses for adipic acid include its use in plasticizers, unsaturated polyesters, and polyester polyols. In addition, applications include applications for the production of polyurethanes, lubricant components, and as food ingredients such as flavoring agents and gelling aids.

0004Historically, adipic acid was prepared from a variety of fats using oxidation. Current commercial processes for adipic acid synthesis use excess strong nitric acid, in KA oil, which is a mixture of cyclohexanone (ketone or K component) and cyclohexanol (alcohol or A component), or in pure cyclohexanol. Depends on oxidation. There are several variants of this theme that differ in the production pathways of KA or cyclohexanol. For example, phenol is an alternative raw material in the production of KA oil and describes the process of synthesizing adipic acid from phenol. Other variants of this process tend to use oxidizing agents other than nitric acid, such as hydrogen peroxide, air or oxygen.

0005Caprolactam is an organic compound that is a lactam of 6-aminohexanoic acid (ε-aminocaproic acid, aminocaproic acid). Alternatively, it can be thought of as a cyclic amide of caproic acid. The main industrial use of caprolactam is as a monomer in the production of nylon-6. The majority of caprolactam is synthesized from cyclohexanone by an oxime process using hydroxyammonium sulfate followed by a catalytic rearrangement using the Beckmann rearrangement process step.

Therefore, alternative methods are needed to effectively produce commercial amounts of compounds such as adipic acid and caprolactam. The present invention meets this requirement and also provides related advantages.

The present invention provides microbial organisms having an adipate, 6-aminocaproic acid or caprolactam pathway. The microorganism contains at least one exogenous nucleic acid encoding an enzyme in its respective adipate, 6-aminocaproic acid or caprolactam pathway. In addition, the present invention provides methods for producing adipate, 6-aminocaproic acid or caprolactam. This method is an adipate, 6-aminocaproic acid or caprolactam-producing microorganism in an amount sufficient to produce at least one exogenous nucleic acid encoding the adipate, 6-aminocaproic acid or caprolactam pathway enzyme. The microorganism to be expressed may include culturing under conditions producing adipate, 6-aminocaproic acid or caprolactam and for a sufficient period of time.The present invention also provides the following items.(Item 1)A non-naturally occurring microorganism comprising a microorganism having an adipate pathway containing at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in an amount sufficient to produce adipate, wherein the adipate pathway is succinyl. -CoA: Acetyl-CoA acyltransferase; 3-Hydroxyacyl-CoA dehydrogenase; 3-Hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phosphotransadipylase / adipine Acid kinase, adipyl-CoA: Non-naturally occurring microorganisms, including acetyl-CoA transferase or adipyl-CoA hydrolase.(Item 2)The non-naturally occurring microorganism according to item 1, wherein the microorganism comprises two exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 3)The non-naturally occurring microorganism according to item 1, wherein the microorganism comprises three exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 4)The non-naturally occurring microorganism according to item 1, wherein the microorganism comprises four exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 5)The non-naturally occurring microorganism according to item 1, wherein the microorganism comprises five exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 6)The above five exogenous nucleic acids are succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA. The non-naturally occurring microorganism according to item 5, which encodes a synthesizer.(Item 7)The above five exogenous nucleic acids are succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and phosphotransazidine. The non-naturally occurring microorganism according to item 5, which encodes a pyrase / adipate kinase.(Item 8)The above five exogenous nucleic acids are succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA. : A non-naturally occurring microorganism according to item 5, which encodes an acetyl-CoA transferase.(Item 9)The above five exogenous nucleic acids are succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrolase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA. The non-naturally occurring microorganism according to item 5, which encodes a hydrolase.(Item 10)The non-naturally occurring microorganism according to item 1, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.(Item 11)The non-naturally occurring microorganism according to item 1, which is present in a substantially anaerobic culture medium.(Item 12)A method for producing adipate, which is a non-naturally occurring microorganism having an adipate pathway containing at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in sufficient quantity to produce adipate. The adipate pathway includes succinyl-CoA: acetyl-CoA acyltransferase; 3-hydroxyacyl-CoA dehydrogenase; 3-hydroxyadipyl-CoA dehydratase; 5 A method comprising -carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phosphotransadipylase / adipate kinase, adipyl-CoA: acetyl-CoA transferase or adipyl-CoA hydrolase.(Item 13)Item 12. The method of item 12, wherein the non-naturally occurring microorganism is present in a substantially anaerobic culture medium.(Item 14)The method of item 12, wherein the microorganism comprises two exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 15)The method of item 12, wherein the microorganism comprises three exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 16)The method of item 12, wherein the microorganism comprises four exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 17)The method of item 12, wherein the microorganism comprises five exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 18)The above five exogenous nucleic acids are succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA. The method of item 17, which encodes a synthesizer.(Item 19)The above five exogenous nucleic acids are succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and phosphotransazidase. 17. The method of item 17, which encodes a pyrase / adipic acid kinase.(Item 20)The above five exogenous nucleic acids are succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA. : The method of item 17, which encodes an acetyl-CoA transferase.(Item 21)The above five exogenous nucleic acids are succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrolase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA. The method of item 17, wherein the hydrolase is encoded.(Item 22)The method of item 12, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.(Item 23)A non-naturally occurring microorganism comprising a microorganism having an adipate pathway containing at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in sufficient quantity to produce adipate, wherein the adipate pathway is succinyl. -CoA: Non-naturally occurring microorganisms, including acetyl-CoA acyltransferase; 3-oxoadipyl-CoA transferase; 3-oxoadipate reductase; 3-hydroxyadipate dehydratase; and 2-enoate reductase.(Item 24)23. The non-naturally occurring microorganism of item 23, wherein the microorganism comprises two exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 25)23. The non-naturally occurring microorganism according to item 23, wherein the microorganism comprises three exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 26)23. The non-naturally occurring microorganism according to item 23, wherein the microorganism comprises four exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 27)23. The non-naturally occurring microorganism according to item 23, wherein the microorganism comprises five exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 28)Items in which the above five exogenous nucleic acids encode succinyl-CoA: acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase. Non-naturally occurring microorganisms described in 27.(Item 29)The non-naturally occurring microorganism according to item 23, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.(Item 30)The non-naturally occurring microorganism according to item 23, which is present in a substantially anaerobic culture medium.(Item 31)Adipate a non-naturally occurring microorganism that has an adipate pathway that contains at least one exogenous nucleic acid that is a method for producing adipate and that encodes an adipate pathway enzyme that is expressed in sufficient quantity to produce adipate. The adipate pathway includes succinyl-CoA: acetyl-CoA acyltransferase; 3-oxoadipyl-CoA transferase; 3-oxoadipate reductase; 3-hydroxyadipine. Acid dehydratase; and methods that include 2-enoate reductase.(Item 32)31. The method of item 31, wherein the non-naturally occurring microorganism is present in a substantially anaerobic culture medium.(Item 33)31. The method of item 31, wherein the microorganism comprises two exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 34)31. The method of item 31, wherein the microorganism comprises three exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 35)31. The method of item 31, wherein the microorganism comprises four exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 36)31. The method of item 31, wherein the microorganism comprises five exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 37)The four exogenous nucleic acids encode succinyl-CoA: acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase. The method described in 36.(Item 38)31. The method of item 31, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.(Item 39)In non-naturally occurring microorganisms, including microorganisms with a 6-aminocaproic acid pathway that contain at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in sufficient quantity to produce 6-aminocaproic acid. A non-naturally occurring microorganism in which the 6-aminocaproic acid pathway described above comprises a CoA-dependent aldehyde dehydrogenase; and a transaminase or 6-aminocaproic acid dehydrogenase.(Item 40)The non-naturally occurring microorganism according to item 39, further comprising the adipyl-CoA pathway.(Item 41)Item 40, wherein the adipyl-CoA pathway comprises succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and 5-carboxy-2-pentenoyl-CoA reductase. The listed non-naturally occurring microorganisms.(Item 42)Item 40, wherein the adipyl-CoA pathway comprises an adipate pathway and an enzyme selected from adipyl-CoA synthesizer, phosphotransadipylase / adipic acid kinase, adipyl-CoA: acetyl-CoA transferase and adipyl-CoA hydrolase. Non-naturally occurring microorganisms described in.(Item 43)42. The adipate pathway comprises succinyl-CoA: acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase. Microorganisms that do not exist in nature.(Item 44)39. The non-naturally occurring microorganism according to item 39, wherein the microorganism comprises two exogenous nucleic acids, each encoding a 6-aminocaproic acid pathway enzyme.(Item 45)The non-naturally occurring microorganism according to item 44, wherein the two exogenous nucleic acids encode a CoA-dependent aldehyde dehydrogenase and a transaminase.(Item 46)The non-naturally occurring microorganism according to item 44, wherein the two exogenous nucleic acids encode a CoA-dependent aldehyde dehydrogenase and a 6-aminocaproic acid dehydrogenase.(Item 47)The non-naturally occurring microorganism according to item 39, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.(Item 48)The non-naturally occurring microorganism according to item 39, which is present in a substantially anaerobic culture medium.(Item 49)A method for producing 6-aminocaproic acid, a 6-aminocapron containing at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in sufficient quantity to produce 6-aminocaproic acid. Non-naturally occurring microorganisms having an acid pathway, including culturing under conditions producing 6-aminocaproic acid and for a sufficient period of time, the 6-aminocaproic acid pathway described above is a CoA-dependent aldehyde dehydrogenase; and nucleic acid.A method comprising lanceaminase or 6-aminocaproic acid dehydrogenase.(Item 50)49. The method of item 49, further comprising an adipyl-CoA pathway.(Item 51)Item 50, wherein the adipyl-CoA pathway comprises succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, and 5-carboxy-2-pentenoyl-CoA reductase. The method described in.(Item 52)Item 50, wherein the adipyl-CoA pathway comprises an adipate pathway and an enzyme selected from adipyl-CoA synthesizer, phosphotransadipylase / adipic acid kinase, adipyl-CoA: acetyl-CoA transferase and adipyl-CoA hydrolase. The method described in.(Item 53)52. The adipate pathway comprises succinyl-CoA: acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase. Method.(Item 54)49. The method of item 49, wherein the non-naturally occurring microorganism is present in a substantially anaerobic culture medium.(Item 55)49. The method of item 49, wherein the microorganism comprises two exogenous nucleic acids, each encoding a 6-aminocaproic acid pathway enzyme.(Item 56)55. The method of item 55, wherein the two exogenous nucleic acids encode a CoA-dependent aldehyde dehydrogenase and a transaminase.(Item 57)55. The method of item 55, wherein the two exogenous nucleic acids encode a CoA-dependent aldehyde dehydrogenase and 6-aminocaproic acid dehydrogenase.(Item 58)49. The method of item 49, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.(Item 59)A non-naturally occurring microorganism comprising a caprolactam pathway containing at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in sufficient quantity to produce caprolactam, wherein the caprolactam pathway is the CoA. Dependent aldehyde dehydrogenase; transaminase or 6-aminocaproic acid dehydrogenase; and amide hydrolase, non-naturally occurring microorganisms.(Item 60)The non-naturally occurring microorganism according to item 59, further comprising the adipyl-CoA pathway.(Item 61)Item 60, wherein the adipyl-CoA pathway comprises succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and 5-carboxy-2-pentenoyl-CoA reductase. The listed non-naturally occurring microorganisms.(Item 62)Item 60, wherein the adipyl-CoA pathway comprises an adipate pathway and an enzyme selected from adipyl-CoA synthesizer, phosphotransadipylase / adipic acid kinase, adipyl-CoA: acetyl-CoA transferase and adipyl-CoA hydrolase. Non-naturally occurring microorganisms described in.(Item 63)62. The adipate pathway comprises succinyl-CoA: acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase. Microorganisms that do not exist in nature.(Item 64)The non-naturally occurring microorganism according to item 59, wherein the microorganism comprises two exogenous nucleic acids, each encoding a caprolactam pathway enzyme.(Item 65)The non-naturally occurring microorganism according to item 59, wherein the microorganism comprises three exogenous nucleic acids, each encoding a caprolactam pathway enzyme.(Item 66)The non-naturally occurring microorganism according to item 65, wherein the above three exogenous nucleic acids encode a CoA-dependent aldehyde dehydrolase, transaminase and amide hydrolase.(Item 67)The non-naturally occurring microorganism according to item 65, wherein the three exogenous nucleic acids encode a CoA-dependent aldehyde dehydrolase, 6-aminocaproic acid dehydrolase and amide hydrolase.(Item 68)The non-naturally occurring microorganism according to item 59, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.(Item 69)The non-naturally occurring microorganism according to item 59, which is present in a substantially anaerobic culture medium.(Item 70)Caprolactam is a method for producing caprolactam, a non-naturally occurring microorganism having a caprolactam pathway that contains at least one exogenous nucleic acid that encodes a caprolactam pathway enzyme that is expressed in sufficient quantity to produce caprolactam. A method comprising culturing under conditions for producing and for a sufficient period of time, wherein the caprolactam pathway comprises a CoA-dependent aldehyde dehydrogenase; transaminase or 6-aminocaproic acid dehydrogenase; and an amide hydrolase.(Item 71)The method of item 70, further comprising an adipyl-CoA pathway.(Item 72)Item 71, wherein the adipyl-CoA pathway comprises succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and 5-carboxy-2-pentenoyl-CoA reductase. The method described.(Item 73)Item 71, wherein the adipyl-CoA pathway comprises an adipate pathway and an enzyme selected from adipyl-CoA synthesizer, phosphotransadipylase / adipic acid kinase, adipyl-CoA: acetyl-CoA transferase and adipyl-CoA hydrolase. The method described in.(Item 74)23. The adipate pathway comprises succinyl-CoA: acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase. Microorganisms that do not exist in nature.(Item 75)The method of item 70, wherein the non-naturally occurring microorganism is present in a substantially anaerobic culture medium.(Item 76)The method of item 70, wherein the microorganism comprises two exogenous nucleic acids, each encoding a caprolactam pathway enzyme.(Item 77)The method of item 70, wherein the microorganism comprises three exogenous nucleic acids, each encoding a caprolactam pathway enzyme.(Item 78)77. The method of item 77, wherein the three exogenous nucleic acids encode a CoA-dependent aldehyde dehydrolase, transaminase and amide hydrolase.(Item 79)77. The method of item 77, wherein the three exogenous nucleic acids encode a CoA-dependent aldehyde dehydrolase, 6-aminocaproic acid dehydrolase and amide hydrolase.(Item 80)The method of item 70, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.(Item 81)A non-naturally occurring microorganism comprising an adipate pathway containing at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in sufficient quantity to produce adipate, wherein the adipate pathway is alpha. -Ketoadipyl-CoA synthesizer, phosphotransketoadipylase / alpha-ketoadipate kinase or alpha-ketoadipyl-CoA: acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydrogenase; 2-hydroxyadipyl-CoA dehydratase; 5- Carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phosphotransadipylase / adipate kinase, adipyl-CoA: non-naturally occurring microorganisms including acetyl-CoA transferase or adipyl-CoA hydrolase.(Item 82)The non-naturally occurring microorganism of item 81, wherein the microorganism comprises two exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 83)The non-naturally occurring microorganism according to item 81, wherein the microorganism comprises three exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 84)The non-naturally occurring microorganism according to item 81, wherein the microorganism comprises four exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 85)The non-naturally occurring microorganism according to item 81, wherein the microorganism comprises five exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 86)The above five exogenous nucleic acids are alpha-ketoadipyl-CoA synthesizer, phosphotransketoadipylase / alpha-ketoadipate kinase or alpha-ketoadipyl-CoA: acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydrogenase; 2- It encodes hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phosphotransadipylase / adipate kinase, adipyl-CoA: acetyl-CoA transferase or adipyl-CoA hydrolase. , Item 85, a non-naturally occurring microorganism.(Item 87)The non-naturally occurring microorganism according to item 81, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.(Item 88)The non-naturally occurring microorganism according to item 81, which is present in a substantially anaerobic culture medium.(Item 89)A method for producing adipate, a non-naturally occurring microorganism having an adipate pathway containing at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in sufficient quantity to produce adipate, adipate. The adipate pathway includes alpha-ketoadipyl-CoA synthesizer, phosphotransketoadipylase / alpha-ketoadipate kinase or alpha-ketoadipyl-CoA: acetyl- CoA transferase; 2-hydroxyadipyl-CoA dehydrogenase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phosphotransadipylase / adipate kinase, adipyl- CoA: A method comprising an acetyl-CoA transferase or an adipyl-CoA hydrolase.(Item 90)89. The method of item 89, wherein the non-naturally occurring microorganism is present in a substantially anaerobic culture medium.(Item 91)89. The method of item 89, wherein the microorganism comprises two exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 92)89. The method of item 89, wherein the microorganism comprises three exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 93)89. The method of item 89, wherein the microorganism comprises four exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 94)89. The method of item 89, wherein the microorganism comprises five exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 95)The above five exogenous nucleic acids are alpha-ketoadipyl-CoA synthesizer, phosphotransketoadipylase / alpha-ketoadipate kinase or alpha-ketoadipyl-CoA: acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydrogenase; 2- It encodes hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phosphotransadipylase / adipate kinase, adipyl-CoA: acetyl-CoA transferase or adipyl-CoA hydrolase. , Item 94.(Item 96)89. The method of item 89, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.(Item 97)A non-naturally occurring microorganism, comprising a microorganism having an adipate pathway containing at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in sufficient quantity to produce adipate, wherein the adipate pathway is 2 -Hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthesizer, phosphotranshydroxyadipylase / 2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA: acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase Non-naturally occurring microorganisms, including 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phosphotransadipylase / adipate kinase, adipyl-CoA: acetyl-CoA transferase or adipyl-CoA hydrolase.(Item 98)The non-naturally occurring microorganism according to item 97, wherein the microorganism comprises two exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 99)The non-naturally occurring microorganism according to item 97, wherein the microorganism comprises three exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 100)The non-naturally occurring microorganism according to item 97, wherein the microorganism comprises four exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 101)The non-naturally occurring microorganism according to item 97, wherein the microorganism comprises five exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 102)The above five exogenous nucleic acids are 2-hydroxyadipyl dehydrogenase; 2-hydroxyadipyl-CoA synthesizer, phosphotranshydroxyadipylase / 2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA: acetyl-CoA transferase. 2-Hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phosphotransadipylase / adipate kinase, adipyl-CoA: acetyl-CoA transferase or adipyl-CoA hydrolase The non-naturally occurring microorganism according to item 101, which encodes.(Item 103)The non-naturally occurring microorganism according to item 97, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.(Item 104)The non-naturally occurring microorganism according to item 97, which is present in a substantially anaerobic culture medium.(Item 105)Adipate a non-naturally occurring microorganism that has an adipate pathway that contains at least one exogenous nucleic acid that encodes an adipate pathway enzyme that is a method for producing adipate and is expressed in sufficient quantity to produce adipate. The adipate pathway includes 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA kinase, phosphotranshydroxyadipylase / 2-hydroxyadic acid. Kinase or 2-hydroxyadipyl-CoA: acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phosphotransadipylase / adipic acid kinase , Adipyl-CoA: A method comprising an acetyl-CoA kinase or an adipyl-CoA hydrolase.(Item 106)The method of item 105, wherein the non-naturally occurring microorganism is present in a substantially anaerobic culture medium.(Item 107)105. The method of item 105, wherein the microorganism comprises two exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 108)105. The method of item 105, wherein the microorganism comprises three exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 109)105. The method of item 105, wherein the microorganism comprises four exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 110)105. The method of item 105, wherein the microorganism comprises five exogenous nucleic acids, each encoding an adipate pathway enzyme.(Item 111)The above five exogenous nucleic acids are 2-hydroxyadipyl dehydrogenase; 2-hydroxyadipyl-CoA synthesizer, phosphotranshydroxyadipylase / 2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA: acetyl-CoA transferase. 2-Hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phosphotransadipylase / adipate kinase, adipyl-CoA: acetyl-CoA transferase or adipyl-CoA hydrolase The method of item 110, which encodes.(Item 112)The method of item 105, wherein the at least one exogenous nucleic acid is a heterologous nucleic acid.

0008<figref num="1">FIG. 1 is a diagram showing an exemplary adipate degradation pathway in peroxisomes of Penicillium chrysogenum.</figref><figref num="2">FIG. 2 is a diagram showing an exemplary adipate formation pathway by the reverse decomposition pathway. Adipyl-Provides several options for the final conversion of CoA to adipate.</figref><figref num="3">FIG. 3 is a diagram showing an exemplary adipate formation pathway by the 3-oxoadipate pathway.</figref><figref num="4">FIG. 4 shows similar enzymatic chemistry for the 3-oxoadipate pathway for adipate synthesis and the last three steps of the reducing TCA cycle.</figref><figref num="5">FIG. 5 shows an exemplary synthetic pathway of adipic acid from glucose via cis, cis-muconic acid. Biosynthetic intermediates (abbreviation): D-erythrose 4-phosphate (E4P), phosphoenolpyruvate (PEP), 3-deoxy-D-arabinohepturosonic acid 7-phosphate (DAHP), 3-dehydroquinic acid (DHQ), 3-dehydroshikimic acid (DHS), protocatechuic acid (PCA). Enzyme (coding gene) or reaction conditions: (a) DAHP synthase (aroFFBR), (b) 3-dehydroquinate synthase (aroB), (c) 3-dehydroquinate dehydratase (aroD), (d) DHS dehydratase (aroZ) , (E) Protocatechuate decarboxylase (aroY), (f) Catechol 1,2-dioxygenase (catA), (g) 10% Pt / C, H2, 3400kPa, 25 ° C. Figure taken from Niu et al., Biotechonol.Prog. 18: 201-211 (2002).</figref><figref num="6">FIG. 6 shows an exemplary adipate synthesis pathway via alpha-ketoadipate using alpha-ketoglutarate as a starting point.</figref><figref num="7">FIG. 7 shows an exemplary synthetic pathway of adipate using lysine as a starting point.</figref><figref num="8">FIG. 8 shows an exemplary caprolactam synthetic pathway using adipyl-CoA as a starting point.</figref><figref num="9">FIG. 9 shows an exemplary adipate synthetic pathway using alpha-ketoadipate as a starting point.</figref>

0009The present invention relates to the design and production of cells and organisms capable of producing biosynthetic production of adipate, 6-aminocaproic acid or caprolactam. The results described herein indicate that metabolic pathways can be designed and recombined in Escherichia coli and other cells or organisms to achieve biosynthesis of adipate, 6-aminocaproic acid or caprolactam. Biosynthetic production of adipate, 6-aminocaproic acid and caprolactam can be confirmed by constructing strains with the designed metabolic genotype. These cells or organisms whose metabolic pathways have been genetically engineered, including those in a state approximately equal to theoretical maximum proliferation, are subjected to adaptive evolution to further increase adipate, 6-aminocaproic acid or caprolactam biosynthesis. You can also do it.

0010As disclosed herein, a number of metabolic pathways for the production of adipate, 6-aminocaproate and caprolactam will be described. The two pathways, the reverse adipic acid degradation pathway and the 3-oxoadipate pathway, are: (i) adipate yield (92% molar yield based on glucol), (ii) lack of oxygen requirements for adipate synthesis, It has proved useful in terms of (iii) incidental energy properties and (iv) the theoretical potential for producing adipate as a monofermented product. It produces lower yields and requires aeration for maximum production, but also describes metabolic pathways for adipate production through α-keto adipate or lysine. Also disclosed herein are routes for producing one or both of 6-aminocaproate and caprolactam from adipyl-CoA (a precursor in the reverse degradation pathway).

0011As disclosed herein, a number of exemplary biosynthetic pathways for adipate will be described. One exemplary pathway involves adipic acid synthesis by a route that relies on the reversibility of adipic acid degradation as described for organisms such as P. chrysogenum (see Examples I and II). The second exemplary pathway requires the formation of 3-oxoadipate followed by its reduction, dehydration and reduction again to form adipate (see Examples III and IV). The yield of adipate using either of these two routes is 0.92 mol per mole of glucose consumed. No oxygen uptake is required to obtain these theoretical maximum yields, and the energy properties under anaerobic conditions are suitable for growth and product secretion. Methods for producing glucose-derived adipate from cis, cis-muconic acid have been previously described (Frost et al., US Pat. No. 5,487,987, issued January 30, 1996) (see Example V). .. The advantages of the embodiments disclosed herein over this previously described method are discussed. Metabolic pathways for adipate production through α-keto adipate (Example VI) or lysine (Example VII) precursors produce lower yields and require aeration for maximum production. A pathway for producing one or both of 6-aminocaproate and caprolactam from adipyl-CoA, a precursor in the reverse degradation pathway, is also described (see Examples VIII and IX). Further production pathways for adipate will be described in Examples X and XI. Illustrative genes and enzymes required to construct microorganisms with these capabilities, as well as cloning and transformation methods, methods of monitoring product formation, and methods of using genetically engineered microorganisms for production will be described.

0012As disclosed herein, six different pathways of adipic acid synthesis using glucose / sucrose as the carbon substrate are described. For all maximum yield calculations, an E. coli stoichiometric network in SimPheny similar to that previously described (Reed et al., Genome Biol. 4: R54 (2003)) of the reaction in a given pathway. Add a deletion. Since adipate is a charged molecule under physiological conditions, it was thought that it would require energy in the form of a proton-based symport system to be secreted out of its network. Such a transport system is thermodynamically viable when fermentation is carried out at neutral or near neutral pH. Low pH adipic acid formation will require an ATP-dependent export mechanism, such as the ABC system, as opposed to proton symports. Reactions in these pathways and methods of executing these pathways will be described in Examples I-XI.

0013As used herein, the term "non-naturally occurring" as used with respect to a microorganism of the invention or microorganism is commonly used in naturally occurring strains of the species, including wild-type strains of the species. Means having at least one genetic modification that cannot be found. Genetic modifications include, for example, modifications that introduce an expressible nucleic acid encoding a metabolic polypeptide, other nucleic acid additions, nucleic acid deletions and / or other functional disruptions of the microbial genetic material. Such modifications include, for example, coding regions for homologous polypeptides, homologous polypeptides, or both homologous and homologous polypeptides of the type and functional fragments thereof. Additional modifications include, for example, non-coding regulatory regions where modification modifies gene or operon expression. Exemplary metabolic polypeptides include adipate, 6-aminocaproic acid or enzymes within the caprolactam biosynthetic pathway.

0014Metabolic modification refers to a biochemical reaction that is modified from its naturally occurring state. Thus, non-naturally occurring microorganisms may have genetic modifications to nucleic acids encoding metabolic polypeptides and their functional fragments. Illustrative metabolic modifications are disclosed herein.

0015As used herein, the term "isolated" as used with respect to a microorganism is construed to mean an organism that is substantially free of at least one component when the microorganism is found in nature. The term includes microorganisms extracted from some or all components where it is found in its natural environment. The term also includes microorganisms that are extracted from some or all components where the microorganism is found in a non-naturally occurring environment. Thus, isolated microorganisms are mildly or completely isolated from other substances if they are found in nature, or if they are grown, stored or survived in a non-naturally occurring environment. Has been done. Specific examples of isolated microorganisms include mildly pure microorganisms, substantially pure microorganisms, and microorganisms cultured in non-naturally occurring media.

0016As used herein, the terms "microorganism", "microorganism" or "microorganism" are meant to mean any organism that exists as a microscopic cell within the realm of archaea, bacteria or eukaryotes. Be interpreted. Thus, the term is construed to include prokaryotic or eukaryotic cells or organisms of microscopic size and includes all species of bacteria, archaea and eubacteria, as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of biochemical products.

0017As used herein, the term "CoA" or "coenzyme A" is an organic cofactor or organic cofactor whose presence is required for the activity of many enzymes (apoenzymes) to form an active enzyme system. Interpreted to mean the prosthetic group (the non-protein portion of the enzyme). Coenzyme A functions in certain condensing enzymes and acts during acetyl or other acyl group transitions, as well as during fatty acid synthesis and oxidation, Pilbert oxidation, and during other acetylation.

0018As used herein, the chemical formula -OOC- (CH)2)4An "adipate" with -COO- (see Figure 2) (IUPAC name: hexanedioate) is an ionized form of adipic acid (IUPAC name: hexanedioic acid), and adipic acid and adipic acid are any salts thereof. It is understood that it may be used interchangeably throughout to refer to a compound in any neutral or ionized form, including form. It will be understood by those skilled in the art that the specific form depends on its pH.

0019As used herein, the chemical formula -OOC- (CH)2)5-NH2"6-Aminocaproate" with (see Figure 8) is an ionized form of 6-aminocaproate (IUPAC name: 6-aminohexanoic acid), as well as 6-aminocaproate and 6-aminocaproate. , It is understood that it may be used interchangeably throughout to refer to a compound in any neutral or ionized form, including its optional salt form. It will be understood by those skilled in the art that the specific form depends on its pH.

0020As used herein, "caprolactam" (IUPAC name: azepan-2-one) is the lactam of 6-aminohexanoic acid (see Figure 8).

0021As used herein, the term "substantially anaerobic" as used with respect to culture or growth conditions means that the amount of oxygen is less than about 10% saturated with respect to the dissolved oxygen in the liquid medium. Interpreted to mean. The term is also interpreted to include a closed chamber of liquid or solid medium maintained in an atmosphere of less than about 1% oxygen.

0022"Extrinsic" is to be construed as meaning that the molecule or activity is introduced into a host microorganism as it is used herein. The molecule can be introduced, for example, by introduction of a coding nucleic acid into a host genetic material, eg by integration into a host chromosome, or as a non-chromosomal genetic material such as a plasmid. Thus, the term refers to the introduction of a coding nucleic acid into a microorganism in an expressible form when it is used with respect to the expression of the coding nucleic acid. When used with respect to biosynthetic activity, the term refers to activity that is introduced into a host reference organism. The source can be, for example, an allogeneic or heterologous coding nucleic acid that expresses the activity after introduction into a host microorganism. Thus, the term "endogenous" refers to the molecule or activity present in the host. Similarly, when used with respect to the expression of a coding nucleic acid, the term refers to the expression of a coding nucleic acid contained in a microorganism. The term "heterologous" refers to a molecule or nucleic acid derived from a source other than the species, whereas "homogeneous" refers to a molecule or activity derived from a host microorganism. Thus, exogenous expression of the coding nucleic acids of the invention may utilize either heterologous or homologous coding nucleic acids, or both.

0023The non-naturally occurring microorganism of the present invention can have a stable genetic modification, which refers to a microorganism that can be cultured for more than 5 generations without losing the modification. In general, stable genetic modifications include modifications that last longer than 10 generations, in particular stable modifications last longer than about 25 generations, and more particularly stable genetic modifications, including indefinite 50. Will be longer than a generation.

0024Genetic modifications, including the metabolic modifications exemplified herein, are suitable for suitable host organisms, such as E. coli and their corresponding metabolic reactants, or desired genetic material such as genes for the desired metabolic pathway. What is described about the source organism will be understood by those skilled in the art. However, given the high level of technology in the field of complete genome sequencing and genomics for a wide variety of organisms, one of ordinary skill in the art will readily apply the teachings and guidance provided herein to essentially all other organisms. You can do it. For example, the E. coli metabolic modifications exemplified herein can be readily applied to other species by incorporating the same or similar coding nucleic acids from species other than that species. Such gene duplications include, for example, gene duplications of species homologues in general, and ortholog, paralog or non-ortholog gene duplications in detail.

0025Orthologs are genes (s) that are associated by vertical descent and are responsible for substantially the same or identical function in different organisms. For example, mouse epoxide hydrolases and human epoxide hydrolases can be considered orthologs for the biological function of epoxide hydrolysis. Genes are associated by vertical descent, or by evolution from a common ancestor, for example, when they share a sufficient amount of sequence similarity to indicate that they are homologous. Genes do not necessarily share sequence similarity, but can also be considered orthologs if they share a sufficient amount of three-dimensional structure to indicate that they have evolved from a common ancestor to the extent that primary sequence similarity cannot be identified. A gene that is an ortholog can encode a protein that has sequence similarity of about 25% to about 100% amino acid sequence identity. Genes encoding proteins that share less than 25% amino acid similarity, as well as their three-dimensional structure, can be considered to have been caused by vertical descent if they show similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are thought to have arisen from a common ancestor by vertical descent.

0026Orthologs include genes or their encoded gene products that have diverged in terms of structure or overall activity, for example by evolution. For example, if one species encodes a gene product that exhibits two functions, and if such function is divided into different genes in a second generation species, then these three genes and their corresponding products Is considered an ortholog. It will be appreciated by those skilled in the art that in the production of biochemical products, ortholog genes possessing metabolic activity that is introduced or disrupted may be selected for the construction of non-naturally occurring microorganisms. Will. An example of an ortholog showing separable activity is when different activities are divided into different gene products between two or more species or within a single species. Specific examples are elastase proteolysis and separation of plasminogen proteolysis (two types of serine prosthesis activity) into different molecules as plasminogen activator and elastase. The second example is the separation of mycoplasma 5'-3'exonuclease and Drosophila DNA polymerase III activity. DNA polymerases from the primary species can be considered orthologs with either or both of the exonucleases and polymerases from the secondary species, and vice versa.

0027In contrast, paralogs are, for example, homologs associated by duplication and subsequent evolutionary divergence, with non-identical but similar or common functions. Paralogs may, for example, originate from or be derived from the same species, or may arise from or be derived from different species. For example, microsomal epoxide hydrolase (epoxy glucoside hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II), since different reactions in the same species represent two different enzymes co-evolution from a common ancestor with catalytic Mr. different functions, paralogs Can be thought of. Paralogs are proteins from the same species that have significant sequence similarity to each other, suggesting that they are homologues or are related by co-evolution from a common ancestor. The group of paralog protein families includes HipA homologs, luciferase genes, peptidases, and others.

0028A non-ortholog gene replacement is a non-ortholog gene from one species that can replace the gene function in a different species. Substitution includes, for example, being able to perform substantially the same or similar function in the species of origin as compared to that function in different species. In general, non-ortholog gene substitutions could be identified as structurally related to known genes encoding the function, but less structurally related but functionally similar genes and them. The corresponding gene product of is in the meaning of this term, as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of the non-ortholog gene product compared to the gene encoding the function to be replaced. Thus, non-ortholog genes include, for example, paralogs or unrelated genes.

0029Therefore, when identifying and constructing a non-naturally occurring microorganism of the present invention capable of biosynthesizing adipate, 6-aminocaproic acid or caprolactam, applying the teachings and guidance provided herein to a particular species will result in metabolic modification. It will be appreciated by those skilled in the art that identification may include identifying and including or inactivating orthologs. If paralog and / or non-ortholog gene duplications are present in the microorganism encoding an enzyme that catalyzes a similar or substantially similar metabolic reaction, those of skill in the art may also utilize these evolutionarily related genes. it can.

0030Ortholog, paralog and non-ortholog gene duplications can be determined by methods well known to those of skill in the art. For example, testing the nucleic acid or amino acid sequences of two polypeptides will reveal sequence identity and similarity between the sequences to be compared. Based on such similarities, one of ordinary skill in the art can determine if the similarities are high enough to indicate that those proteins are associated by evolution from a common ancestor. .. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine untreated sequence similarity or identity, and also determine the presence or significance of gaps within the sequence that can be assigned to weight or score. Such algorithms are also known in the art and can be similarly applied to the determination of nucleotide sequence similarity or identity. Sufficient similarity to determine relevance based on statistical similarity, or the opportunity to find similar matches in random polypeptides, and well-known methods for calculating the significance of determined matches. Computerize the parameters. Those skilled in the art can also visually optimize computer comparisons of two or more sequences, if desired. The related gene product or protein can be expected to have high similarity, eg 25% to 100% sequence identity. Unrelated proteins can have an identity that is essentially the same as expected to occur by chance when scanned for a sufficiently large database (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the sequences being compared are related. Additional statistical analysis to determine the significance of such matches given the size of the dataset can be performed to determine the relevance of these sequences.

0031Illustrative parameters for determining the relevance of two or more sequences using the BLAST algorithm can be, for example, as shown below. Simply put, amino acid sequence alignment can be done with BLASTP version 2.0.8 (January 5, 1999) and the following parameters: Matrix: 0 BLOSUM62; Gap start: 11; Gap extension: 1; x_ Fall: 50; Expected Value: 10.0; Word Size: 3; Filter: On. Nucleic acid sequence alignment can be performed using BLASTN version 2.0.6 (September 16, 1998) and the following parameters: Match: 1; Mismatch: -2; Gap start: 5; Gap extension: 2; x_Fall: 50; Expected Value: 10.0; Word Size: 11; Filter: Off. For example, those skilled in the art will know what changes can be made to the above parameters to increase or decrease the stringency of the comparison and to determine the relevance of two or more sequences. You will understand.

0032The present invention provides non-naturally occurring microorganisms capable of producing adipate, 6-aminocaproic acid or caprolactam. For example, the adipate pathway may be the inverse adipate degradation pathway (see Examples I and II). In one embodiment, the invention is a non-naturally occurring microorganism having an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in an amount sufficient to produce the adipate. The adipate pathway is succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthesizer or phosphotrans. Adipylase (phosphotransadipylase) / adipate kinase (adipate) Kinase) or adipyl-CoA: The above-mentioned non-naturally occurring microorganisms comprising acetyl-CoA transferase or adipyl-CoA hydrolase. In addition, the adipate pathway may follow the 3-oxoadipate pathway (see Examples III and IV). In another embodiment, the invention is a non-naturally occurring microorganism having an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in an amount sufficient to produce adipate. The adipate pathways are succinyl-CoA: acetyl-CoA acyltransferase, 3-oxoadipyl-CoA reductase, 3-oxoadipate reductase, 3-hydroxyadipate reductase, and 2 -Provides the above-mentioned non-naturally occurring microorganisms, which contain 2-enoate reductase.

0033In yet another embodiment, the invention comprises a 6-aminocaproic acid pathway comprising at least one exogenous nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in an amount sufficient to produce 6-aminocaproic acid. Provided are said non-naturally occurring microorganisms having such non-naturally occurring microorganisms, wherein the 6-aminocaproic acid pathway comprises a CoA-dependent aldehyde dehydrogenase and transaminase (Examples VIII and IX). Alternatively, 6-aminocaproate Dehydrogenase) can be used to convert adipic acid semialdehyde to form 6-aminocaproate (see Figure 8). In a further embodiment, the invention is a non-naturally occurring microorganism having a caprolactam pathway comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in an amount sufficient to produce caprolactam. The above non-naturally occurring microorganisms are provided in which the caprolactam pathway comprises a CoA-dependent aldehyde dehydrogenase, transaminase or 6-aminocaproic acid dehydrogenase, and amide hydrolase (see Examples VIII and IX).

0034As disclosed herein, the 6-aminocaproic acid or caprolactam-producing microorganisms of the present invention can produce 6-aminocaproic acid and / or caprolactam from the adipyl-CoA precursor (FIG. 8 and Example VIII). And IX). Thus, 6-aminocaproic acid or caprolactam-producing microorganisms may further include pathways for producing adipyl-CoA. For example, the adipyl-CoA pathway utilizes succinyl-CoA and acetyl-CoA as precursors for the production of adipyl-CoA, ie there is no enzyme for the final step of converting adipyl-CoA to adipate, figure. May contain 2 enzymes. Thus, one exemplary adipyl-CoA pathway includes succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and 5-carboxy-2-pentenoyl-CoA reductase. May include.

0035In addition, as shown in FIG. 1, the adipate degradation pathway involves the conversion of adipate to adipyl-CoA by adipate-CoA ligase. Thus, the adipyl-CoA pathway is the enzymatic activity that converts adipate to adipyl-CoA, including the adipyl-CoA ligase activity as in the first step of FIG. 1, or the final step of FIG. 2 that takes place in the opposite direction. Any of the enzymes in, for example, adipyl-CoA synthesizer (also called adipyl-CoA ligase), phosphotransadipylase / adipate kinase, adipyl-CoA: acetyl-CoA transferase or adipyl-CoA hydrolase. It may also be an adipate pathway that includes. Enzymes with adipate to adipyl-CoA activity may be endogenous activity, as disclosed herein, or may be supplied as an exogenous nucleic acid encoding the enzyme. Thus, any adipate pathway can be utilized with adipate-to-adipyl-CoA enzymatic activity to yield the adipyl-CoA pathway. Such pathways can be included in 6-aminocaproic acid or caprolactam-producing microorganisms to give rise to 6-aminocaproic acid and / or adipyl-CoA precursors for caprolactam production.

0036An additional exemplary adipate pathway utilizes alpha-ketoadipate as a precursor (see Figure 6 and Example VI). In yet another embodiment, the invention is a non-naturally occurring microorganism having an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in an amount sufficient to produce adipate. Provided with said non-naturally occurring microorganisms, wherein the adipate pathway comprises homocitrate synthase, homoaconitase, homoisocitrate dehydrogenase, 2-ketoadipate reductase, alpha-hydroxyadipate dehydrase and oxidoreductase. A further exemplary adipate pathway utilizes the lysine degradation pathway (FIG. 7 and Example VII). Another embodiment of the invention is a non-naturally occurring microorganism having an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in an amount sufficient to produce adipate. Provided are said non-naturally occurring microorganisms, wherein the adipate pathway comprises carbon-nitrogen lyase, oxidoreductase, transaminase and oxidoreductase.

0037Yet another exemplary adipate pathway utilizes alpha-ketoadipate as a precursor (see Figure 9 and Examples X and XI). Accordingly, the present invention is a non-naturally occurring microorganism having an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in an amount sufficient to produce adipate, wherein the adipate pathway is , Alpha-ketoadipyl-CoA synthetase, phosphotransketoadipylase / alpha-ketoadipate Kinase) or alpha-ketoadipyl-CoA: acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydrogenase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phospho Further provided are the non-naturally occurring microorganisms said to include transadipylase / adipate kinase, adipyl-CoA: acetyl-CoA transferase or adipyl-CoA hydrolase. In yet another embodiment, the invention is a non-naturally occurring microorganism having an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in an amount sufficient to produce adipate. , The adipate pathway is 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthesizer, phosphotranshydroxyadipylase / 2-hydroxyadipate kinase. Kinase) or 2-hydroxyadipyl-CoA: acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phosphotransadipylase / adipic acid Provided are non-naturally occurring microorganisms, including kinases, adipyl-CoA: acetyl-CoA transferase, or adipyl-CoA hydrolase.

0038In additional embodiments, the invention encodes a non-naturally occurring microorganism having an adipate, 6-aminocaproic acid or caprolactam pathway, a polypeptide that converts a substrate into a product, as disclosed herein. It provides a non-naturally occurring microorganism containing at least one exogenous nucleic acid. Accordingly, the present invention is an adipate, 6 such as a non-naturally occurring microorganism containing at least one exogenous nucleic acid encoding a polypeptide, wherein the polypeptide is shown in FIGS. 2, 3, 8 or 9. -Provides the above-mentioned non-naturally occurring microorganisms, which are enzymes or proteins that convert substrates and products of the aminocaproic acid or caprolactam pathway.

0039In one embodiment, the invention is a non-naturally occurring microorganism with an adipate pathway, succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. To produce a substrate selected from; 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; adipyl-CoA to adipate, Provided are said microorganisms containing at least one exogenous nucleic acid encoding a polypeptide that converts to (see Figure 2). In another embodiment, the invention is a non-naturally occurring microorganism having an adipate pathway, succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; 3-oxoadipyl-CoA to oxoadipate; 3- Encodes a polypeptide that converts a substrate selected from oxoadipate to 3-hydroxyadipate; 3-hydroxyadipate to hexa-2-endioate; hexa-2-engioate to adipate, to a product (see Figure 3). The above-mentioned microorganism containing at least one exogenous nucleic acid is provided.

0040In additional embodiments, the invention is a non-naturally occurring microorganism having a 6-aminocaproic acid pathway, adipyl-CoA to adipic acid semialdehyde; and adipic acid semialdehyde to 6-aminocaproate. Provided are said microorganisms containing at least one exogenous nucleic acid encoding a polypeptide (see FIG. 8) that converts a substrate selected from the above into a product. In yet another embodiment, the invention is a non-naturally occurring microorganism with a caprolactam pathway, which converts adipyl-CoA to adipic acid semialdehyde; adipic acid semialdehyde to 6-aminocaproate; and 6-. Provided are said microorganisms containing at least one exogenous nucleic acid encoding a polypeptide that converts an amino caproate into caprolactam and a substrate selected from.

0041In yet another embodiment, the invention is a non-naturally occurring microorganism with an adipate pathway that converts alpha-ketoadipate to alpha-ketoadipyl-CoA; alpha-ketoadipyl-CoA to 2-hydroxyadipyl-CoA. 2-Hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and adipyl-CoA to adipate, producing substrates selected from Provided are said microorganisms containing at least one exogenous nucleic acid encoding a polypeptide that converts to (see FIG. 9). In addition, the present invention is a non-naturally occurring microorganism with an adipate pathway that converts alpha-ketoadipate to 2-hydroxyadipate; 2-hydroxyadipate to 2-hydroxyadipyl-CoA; 2-hydroxyadipyl. -A polypeptide that converts a substrate selected from 5-carboxy-2-pentenoyl-CoA to 5-carboxy-2-pentenoyl-CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and adipyl-CoA to adipate, to the product. Provided are said microorganisms containing at least one exogenous nucleic acid encoding (see Figure 9).

0042In general, with respect to a metabolic reaction, its reactants or products, or specifically, one or more enzymes that accompany or catalyze the metabolic reaction, reactants or products. The present invention is described herein with respect to nucleic acids or genes. It will be appreciated by those skilled in the art that reference to a reaction also refers to a reactant and product of the reaction, unless otherwise stated herein. Similarly, unless otherwise stated herein, reference to a reactant or product is also a reference to that reaction, and reference to any of these metabolic components is a reference to that reaction. It is also a reference to the gene (s) encoding the enzyme that catalyzes the reactant or product. Similarly, given the well-known fields of metabolic biochemistry, enzymology and genomics, references herein to genes or coding nucleic acids refer to the corresponding encoded enzymes and their catalytic reactions and their reactants and reactants of those reactions. It is also a reference to the product.

0043By introducing an expressible nucleic acid encoding one or more adipates, 6-aminocaproic acid or one or more of the enzymes involved in the caprolactam biosynthetic pathway, the non-naturally occurring microorganisms of the invention can be produced. .. Nucleic acids can be expressed for some or all of the specific adipate, 6-aminocaproic acid or caprolactam biosynthetic pathways, depending on the host microorganism selected for biosynthesis. For example, if the host of choice is deficient in one or more enzymes for the desired biosynthetic pathway, the nucleic acids expressible for those (them) deficient enzymes will later be exogenous. Introduced into the host for expression. Alternatively, if the selected host exhibits endogenous expression of some pathway genes but lacks others, then to achieve adipate, 6-aminocaproic acid or caprolactam biosynthesis ( Requires coding nucleic acids for those) deletion enzymes. Thus, by introducing exogenous enzyme activity to obtain the desired biosynthetic pathway, the non-naturally occurring microorganisms of the invention can be produced or desired products such as adipate, 6-aminocaproic acid or caprolactam. The desired biosynthetic pathway can be obtained by introducing one or more enzyme activities that are produced in cooperation with one or more endogenous enzymes.

0044Depending on the host microorganism adipate, 6-aminocaproic acid or caprolactam biosynthetic pathway component of choice, the non-naturally occurring microorganisms of the invention are at least one extrinsically expressed adipate, 6-aminocaproic acid or caprolactam. It will include a pathway-coding nucleic acid and all the coding nucleic acids for one or more adipates, 6-aminocaproic acid or caprolactam biosynthetic pathways. For example, exogenous expression of the corresponding coding nucleic acid in a host lacking a pathway enzyme can establish adipate, 6-aminocaproic acid or caprolactam biosynthesis. For hosts deficient in all enzymes of the adipate, 6-aminocaproic acid or caprolactam pathway, exogenous expression of all enzymes in that pathway can be included, but the host is at least one of those pathway enzymes. It is understood that all enzymes of the pathway can be expressed, even if they contain one.

0045For example, all enzymes in the pathway for adipate production, such as succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl. Extrinsic expression of -CoA reductase and adipyl-CoA synthesizer or phosphotransadipylase / adipate kinase or adipyl-CoA: acetyl-CoA transferase or adipyl-CoA hydrolase can be included in the host organism. Specifically, the host organism is the adipate pathway enzyme succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and May contain adipyl-CoA synthesizer. Alternatively, the host organism can be the adipate pathway enzyme succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and phosphotrans. May contain adipylase / adipate kinase. In addition, host organisms include the adipate pathway enzyme succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl. -CoA: May contain acetyl-CoA transferase. In addition, the host organism is the adipate pathway enzyme succinyl-CoA:

0046For 6-aminocaproic acid-producing microorganisms, exogenous expression of all enzymes in the pathway for the production of 6-aminocaproic acid, such as CoA-dependent aldehyde dehydrogenase and transaminase or CoA-dependent aldehyde dehydrogenase and 6-aminocaproic acid dehydrogenase. Can be included in the host organism. For caprolactam-producing microorganisms, the host organism can include exogenous expression of all enzymes in the pathway for caprolactam production, such as CoA-dependent aldehyde dehydrogenase, transaminase or 6-aminocaproic acid dehydrogenase, and amidohydrolase. ..

0047Given the teachings and guidance provided herein, the number of coding nucleic acids introduced in an expressible form is at least comparable to the lack of adipate, 6-aminocaproic acid or caprolactam pathways of selected host microorganisms. It will be understood by those skilled in the art. Thus, the non-naturally occurring microorganisms of the invention are one, two, three, four or five nucleic acids up to all that encode the enzymes that make up the adipate, 6-aminocaproic acid or caprolactam biosynthetic pathway. May have. In some embodiments, the non-naturally occurring microorganism also comprises other genetic modifications that facilitate adipate, 6-aminocaproic acid or caprolactam biosynthesis, or confer other useful functions on its host microorganism. There is. One such other functionality includes, for example, adipate, 6-aminocaproic acid or caprolactam pathway precursors, eg, the adipate pathway enzyme disclosed herein, succinyl-CoA in the case of adipate synthesis. And / or an increase in the synthesis of one or more of acetyl-CoA, or in the case of 6-aminocaproic acid or caprolactam synthesis, adipyl-CoA.

0048In some embodiments, the non-natural microorganisms of the invention are produced from a host having an enzymatic ability to synthesize adipate, 6-aminocaproic acid or caprolactam. In this particular embodiment, for example, the synthesis or accumulation of adipate, 6-aminocaproic acid or caprolactam pathway products to lead the adipate, 6-aminocaproic acid or caprolactam pathway reaction to adipate, 6-aminocaproic acid or caprolactam production. It may be useful to increase. For example, overexpression of a nucleic acid encoding one or more of the adipate, 6-aminocaproic acid or caprolactam pathway enzymes can carry out synthesis or increased accumulation. Overexpression of adipate, 6-aminocaproic acid or caprolactam pathway enzyme (s) is, for example, by exogenous expression of an endogenous gene (s) or by exogenous expression of a heterologous gene (s). It can happen. Thus, naturally occurring organisms, for example, by overexpressing one, two, three, four, five, or all, nucleic acids encoding adipate, 6-aminocaproic acid or caprolactam biosynthetic pathway enzymes, for example. It can be easily produced to be a non-naturally occurring microorganism of the invention that produces adipate, 6-aminocaproic acid or caprolactam. In addition, mutagenesis of endogenous genes that results in increased activity of enzymes in the adipate, 6-aminocaproic acid or caprolactam biosynthetic pathway can produce non-naturally occurring microorganisms.

0049In a particularly useful embodiment, exogenous expression of the coding nucleic acid is utilized. Exogenous expression provides the host and application with the ability to tailor expression and / or regulatory elements to achieve the desired expression levels controlled by the user. However, in other embodiments, endogenous expression can also be utilized, for example by elimination of negative regulatory effectors, or induction of the promoter of the gene when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene with a naturally occurring inducible promoter can be upregulated by supplying an appropriate inducer, or the regulatory region of the endogenous gene can be genetically engineered to produce an inducible regulatory element. It can be incorporated, which allows regulation of increased expression of the endogenous gene at the desired time point. Similarly, inducible promoters can be included as regulators for exogenous genes that are introduced into non-naturally occurring microorganisms.

0050It is understood that in the methods of the invention, any one or more exogenous nucleic acids can be introduced into the microorganism to produce the non-naturally occurring microorganism of the invention. These nucleic acids can be introduced, for example, to confer the microorganism with an adipate, 6-aminocaproic acid or caprolactam biosynthetic pathway. Alternatively, a coding nucleic acid can be introduced to produce an intermediate microorganism capable of catalyzing some of the reactions required to confer the ability to biosynthesize adipate, 6-aminocaproic acid or caprolactam. For example, a non-naturally occurring microorganism having an adipate, 6-aminocaproic acid or caprolactam biosynthetic pathway may contain at least two exogenous nucleic acids encoding the desired enzyme. For adipate production, the at least two exogenous nucleic acids are enzymes such as succinyl-CoA: acetyl-CoA acyl transferase and 3-hydroxyacyl-CoA dehydrogenase, or succinyl-CoA: acetyl-CoA acyl transferase and 3 -A combination of hydroxyadipyl-CoA dehydratase, or a combination of 3-hydroxyadipyl-CoA and 5-carboxy-2-pentenoyl-CoA reductase, or a combination of 3-hydroxyacyl-CoA and adipyl-CoA synthesizer, and these. May code something similar to. For caprolactam production, the at least two exogenous nucleic acids encode an enzyme, eg, a combination of CoA-dependent aldehyde dehydrogenase and transaminase, or a combination of CoA-dependent aldehyde dehydrogenase and amidohydrolase, or a combination of transaminase and amidohydrolase. May be done. Thus, it is understood that any combination of two or more enzymes in the biosynthetic pathway can be included in the non-naturally occurring microorganisms of the invention.

0051Similarly, if the combination of enzymes in the desired biosynthetic pathway results in the production of the corresponding desired product, then any combination of three or more enzymes in the biosynthetic pathway, eg adipate production. In the case of, the combination of the enzyme succinyl-CoA: acetyl-CoA acyl transferase and 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyadipyl-CoA dehydratase, or the enzyme succinyl-CoA: acetyl-CoA acyl transferase and 3- A combination of hydroxyacyl-CoA dehydrogenase and 5-carboxy-2-pentenoyl-CoA reductase, or a combination of succinyl-CoA: acetyl-CoA acyl transferase and 3-hydroxyacyl-CoA dehydrogenase and adipyl-CoA synthesizer, or 3- The combination of hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and adipyl-CoA: acetyl-CoA transferase, etc., can be included in the non-naturally occurring microorganisms of the invention, if desired. Is understood. Similarly, if the combination of enzymes in the desired biosynthetic pathway results in the production of the corresponding desired product, then for four or more enzymes in the biosynthetic pathway as disclosed herein. Any combination can be included in the non-naturally occurring microorganisms of the invention, if desired.

0052In addition to the biosynthesis of adipate, 6-aminocaproic acid or caprolactam as described herein, the non-naturally occurring microorganisms and methods of the invention can be utilized in various combinations with each other, as well as others. It can also be used in various combinations with other microorganisms and methods well known in the art to achieve root product biosynthesis. For example, other than the use of adipate, 6-aminocaproic acid or caprolactam producers, one alternative for producing adipate, 6-aminocaproic acid or caprolactam is adipate, 6-aminocaproic acid or caprolactam pathway intermediate adipate, 6 -Due to the addition of another microorganism that can be converted to aminocaproic acid or caprolactam. One such procedure involves, for example, fermentation of microorganisms that produce adipate, 6-aminocaproic acid or caprolactam pathway intermediates. The adipate, 6-aminocaproic acid or caprolactam pathway intermediate is then used as a substrate for a second microorganism that converts the adipate, 6-aminocaproic acid or caprolactam pathway intermediate to adipate, 6-aminocaproic acid or caprolactam. can do. The adipate, 6-aminocaproic acid or caprolactam pathway intermediate can be added directly to another culture of the second organism, or from the original culture of adipate, 6-aminocaproic acid or caprolactam pathway intermediate product. These microorganisms can be depleted, for example by cell isolation, and then the subsequent addition of the second organism to the fermentation broth can be used to produce the final product without an intermediate purification step.

0053In other embodiments, the non-naturally occurring microorganisms and methods of the invention can be assembled by a wide variety of subroutes to achieve, for example, biosynthesis of adipate, 6-aminocaproic acid or caprolactam. In these embodiments, the biosynthetic pathway for the desired product of the invention can be separated into different microorganisms and the different microorganisms can be co-cultured to produce the final product. In such a biosynthesis scheme, the product of one microorganism becomes a substrate for the other microorganism until the final product is synthesized. Carrying out the biosynthesis of adipate, 6-aminocaproic acid or caprolactam, for example, by constructing a microorganism that has a biosynthetic pathway for the conversion of one pathway intermediate to another pathway intermediate or its product. Can be done. Alternatively, adipate, 6-aminocaproic acid or caprolactam can be biosynthesized from microorganisms by co-culture or co-fermentation using two organisms in the same container, in which case the first microorganism is adipate, 6-aminocapron. It produces an acid or caprolactam intermediate, and a second microorganism converts the intermediate to adipate, 6-aminocaproic acid or caprolactam.

0054Given the teachings and guidance provided herein, co-cultures of non-naturally occurring microorganisms and methods of the invention with other microorganisms, as well as other non-naturally occurring microorganisms having sub-permutations, and. The existence of a wide variety of combinations and permutations with combinations of other chemical and / or biochemical procedures well known in the art for producing adipate, 6-aminocaproic acid or caprolactam is to those skilled in the art. Will be understood.

0055Sources that encode nucleic acids for adipate, 6-aminocaproic acid or caprolactam pathway enzymes include, for example, any species in which the encoded gene product can catalyze the reaction. Such species include, but are not limited to, bacteria including archaea and eubacteria, as well as eukaryotes including, but not limited to, yeasts, plants, insects, animals and mammals (including and humans). ), Includes both prokaryotes and eukaryotes. Illustrative species for such sources include, for example, Escherichia coli, Pseudomonas knackmussii, Pseudomonas putida, Pseudomonas fluorescens, Klebsiella pneumoniae, Serratia proteamaculans, Streptomyces sp.2065, Pseudomonas aeruginosa, Ralstonia denticola, Clostridium kluyveri, Homo sapiens, Rattus norvegicus, Acinetobacter sp.ADP1, Streptomyces coelicolor, Eubacterium barkeri, Peptostreptococcus asaccharolyticus, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium Arthrobacter aurescens, Penicillium chrysogenum, Aspergillus niger, Aspergillus nidulans, Bacillus subtilis, Saccharomyces cerevisiae, Zymomonas mobilis, Mannheimia succiniciproducens, Clostridium ljungdahlii, Clostridium carboxydivorans, Geobacillus stearothermophilus, Agrobacterium tumefaciens, Achromobacter denitrificans, Arabidopsis thaliana, Haemophilus influenzae, Acidaminococcus fermentans, Clostridium sp Nucleatum, as well as other exemplary species (see Examples) that can be used as source organisms for the genes disclosed or corresponding herein. However, complete genome sequences currently available for more than 550 species, including 395 microbial genomes and various yeast, fungal, plant and mammalian genomes (more than half of these are available in public databases such as NCBI). Required adipates for one or more genes in related or distantly related species, including, for example, homologues of known genes, orthologs, paralogs and non-orthologs gene duplications, and mutual exchange of gene duplications between organisms, 6 -Identification of genes encoding aminocaproic acid or caprolactam biosynthetic activity is routine and well known in the art. Thus, metabolic modifications that allow the biosynthesis of adipate, 6-aminocaproic acid or caprolactam described herein for certain organisms such as E. coli to other microorganisms, including prokaryotes and eukaryotes, as well. Easy to apply. Given the teachings and guidance provided herein, one of ordinary skill in the art will appreciate that the metabolic modifications exemplified in one organism can be applied equally to other organisms.

0056In some cases, for example, if an alternative adipate, 6-aminocaproic acid or caprolactam pathway is present in an unrelated species, for example, a paralog from that unrelated species that catalyzes a non-identical but similar metabolic reaction that replaces the reaction. Extrinsic expression of (or more) can result in adipate, 6-aminocaproic acid or caprolactam biosynthesis in the host species. It will be appreciated by those skilled in the art that the actual gene utilization may differ between different organisms, as there are certain differences between metabolic networks between different organisms. However, given the teachings and guidance provided herein, those exemplified herein to construct microorganisms within a species of interest that will synthesize adipate, 6-aminocaproic acid or caprolactam. It will also be appreciated by those skilled in the art that the teachings and methods of the present invention can be applied to all microorganisms using the cognate metabolic modification to.

0057The host microorganism can be selected from, for example, bacteria, yeast, fungi, or various other microorganisms applicable to the fermentation process, and, for example, bacteria, yeast, fungi, or various other microorganisms applicable to the fermentation process. It may be a non-naturally occurring microorganism produced in. Illustrative bacteria include Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans Species selected from fluorescens and Pseudomonas putida can be mentioned. Illustrative yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger and Pichia pastoris. For example, E. coli is a particularly useful host organism as it is a well-characterized microorganism suitable for genetic engineering. Other particularly useful host organisms include yeast, such as Saccharomyces cerevisiae.

0058Methods for constructing and expressing levels of non-naturally occurring adipate, 6-aminocaproic acid or caprolactam-producing hosts can be performed, for example, by recombinant and detection methods well known in the art. Such methods include, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, You can find it in MD (1999).

0059Adipate, 6-aminocapron, using techniques well known in the art, including (but not limited to) conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasonic transformation. Exogenous nucleic acid sequences involved in the pathway for the production of acid or caprolactam can be stably or transiently introduced into the host cell. Due to exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids are, if desired, prior to transformation into prokaryotic host cells. It may encode a targeting signal that can be eliminated, such as an N-terminal mitochondria or other targeting signal. For example, removal of the mitochondrial leader sequence resulted in increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280: 4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, the gene can be expressed in the cytosol without the addition of leader sequences, or suitable such as mitochondrial targeting or secretory signals suitable for their host cells. Addition of targeting sequences can target mitochondria or other organelles or target secretion. Therefore, it will be appreciated that appropriate modifications to the nucleic acid sequence to remove or include the targeting sequence can be introduced into the exogenous nucleic acid sequence to confer the desired properties. Furthermore, optimized expression of proteins can be achieved by attaching genes to codon optimization in techniques well known in the art.

0060Expression vectors such as containing one or more adipates, 6-aminocaproic acid or caprolactam biosynthetic pathways encoding nucleic acids as exemplified herein operably linked to expression control sequences capable of functioning in the host organism. You can build one or more). Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, including vectors that can be used for stable integration into the host chromosome and selective sequences or markers. , Episomes and artificial chromosomes. In addition, the expression vector may contain one or more selectable marker genes and appropriate expression control sequences. For example, selectable marker genes that provide resistance to antibiotics or toxins, supplement auxotrophic deficiencies, or supply important nutrients that are not in the culture medium can also be included. Expression control sequences include constitutive and inducible promoters, transcription enhancers, transcription terminators and the like, which are well known in the art. When attempting to co-express two or more exogenous nucleic acids encoding, both nucleic acids can be inserted, for example, into a single expression vector or into separate expression vectors. For single vector expression, the coding nucleic acid can be operably linked to one common expression control sequence, or to a different expression control sequence, eg, one inducible promoter and one constitutive promoter. Can be done. Conversion of exogenous nucleic acid sequences involved in metabolic or synthetic pathways can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, eg, Northern blot or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of a gene product, or an introduced nucleic acid sequence or its corresponding gene product. Other suitable analytical methods for testing expression include. Exogenous nucleic acid

0061In addition, the present invention provides a method for producing the desired product, such as adipate, 6-aminocaproic acid or caprolactam. In one embodiment, the invention is a method for producing adipate, which comprises an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in sufficient quantity to produce adipate. The adipate pathway comprises succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3 which involves culturing the non-naturally occurring microorganisms with adipate under conditions and for a sufficient period of time. Includes -hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthesizer or phosphotransadipylase / adipate kinase or adipyl-CoA: acetyl-CoA transferase or adipyl-CoA hydrolase Provide a method that is irrelevant. In another embodiment, the invention is a method for producing adipate, an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in sufficient quantity to produce adipate. The adipate pathway comprises succinyl-CoA: acetyl-CoA acyltransferase, 3-oxoadipyl-CoA reductase, 3 which comprises culturing a non-naturally occurring microorganism having an adipate under conditions producing adipate and for a sufficient period of time. -Provides methods that include oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate reductase.

0062In yet another embodiment, the invention encodes a 6-aminocaproic acid pathway enzyme that is a method for producing 6-aminocaproic acid and is expressed in an amount sufficient to produce 6-aminocaproic acid. The 6-aminocaproic acid pathway comprises culturing a non-naturally occurring microorganism having a 6-aminocaproic acid pathway containing at least one exogenous nucleic acid under conditions producing 6-aminocaproic acid and for a sufficient period of time. , CoA-dependent aldehyde dehydrogenase and transaminase or 6-aminocaproic acid dehydrogenase. In a further embodiment, the invention is a method for producing caprolactam, the caprolactam pathway comprising at least one exogenous nucleic acid encoding a caprolactam pathway enzyme expressed in sufficient quantity to produce caprolactam. The caprolactam pathway comprises coA-dependent aldehyde dehydrogenase, transaminase, 6-aminocaproic acid dehydrogenase, and amide hydrolase, comprising culturing a non-naturally occurring microorganism having caprolactam under conditions that produce caprolactam and for a sufficient period of time. Provide a way to be ridiculous.

0063In addition, the present invention is a method for producing adipate, a natural having an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in sufficient quantity to produce adipate. The adipate pathway comprises culturing a microorganism that is not present in the drug under conditions that produce adipate and for a sufficient period of time, the adipate pathway being alpha-ketoadipyl-CoA synthesizer, phosphotransketoadipylase / alpha-ketoadipate kinase or alpha. -Ketoadipyl-CoA: Acetyl-CoA transferase; 2-Hydroxyadipyl-CoA dehydrogenase; 2-Hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer, phosphotransadipylase / Adipate kinase, adipyl-CoA: Provided is a method comprising an acetyl-CoA transferase or adipyl-CoA hydrolase.

0064In yet another embodiment, the invention is a method for producing adipate, which comprises at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in an amount sufficient to produce adipate. Including culturing non-naturally occurring microorganisms with adipic acid under conditions producing adipate and for a sufficient period of time, the adipate pathway is 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA kinase, phosphotrans. Hydroxyadipylase / 2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA: Acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthesizer , Phosphotransadipylase / adipic acid kinase, adipyl-CoA: acetyl-CoA transferase or adipyl-CoA hydrolase.

0065Assays for testing for suitable purification and / or production of adipate, 6-aminocaproic acid or caprolactam can be performed using well-known methods. For each of the genetically engineered strains to be tested, grow suitable replicas, such as triple repeat cultures. For example, product and by-product formation in genetically engineered production hosts can be monitored. By methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectrometry) and LC-MS (Liquid Chromatography-Mass Spectrometry), using routine procedures well known in the art. The final product and intermediates, as well as other organic compounds, can be analyzed. The culture supernatant can also be used to test for release of the product into the fermentation broth. For example, HPLC using a refractive index for glucose and alcohol, and a UV detector for organic acids (Lin et al., Biotechnol.Bioeng. 90: 775-779 (2005)), or other well known in the art. By-products and residual glucose can be quantified by suitable assays and detection methods. Individual enzymatic activity from exogenous DNA sequences can also be assayed using methods well known in the art.

0066Various methods well known in the art can be used to separate adipate, 6-aminocaproic acid or caprolactam from other components in the culture. Such separation methods include, for example, extraction procedures, as well as continuous liquid-liquid extraction, dialysis evaporation, membrane filtration, membrane separation, backpenetration, electrodialysis, distillation, crystallization, centrifugation, extraction filtration, ion exchange chromatography. , Size exclusion chromatography, adsorption chromatography, and methods including ultrafiltration. All of the above methods are well known in the art.

0067Any non-naturally occurring microorganism described herein can be cultivated to produce and / or secrete the biosynthetic product of the invention. For example, adipate, 6-aminocaproic acid or caprolactam producers can be cultivated for biosynthetic production of adipate, 6-aminocaproic acid or caprolactam.

0068Cultivate the recombinant strain in medium with a carbon source and other essential nutrients for the production of adipate, 6-aminocaproic acid or caprolactam. It is highly desirable to maintain anaerobic conditions in the fermenter in order to reduce the cost of the entire process. Such conditions can be obtained, for example, by first spraying the medium with nitrogen and then sealing the flasks with septams and crimp caps. For strains in which growth is not observed anaerobic, microaerobic conditions can be utilized by providing small holes in the septum for restricted ventilation. The exemplary anaerobic conditions have been described above and are well known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in US Patent Application No. 11 / 891,602 filed August 10, 2007. Fermentation can be carried out in batches, fed-batch batches or continuous modes as disclosed herein.

0069If desired, when it is necessary to maintain the culture medium at the desired pH, the pH of the medium can be adjusted to the desired pH, particularly neutral pH, by adding a base, such as NaOH or other base, or an acid. It can be maintained at a pH of, for example, about 7. The growth rate can be determined by measuring the optical density using a spectrophotometer (600 nm), and the glucose uptake rate can be determined by monitoring carbon source depletion over time.

0070The growth medium may be, for example, any carbohydrate source capable of supplying a carbon source to non-developing microorganisms. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Other sources of carbohydrates include, for example, renewable feedstocks and biomass. Illustrative types of biomass that can be used as feedstock in the methods of the invention include cellulosic biomass, hemicellulose biomass and some of the lignin feedstocks or feedstocks. Such biomass feeds contain, for example, carbohydrate substrates useful as carbon sources, such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, renewable feedstocks and biomasses other than those exemplified above are also used in culturing the microorganisms of the invention for the production of adipate, 6-aminocaproic acid or caprolactam. What can be done will be understood by those skilled in the art.

0071In addition to renewable feedstocks such as those exemplified above, the adipate, 6-aminocaproic acid or caprolactam microorganisms of the invention can also be modified for growth using syngas as their carbon source. In this particular embodiment, one or more proteins or enzymes are expressed in adipate, 6-aminocaproic acid or caprolactam-producing organisms to provide a metabolic pathway for the utilization of syngas or other gaseous carbon sources.

0072Syngas, also known as thin gas or generator gas, is the main product of the vaporization of coal and the vaporization of carbonaceous materials such as biomass materials such as agricultural products and residues. Syngas is mainly H2It is a mixture of and CO, and can be obtained from the vaporization of any organic feedstock, including, but not limited to, coal, coal petroleum, natural gas, biomass and organic waste. Vaporization is generally carried out under a high fuel to oxygen ratio. Mostly H2And CO, but Syngas has less CO2And may contain other gases. Therefore, syngas is CO and, moreover, CO.2It becomes a cost-effective gaseous carbon source.

0073The Wood-Ljungdahl pathway provides CO and H to acetyl-CoA and other products such as acetate.2Catalyze the conversion of. Organisms that can utilize CO and syngas have CO by the same basic set of enzymes and conversions contained in the Wood-Ljungdahl pathway.2And CO2/ H2It also generally has the ability to utilize the mixture. CO to acetate by microorganisms2H2Dependent transformations have been recognized long before it became clear that CO could also be used by the same organism and that the same pathways were involved. Many acetic acid-producing bacteria are CO2It has been shown to grow in the presence of hydrogen and, in the presence of hydrogen, produce compounds such as acetate to supplement the required reduction equivalent (eg, Drake, Acetogenesis, pp.3-60 Chapman and Hall, See New York, (1994)). This can be summarized by the following equation: 2CO2+ 4H2+ nADP + nPi CH3COOH + 2H2O + nATP Therefore, non-naturally occurring microorganisms with the Wood-Ljungdah1 pathway also produce CO for the production of acetyl-CoA and other desired products.2And H2A mixture of can be used.

0074The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions, which can be divided into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofuric acid (methyl-THF), whereas the carbonyl branch converts methyl-THF to acetyl-CoA. Reactions at the methylbranch are catalyzed in sequence by the following enzymes: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthesizer, metenyltetrahydrofolate cyclodehydrase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reaction at the carbonyl branch is catalyzed in sequence by the following enzymes: cobalamide corinoid / iron-sulfur protein, methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase, acetyl-CoA synthase disulfide reductase and hydrogenase. Introduce at least the nucleic acid encoding the Wood-Ljungdahl enzyme absent from the host organism according to the teachings and guidance provided above for introducing a sufficient number of coding nucleic acids to generate the adipate, 6-aminocaproic acid or caprolactam pathway. Those skilled in the art will appreciate that the same engineering design can be done in terms of doing. Therefore, introduction of one or more coding nucleic acids into the microorganisms of the invention such that the modified organism contains the complete Wood-Ljungdahl pathway will result in syngas utilization.

0075Given the teachings and guidance provided herein, one of ordinary skill in the art will be able to produce non-naturally occurring microorganisms that secrete the biosynthetic compounds of the invention when grown using carbon sources such as carbohydrates. Will be understood. Such compounds include, for example, adipate, 6-aminocaproic acid or caprolactam, and any intermediate metabolites in the adipate, 6-aminocaproic acid or caprolactam pathway. All that is required is to achieve the biosynthesis of the desired compound or intermediate with respect to one or more of the required enzymatic activities, including including, for example, some or all of the adipate, 6-aminocaproic acid or caprolactam biosynthetic pathways. It's just a matter of genetic engineering. Thus, the present invention produces adipate, 6-aminocaproic acid or caprolactam when grown with carbohydrates, and intermediates shown in the adipate, 6-aminocaproic acid or caprolactam pathway when grown with carbohydrates. Provides non-naturally occurring microorganisms that produce and / or secrete any of the metabolites. For example, adipate-producing microorganisms can optionally be from intermediates such as 3-oxoadipyl-CoA, 3-hydroxyadipyl-CoA, 5-carboxy-2-pentenoyl-CoA, or adipyl-CoA (see Figure 2). The synthesis can be started. In addition, adipate-producing microorganisms can initiate synthesis from 3-oxoadipyl-CoA, 3-oxoadipate, 3-hydroxyadipate, or hexa-2-endioate (see Figure 3). The 6-aminocaproic acid-producing microorganisms of the present invention can be initiated from an intermediate such as adipic acid semialdehyde (see FIG. 8). The caprolactam-producing microorganisms of the present invention can initiate synthesis from intermediates such as adipic acid semialdehyde or 6-aminocaproic acid (see FIG. 8), if desired.

0076Non-naturally occurring microorganisms of the invention use adipate, 6-aminocaproic acid or at least one nucleic acid encoding a caprolactam pathway enzyme, adipate, 6 using methods well known in the art as exemplified herein. -It is constructed to be extrinsically expressed in an amount sufficient to produce aminocaproic acid or caprolactam. It is understood that the microorganisms of the invention are cultured under conditions sufficient to produce adipate, 6-aminocaproic acid or caprolactam. According to the teachings and guidance provided herein, the non-naturally occurring microorganisms of the invention are biosynthetic of adipate, 6-aminocaproic acid or caprolactam resulting in intracellular concentrations between about 0.1 and 200 mM or higher. Synthesis can be achieved. In general, the intracellular concentration of adipate, 6-aminocaproic acid or caprolactam is between about 3 to 150 mM, especially between about 5 and 125 mM, and even more particularly about 8 to 100 mM, including 10 mM, 20 mM, 50 mM, 80 mM and above. Between. Intracellular concentrations between and above each of these exemplary ranges can also be achieved from the non-naturally occurring microorganisms of the invention.

0077In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. The exemplary anaerobic conditions have been described above and are well known in the art. Illustrative anaerobic conditions for the fermentation process are described herein, eg, in US Patent Application No. 11 / 891,602, filed August 10, 2007. Any of these conditions can be used with non-naturally occurring microorganisms as well as other anaerobic conditions well known in the art. Under such anaerobic conditions, adipate, 6-aminocaproic acid or caprolactam producers at 5-10 mM or higher intracellular concentrations as well as all other concentrations exemplified herein, adipate, 6-aminocaproic acid. Alternatively, caprolactam can be synthesized. Even if the above description is related to intracellular concentration, adipate, 6-aminocaproic acid or caprolactam-producing microorganisms may produce adipate, 6-aminocaproic acid or caprolactam intracellularly, and / or their products. It is understood that may be secreted into the culture medium.

0078Culturing conditions include, for example, liquid culturing procedures as well as fermentation and other large-scale culturing procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.

0079As described herein, exemplary growth conditions for achieving biosynthesis of adipate, 6-aminocaproic acid or caprolactam include anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microorganisms of the present invention can be maintained, cultured or fermented under anaerobic or substantially anaerobic conditions. Simply put, anaerobic conditions refer to an environment that is completely oxygen-free. Substantially anaerobic conditions include, for example, culturing, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0% and 10% of saturation. Substantially anaerobic conditions also include cell proliferation or quiescence in liquid medium or on solid agar in a sealed chamber maintained in an atmosphere of less than 1% oxygen. This percentage of oxygen is, for example, N2/ CO2It can be maintained by spraying the culture with a mixture or other suitable non-oxygen gas (s).

0080The culture conditions described herein can be scaled up and continuously grown for the production of adipate, 6-aminocaproic acid or caprolactam. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial amounts of adipate, 6-aminocaproic acid or caprolactam. In general, and as in discontinuous culture procedures, continuous and / or quasi-continuous production of adipate, 6-aminocaproic acid or caprolactam is sufficient nutrition and medium to sustain and / or nearly sustain growth during the exponential phase. Will include culturing non-naturally occurring adipates, 6-aminocaproic acid or caprolactam-producing organisms of the present invention. Continuous culture under such conditions can include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more. In addition, continuous culture can include one week, two, three, four or five weeks or more, and several months or less. Alternatively, the organism of the invention can be cultured for several hours if suitable for a particular application. It should be understood that the above continuous and / or quasi-continuous culture conditions may also include all time intervals during these exemplary periods. It is further understood that the culture time of the microorganisms of the present invention extends long enough to produce a sufficient amount of product for the desired purpose.

0081Fermentation procedures are well known in the art. Simply put, for biosynthetic production of adipate, 6-aminocaproic acid or caprolactam, for example, fed-batch fermentation and batch separation; utilizing fermentation in fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Can be done. Examples of batch and continuous fermentation procedures are well known in the art.

0082In addition to the above fermentation procedure using adipate, 6-aminocaproic acid or caprolactam products of the invention for continuous production of substantial amounts of adipate, 6-aminocaproic acid or caprolactam, adipate, 6-aminocaproic acid or caprolactam. The product, if desired, is simultaneously subjected to a chemical synthesis procedure to convert the fermented product into another compound or product that can be separated from the fermented culture, followed by sequential chemical conversion. The product can be converted to other compounds. As described herein, intermediates in the adipate pathway utilizing 3-oxoadipate, hexa-2-engioate can be converted to adipate, for example, by chemical hydrogenation with a platinum catalyst (Example). See III).

0083Metabolic modeling can be used to optimize growth conditions to produce better producers. Modeling can also be used to design gene knockouts that further optimize the use of that pathway (eg, US Patent Publication US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, See US 2003/0059792, US 2002/0168654 and US 2004/0009466, and US Pat. No. 7,127,379). Modeling analysis allows reliable prediction of effects on cell proliferation that shift metabolism to more efficient production of adipate, 6-aminocaproic acid or caprolactam.

0084One computer computing method for identifying and designing metabolic modifications suitable for biosynthesis of the desired product is the OptKnock computer computing framework, Burgard et al., Biotechnol. Bioeng, 84,647-57 (2003). OptKnock is a metabolic modeling and simulation program that suggests gene deletion strategies that result in genetically stable microorganisms that overproduce target products. Specifically, this framework investigates the complete metabolism and / or biochemical network of microorganisms and suggests genetic manipulations that force the desired biochemical product to be a by-product of cell proliferation. Proliferation selection applied to genetically engineered strains after a long period of time in a bioreactor by linking cell proliferation and biochemical production by strategically imposing gene deletions or other functional gene disruptions. Pressure results in improved performance as a result of its forced growth-linked biochemical production. Finally, when constructing a gene deletion, the genes selected by OptKnock will be completely removed from the genome, ignoring the possibility that the designed strain will revert to their wild-type state. Good. Therefore, this computer computational methodology can be used to identify alternative pathways leading to the biosynthesis of the desired product, or to use for non-naturally occurring microorganisms for further optimization of the biosynthesis of the desired product. Can be done.

0085Simply put, OptKnock is a term used herein to refer to computer computational methods and systems for modeling cell metabolism. The OptKnock program involves a model and method framework that incorporates specific constraints into a flux equilibrium analysis (FBA) model. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and / or DNA microarray experimental data. OptKnock also estimates solutions to various metabolic problems, for example by narrowing the flux range obtained by the flux equilibrium model and then scrutinizing the performance limits of the metabolic network in the presence of gene additions or deletions. The OptKnock computer computing framework enables the creation of model equations that allow effective queries of the performance limits of metabolic networks and provides a solution to the resulting mixed integer linear programming problem. The metabolic modeling and simulation method referred to herein as OptKnock is described, for example, in US Patent Publication No. 2002/0168654, filed January 10, 2002, and International Patent No. PCT / US02 /, filed January 10, 2002. It is described in 00660 and in US Patent Application No. 11 / 891,602 filed August 10, 2007.

0086Another computer computing method for identifying and designing metabolic modifications suitable for biosynthetic production of products is a metabolic modeling and simulation system called SimPheny®. This computer calculation method and system is described, for example, in US Patent Publication No. 2003/0233218 filed June 14, 2002, and International Patent Application No. PCT / US03 / 18838 filed June 13, 2003. There is. SimPheny® can be used to generate network models in Incilico, and can be used to simulate mass, energy, or charge flux from chemical reactions in a biological system, and the chemistry in that system. A computer computing system that can define a solution space that includes any and all possible functionality of a reaction, thereby determining the range of permissible activity for that ecosystem. This approach is called constraint-based modeling. This is because the solution space is defined by constraints, such as the known stoichiometry of the reactions involved, as well as the reaction thermodynamics and capacitance constraints associated with the maximum flux of the reaction. The space defined by these constraints can be questioned to determine the phenotypic capacity and behavior of the biological system or its biochemical components.

0087These computer computational approaches are consistent with biological reality, as biological systems are adaptable and can achieve the same results in many different ways. Biological systems are designed by evolutionary mechanisms constrained by the fundamental constraints faced by all living systems. Therefore, constraint-based modeling strategies embrace these general realities. In addition, tighter constraints can continuously impose additional constraints on the network model, reducing the size of the solution space and, as a result, improving the accuracy with which physiological performance or phenotype can be predicted. To.

0088Given the teachings and guidance provided herein, one of ordinary skill in the art will utilize various computer computing frameworks for metabolic modeling and simulation to design and perform biosynthesis of the desired compound in host microorganisms. Will be able to. Such metabolic modeling and simulation methods include, for example, the computer computing systems exemplified above as SimPheny® and OptKnock. For illustration purposes of the present invention, some methods are described herein with respect to the OptKnock computer computing framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and execution of metabolic modifications using OptKnock to any other such metabolic modeling and simulation computer computing frameworks and methods well known in the art. ..

0089The method described above will result in a set of metabolic reactions to be disrupted. Removal of each reaction or metabolic modification in the set will give the desired product as an essential product during the growth phase of the organism. Since these reactions are known, the solution to the bilayer OptKnock problem will also result in related genes (s) encoding one or more enzymes that catalyze each reaction in the reaction set. The identification of a set of reactions and their corresponding genes encoding the enzymes involved in each reaction is generally performed automatically by the correlation of the reactions with the reaction database that has the relationship between the enzyme and the coding gene. It is a process.

0090Once identified, a set of reactions that will be disrupted to achieve the desired product production is the function of at least one gene in the target cell or organism that encodes each metabolic response within that set. Performed by destruction. One particularly useful means of achieving functional disruption of this reaction set is by deletion of each coding gene. However, in some cases, the coding sequence may be due to other genetic abnormalities, including, for example, mutations, deletions of regulatory regions, eg promoters, or cis binding sites for regulators, or at any of multiple positions It may be beneficial to disrupt the reaction by truncation. These latter abnormalities, which result in less than complete deletions of the gene set, can be useful, for example, when rapid evaluation of product linkage is desired or when reversion mutations are unlikely to occur. ..

0091To identify additional generative solutions to the above two-layer OptKnock problem that result in an additional set of disruptive reactions or metabolic modifications that can result in biosynthesis, including proliferation-linked biosynthesis of the desired product. An optimization method called integer cut can be performed. This method is done by iteratively solving the OptKnock problem illustrated above, incorporating an additional constraint called an integer cut at each iteration. The integer cut constraint effectively prevents the solution procedure from selecting the exact same set of reactions identified in any previous iteration that forces the biosynthesis of the product to grow. For example, if the previously identified growth-linked metabolic modification specifies reactions 1, 2 and 3 for disruption, the following constraints prevent the same reaction from being considered simultaneously in subsequent answers. .. This integer cutting method is well known in the art and can be found, for example, described in Burgard et al., Biotechnol.Prog, 17: 791-797 (2001). Similar to all methods described herein with respect to the OptKnock computer computing framework for metabolic modeling and simulation and their combination, an integer cutting method that reduces duplication in iterative computer computational analysis is, for example, SimPheny®. It can also be used with other computer computing frameworks well known in the art, including.

0092The methods exemplified herein allow the construction of cells and organisms that biosynthesize and produce the desired product, which methods have been genetically engineered to have identified genetic modifications. Includes linking the production of target biochemical products to the growth of cells or organisms. Accordingly, the computer computational methods described herein allow the identification and execution of metabolic modifications identified by in silico methods selected from OptKnock or SimPheny®. The set of metabolic modifications can include functional disruption of one or more metabolic reactions, including, for example, addition of one or more biosynthetic pathway enzymes and / or disruption due to, for example, a gene deletion.

0093As discussed above, the OptKnock methodology was developed on the premise that mutant microbial networks can evolve towards their computer-calculated predicted maximum growth phenotype when subjected to long growth selection periods. In other words, this approach affects an organism's ability to self-optimize under selective pressure. The OptKnock framework allows a comprehensive enumeration of gene deletion combinations that force biochemical production and cell proliferation based on network stoichiometry. Identifying the optimal gene / reaction disruption requires a solution to a two-layer optimization problem that selects the set of active reactions to overproduce the biochemical product of interest for the optimal growth solution for the resulting network. (Burgard et al., Biotechnol Bioeng. 84: 647-657 (2003)).

0094Previously exemplified and, for example, in US Patent Publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and As described in US Pat. No. 7,127,379, an insilico chemotherapeutic model of E. coli metabolism can be used to identify essential genes in the metabolic pathway. As disclosed herein, the OptKnock mathematical network can be used to pinpoint the location of gene deletions that result in growth-linked production of the desired product. Moreover, the solution to the two-layer OptKnock problem gives only one set of deletions. An optimization technique called integer cut can be performed to enumerate all meaningful solutions, that is, all sets of knockouts that lead to proliferation-linked production formation. This inevitably involves solving the OptKnock problem iteratively, incorporating additional constraints called integer cuts at each iteration, as discussed above.

0095It will be appreciated that modifications that do not substantially affect the activity of various embodiments of the invention are also included in the definitions of the invention provided herein. Therefore, the following examples are for illustration purposes only and are not intended to limit the present invention.

Example I Inverse adipate degradation pathway This example illustrates an exemplary adipate synthesis pathway by the reverse adipate degradation pathway. Organisms such as Penicillium chrysogenum have the ability to decompose adipate naturally (Thykaer et al., Metab. Eng. 4: 151-158. (2002)). This mechanism is similar to the oxidation of fatty acids (see Figure 1). The first step in adipate degradation is an ATP-dependent reaction that activates adipate with CoA. The second reaction is catalyzed by a dehydrogenase that forms 5-carboxy-2-pentenoyl-CoA from adipyl-CoA. During the adipate degradation of peroxisomes, the dehydrogenase enzyme contains FAD, which accepts electrons and then transfers them directly to oxygen. Catalase enzyme is H formed by the reduction of oxygen2O2Dissipate. In mitochondrial fatty acid oxidation, FAD from dehydrogenase transfers electrons directly to the electron transport chain. In eukaryotes such as S. cerevisiae and P. chrysogenum, polyfunctional fatty acid oxidized proteins carry out subsequent hydratase and dehydrogenase steps. The final step is an acetyltransferase that divides 3-oxoadipyl CoA into acetyl-CoA and succinyl-CoA. A highly efficient pathway for the production of adipate is achieved by genetically engineering the microorganism to utilize a similar enzymatic reaction for the synthesis of adipate from succinyl-CoA and acetyl-CoA (see Figure 2). The realization of this successful result is to express the appropriate genes, regulate their expression, and follow this pathway by high acetyl-CoA, succinyl-CoA, and / or redox (eg, NADH / NAD +) ratios. It is necessary to modify the culture conditions so that the passing metabolic flux is directed to adipate synthesis rather than degradation. Significant resemblance to butyrate formation in Clostridia (Kanehisa and Goto, Nucl. Acids Res. 28: 27-30 (2000)) is that each step in the adipate synthesis pathway is thermodynamically feasible and the direction of the reaction. We support that it depends on the concentration of metabolites involved. The final step in forming adipate from adipyl-CoA can be by either a synthesizer, a phosphotransadipylase / kinase, a transferase, or a hydrolase mechanism. The maximum theoretical yield of adipate using this pathway was calculated both in the presence and absence of external electron acceptors such as oxygen. These calculations show that this pathway adipates glucose and CO under anaerobic conditions.2It is shown that conversion can be performed efficiently with a molar yield of 92% (Table I). Production of adipate using this pathway does not require oxygen uptake, as NAD + can be regenerated at the two hydrogenase steps that form 3-hydroxyadipyl-CoA and adipyl-CoA (see Figure 2). In addition, this pathway forms up to 1.55 moles of glucose per mole of glucose consumed at the maximum theoretical yield of adipate, assuming either the synthesizer, phosphotransadipylase / kinase, or transferase mechanism for the final conversion step. Therefore, it is energetically suitable. Assuming that phosphoenolpyruvate carboxylase (PPCK) functions in the direction of ATP production towards oxaloacetate formation, the ATP yield can be further improved to 2.47 moles of ATP produced per mole of glucose. The maximum ATP yield was then calculated assuming that the conversion of adipyl-CoA to adipate was the hydrolysis step. Assuming that PPCK is irreversible and reversible, the maximum ATP yield during maximum adipate production is ATP per mole of glucose consumed. It is reduced to 0.85 and 1.77 mol, respectively. Nevertheless, these ATP yields are sufficient for cell proliferation, maintenance and production.<tables num="1"><img id="000002" he="38" wi="116" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> To successfully reshape this pathway in genetic engineering, it is necessary to identify the appropriate set of enzymes with sufficient activity and specificity. This requires identification of the appropriate set of enzymes, cloning of their corresponding genes into the production host, optimization of fermentation conditions, and assays for post-fermentation product formation. One or more exogenous DNA sequences are expressed in the appropriate host microorganism to genetically engineer the production host for the production of adipate. In addition, the microorganism may carry a functionally deleted endogenous gene (s). These modifications allow the production of adipates using renewable feedstocks. A number of biochemically characterized candidate genes encoding enzymes that catalyze each step of the reverse adipate degradation pathway in the production host are described below. E. coli is used to describe genetic engineering remodeling of pathways, but essentially any suitable host organism may be used. Specifically, we list the genes inherent in E. coli as well as genes in other organisms that can be used to catalyze proper conversion when properly cloned and expressed. With reference to FIG. 2, step 1 comprises succinyl-CoA: acetyl-CoA acyltransferase (β-ketothiolase). The first step in this pathway is to combine acetyl-CoA with succinyl-CoA to form 3-oxoadipyl-CoA. PcaF in Pseudomonas strain B13 (Kaschabek et al., J. Bacteriol. 184: 207-215 (2002)), phaD in Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci. USA 95: 6419-6424 (1998)) , PaaE (Di) in Pseudomonas fluorescens ST The gene products encoded by Gennaro et al., Arch.Microbiol.188: 117-125 (2007)) and paaJ from E. coli (Nogales et al., Microbiol.153: 357-365 (2007)) are phenylacetates. Alternatively, it catalyzes the conversion of 3-oxoadipyl-CoA to succinyl-CoA and acetyl-CoA during the decomposition of aromatic compounds such as styrene. Since β-ketothiolase enzymes catalyze reversible conversion, these enzymes can be utilized in the first step of adipate synthesis shown in FIG. For example, ketothiolase phaA from R. eutropha is combined with two acetyl-CoA molecules to form acetoacetyl-CoA (Sato et al., J. Biosci. Bioengineer. 103: 38-44 (2007)). Similarly, β-ketothiolase (bktB) has been reported to catalyze the condensation of acetyl-CoA and propynyl-CoA to form β-ketovaleryl-CoA in R. eutropha (Slater et al., J. et al. Bacteriol. 180: 1979-1987 (1998)). Protein sequences for the above gene products are well known in the art and are available in public databases such as GenBank using the accession numbers below.<maths num="1"><img id="000003" he="31" wi="109" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> These exemplary sequences can be used to identify homologous proteins by sequence similarity search (eg, BLASTp) in GenBank or other databases. The resulting homologous proteins and their corresponding gene sequences provide additional exogenous DNA sequences for transformation into E. coli or other suitable host microorganisms to produce the production host. For example, the paaJ ortholog from Escherichia coli K12 can be found using the GenBank accession number below:<maths num="2"><img id="000004" he="18" wi="68" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> An example of a pcaF ortholog from Pseudomonas knackmussii can be found using the GenBank accession number below:<maths num="3"><img id="000005" he="17" wi="72" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> Additional natural candidate genes for the ketothiolase step are atoB (Sato et al., J. Biosci. Bioengineer. 103: 38-44), which can catalyze the reversible condensation of two acetyl-CoA molecules, and its homolog yqeF. Can be mentioned. Non-natural gene candidates include phaA from R. eutropha (Sato et al., Supra, 2007) and bktB (Slater et al., J. Bacteriol. 180: 1979-1987 (1998)), and two ketothiolase from Clostridium acetobutylicum. , ThiA and thiB (Winzer et al., J. Mol. Microbiol. Biotechnol. 2: 531-541 (2000)). Protein sequences for each of these exemplary gene products can be found using the GenBank accession numbers below:<maths num="4"><img id="000006" he="35" wi="82" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> With reference to FIG. 2, step 2 comprises 3-hydroxyacyl-CoA dehydrogenase. The second step in this pathway involves the reduction of 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. PhaC in Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci. USA 95: 6419-6424 (1998)) and paaC in Pseudomonas Fluorescens ST (DiGennaro et al., Arch. Microbiol. 188: 117-125 (2007)) The gene product encoded by catabolizes the reverse reaction during catabolism of phenylacetate or styrene, namely the oxidation of 3-hydroxyadipyl-CoA, which forms 3-oxoadipyl-CoA. Such dehydrogenase-catalyzed reactions are reversible and therefore these genes are candidates for the second step of adipate synthesis as shown in FIG. Clostridium Similar transformations occur with the hbd gene product in acetobutylicum (Atsumi et al., Metab. Eng. (Epub Sep. 14, 2007); Boynton et al., J. Bacteriol. 178: 3015-3024 (1996)). This enzyme converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA. Finally, the E. coli proximity of paaH to other genes in phenylacetate-degrading operons (Nogales et al., Microbiol. 153: 357-365 (2007)) and the fact that paaH mutants cannot grow on phenylacetate ( Given Ismail et al., Eur.J.Biochem.270: 3047-3054 (2003)), the E. coli paaH gene is expected to encode a 3-hydroxyacyl-CoA dehydrogenase. Protein sequences for each of these exemplary gene products can be found using the GenBank accession numbers below:<maths num="5"><img id="000007" he="23" wi="83" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> With reference to FIG. 2, step 3 comprises 3-hydroxyadipyl-CoA dehydratase. The gene product of crt from C. acetobutylicum catalyzes the dehydration of 3-hydroxybutyryl-CoA to Clostridium-CoA (see Figure 2) (Atsumi et al., Supra, 2007; Boynton et al., J. Bacteriol.178). 3015-3024 (1996)). The homologue of this gene is a strong candidate for performing the third step in the adipate synthesis pathway illustrated in Figure 2. In addition, genes known to catalyze double bond hydroxylation in enoyl-CoA compounds are additional candidates given the reversibility of such enzymatic conversion. For example, P. putida's enoyl-CoA hydratase, phaA and phaB, are thought to undergo double bond hydroxylation during phenylacetate catabolism (Olivera et al., Proc. Natl. Acad. Sci. USA). 95: 6419-6424 (1998)) Therefore, it can be said that it is an additional candidate to be incorporated into E. coli. Deletion of these genes in P. putida prevents phenylacetate degradation. PaaA and paaB from P. fluorescens catalyze similar conversions (Olivera et al., Supra, 1998). Finally, maoC (Park and Lee, J.Bacteriol.185: 5391-5397 (2003)), paaF (Ismail et al., Eur.J.Biochem.270: 3047-3054 (2003); Park and Lee, Biotechnol.Bioeng .86: 681-686 (2004); Park and Lee, Appl.Biochem. Biotechnol.113-116: 335-346 (2004)), and paaG (Ismail et al., 2003; Park and Lee, 2004; Park and Lee, 2004) and many others. The coli gene has been shown to demonstrate the functionality of enoyl-CoA hydratase. Protein sequences for each of these exemplary genes can be found using the GenBank accession numbers below:<maths num="6"><img id="000008" he="47" wi="83" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> Alternatively, the β-oxidizing gene is a candidate for the first three steps in adipate synthesis. Candidate genes for the proposed adipate synthetic pathway also include E. coli's natural fatty acid oxidation genes and their homologues in other organisms. The E. coli genes fadA and fadB encode a multienzyme complex exhibiting ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydratase activity (Yang et al., Biochem. 30: 6788-6795 (1991). ); Yang et al., J.Biol.Chem.265: 10424-10429 (1990); Yang et al., J.Biol.Chem.266: 16255 (1991); Nakahigashi and Inokuchi, Nucl.Acids Res. 18: 4937 (1990)). These activities are mechanically similar to the first three conversions shown in Figure 2. The fadI and fadJ genes encode similar functions and are spontaneously expressed only anaerobically (Campbell et al., Mol. Microbiol. 47: 793-805 (2003)). These gene products convert short, medium and long chain fat-acyl-CoA compounds rather than converting succinyl-CoA and acetyl-CoA to 5-carboxy-2-pentenoyl-CoA as shown in Figure 2. It originally acts to break down into acetyl-CoA. However, it is well known that ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase enzymes catalyze reversible conversion. In addition, directional evolution and related approaches can be used to regulate the substrate specificity of E. coli's native β-oxidation machines. Therefore, these enzymes or their homologues can be utilized for adipate production. If the native gene acts to degrade adipate or its precursors in vivo, appropriate gene modifications are made to mitigate or eliminate these functions. But that wouldn't be necessary. Poly [(R) -3- in E. coli, including activating fadB by knocking out a negative regulator, fadR, and co-expressing the unnatural ketothiolase, phaA, from Ralstonia eutropha. This is because the production method of [hydroxybutyrate] is described (Sato et al., J. Biosci. Bioeng. 103: 38-44 (2007)). In this study, β-oxidizing enzymes, especially the fadB gene product encoding both 3-hydroxyacyl-CoA dehydrogenase activity and enoyl-CoA hydratase activity, produced longer-chain molecules from acetyl-CoA precursors. It has been clearly demonstrated that it can function as part of the pathway. Protein sequences for each of these exemplary gene products can be found using the GenBank accession numbers below:<maths num="7"><img id="000009" he="31" wi="68" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> With reference to FIG. 2, step 4 comprises 5-carboxy-2-pentenoyl-CoA reductase. The enoyl-CoA hydratase steps are generally reversible, whereas the enoyl-CoA reductase steps are almost always oxidative and irreversible under physiological conditions (Hoffmeister et al., J. Biol. Chem. 280: 4329-4338 (2005)). FadE catalyzes this probably irreversible transformation in E. coli (Campbell and Cronan, J. Bacteriol. 184: 3759-3764 (2002)). This pathway requires an enzyme capable of reducing the 2-enoyl-CoA intermediate, rather than an enzyme such as FadE that would simply oxidize acyl-CoA to a 2-enoyl-CoA compound. Furthermore, it has been suggested that E. coli originally possesses an enzyme for enoyl-CoA reduction (Mizugaki et al., J. Biochem. 92: 1649-1654 (1982); Nishimaki et al., J. Biochem. .95: 1315-1321 (1984)), but no E. coli gene with this function has been biochemically characterized. One candidate gene for the enoyl-CoA reductase step is the gene product of bcd from C. acetobutylicum (Atsumi et al., Supra, 2007; Boynton et al., J. Bacteriol. 178: 3015-3024 (1996)). This naturally results in the reduction of crotonyl-CoA to butyryl-CoA, a reaction that is mechanically similar to the desired reduction of 5-carboxy-2-pentenoyl-CoA to adipyl-CoA in the adipate synthesis pathway. Catalyze. The activity of this enzyme is C. acetobutylicum, which encodes an electron transport flavoprotein. It can be enhanced by expressing bcd along with the expression of the etfAB gene. An additional candidate for the enoyl-CoA reductase step is mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et al., J. Biol. Chem. 280: 4329-4338 (2005)). After removal of the mitochondrial targeting leader sequence, constructs derived from this sequence were cloned in E. coli, resulting in active enzymes (Hoffmeister et al., Supra, 2005). This approach is well known to engineers in the art of expressing eukaryotic genes, particularly those having leader sequences capable of targeting gene products to specific intracellular compartments in prokaryotes. A close homologue of this gene from the prokaryote Treponema denticola, TDE0597, is representative of a third enoyl-CoA reductase cloned and expressed in E. coli (Tucci and Martin, FEBS). Lett.581: 1561-1566 (2007)). Protein sequences for each of these exemplary gene products can be found using the following GenBank accession numbers:<maths num="8"><img id="000010" he="30" wi="94" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> With reference to Figure 2, step 5 contains adipyl-CoA synthesizer (also called adipic acid-CoA ligase), phosphotransadipylase / adipic acid kinase, adipyl-CoA: acetyl-CoA hydrolase, or adipyl-CoA hydrolase. Including. From an energetic point of view, the final step in this adipate synthesis pathway is preferably catalyzed by an enzyme or enzyme pair capable of conserving the ATP equivalent stored in the thioester bond of adipyl-CoA. E. coli sucC and sucD genes or their homologous products, which may be able to catalyze the final conversion shown in Figure 2, should be active against adipyl-CoA. The sucCD gene naturally forms a succinyl-CoA synthesizer complex, which forms succinyl-CoA from succinate with concaminant consumption of one ATP (a reaction that is reversible in vivo). (Buck et al., Biochem. 24: 6245-6252 (1985)). Given the structural similarity between succinate and adipate, that is, both are linear dicarboxylic acids, it makes sense to expect some activity of the sucCD enzyme on adipyl-CoA. Enzymes exhibiting adipyl-CoA ligase activity, when acting in the antiphysiological direction as shown in Figure 1, use ATP-producing production of adipate from adipyl-CoA (in this case using AMP and PPi as cofactors). Can be done equally. Two exemplary CoA-ligases are characterized from the rat dicarboxylate-CoA ligase whose sequence has not yet been characterized (Vamecq et al., Biochem.J. 230: 683-693 (1985)) and P. chrysogenum. One of the phenylacetate-CoA ligases (Lamas-Maceiras et al., Biochem.J. 395, 147-155 (2005); Wang et al., Biochem. Biophy. Res.Commun.360: 453-458 (2007)), Phenylacetate-CoA ligase from Pseudomonas Putida (Martinez-Blanco et al., J.Biol.Chem.265: 7084-7090 (1990)), and Bacilis subtilis. 6-carboxyhexanoate-CoA ligase (Bower et al., J. Bacteriol. 178: 4122-4130 (1996)) can be mentioned. Protein sequences for each of these exemplary gene products can be found using the following GenBank accession numbers:<maths num="9"><img id="000011" he="13" wi="79" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> Another option to use phosphotransadipylase / adipic acid kinase is the gene products of buk1, buk2 and ptb from C. acetobutylicum (Walter et al., Gene 134: 107-111 (1993); Huang et al., J. .Mol.Microbiol.Biotechnol.2: 33-38 (2000)) or their homologs are catalyzed. The ptb gene encodes an enzyme capable of converting butyryl-CoA to butyryl-phosphate, which is then converted to butyrate by any of the buk gene products with the concomitant development of ATP. Will be done. A similar set of conversions, namely the conversion of adipyl-CoA to adipyl-phosphate, followed by the conversion of adipyl-phosphat to adipate, can be carried out by the buk1, buk2 and ptb gene products. Protein sequences for each of these exemplary gene products can be found using the following GenBank accession numbers:<maths num="10"><img id="000012" he="19" wi="91" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> Alternatively, an acetyltransferase capable of transferring the CoA group from adipyl-CoA to acetate can be utilized. The gene products of cat1, cat2 and cat3 of Clostridium kluyveri, which have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activities, respectively (Sohling and Gottschalk, J. Bacteriol.178: 871-880 (1996); Seedorf et al., Proc.Natl.Acad.Sci.USA 105: 2128-2133 (2008)) catalyze similar conversions. Protein sequences for each of these exemplary gene products can be found using the following GenBank accession numbers:<maths num="11"><img id="000013" he="18" wi="84" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> Finally, although seemingly undesirable from an energetic point of view, the conversion of adipyl-CoA to adipate can also be done by acyl-CoA hydrolase or equivalent thioesterase. The top E. coli gene candidate is highly similar to human acot8 (Westin et al., J. Biol. Chem. 280: 38125-38132 (2005)), a dicarboxylic acid acetyltransferase that is active against adipyl-CoA. It is tesB (Naggert et al., J.Biol.Chem.266: 11044-11050 (1991)) showing sex. This activity has also been characterized in rat liver (Deana, Biochem. Int. 26: 767-773 (1992)). Protein sequences for each of these exemplary gene products can be found using the following GenBank accession numbers:<maths num="12"><img id="000014" he="18" wi="80" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> Other natural candidate genes include tesA (Bonner and Bloch, J.Biol.Chem.247: 3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol.Rev.29: 263-279 (2005); Zhuang Et al., FEBS Lett. 516: 161-163 (2002)), paaI (Song et al., J.Biol.Chem.281: 11028-11038 (2006)), and ybdB (Leduc et al., J.Bacteriol.189: 7112- 7126 (2007)). Protein sequences for each of these exemplary gene products can be found using the following GenBank accession numbers:<maths num="13"><img id="000015" he="24" wi="78" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> The above description provides an exemplary adipate synthesis pathway by the reverse adipate degradation pathway. Example II Production of adipate-producing microorganisms with reverse degradation pathway This example illustrates the production of microorganisms capable of producing adipate using the reverse degradation pathway. Escherichia coli is used as a target organism for genetically engineering the reverse adipate degradation pathway as shown in Fig. 2. E. coli is a good host for producing non-naturally occurring microorganisms capable of producing adipate. E. coli is easy to genetically engineered, and it is known that E. coli can produce various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid effectively under anaerobic or microaerobic conditions. Is. Nucleic acids encoding enzymes used in the reverse degradation pathway to produce E. coli strains genetically engineered to produce adipate have been developed using well-known molecular biological techniques (eg, Sambrook, supra). (See literature, 2001; Ausubel, supra, 1999) for expression in E. coli. Specifically, succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyadipyl-CoA dehydratase activity encoding paaJ (NP_415915.1), paaH (NP_415913.1), and maoC. The (NP_415905.1) gene is cloned into the pZE13 vector (Expressys in Ruelzheim, Germany) under the PA1 / lacO promoter. In addition, the bcd (NP_349317.1), etfAB (349315.1 and 349316.1) and sucCD (NP_415256.1 and AAC73823.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthesizer activity, PA1 Under the / lacO promoter, clone into the pZA33 vector (Expressys in Ruelzheim, Germany), respectively. These two sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for adipate synthesis by the reverse degradation pathway. The resulting genetically engineered organism is cultured in glucose-containing medium according to procedures well known in the art (see, eg, Sambrook et al., Supra, 2001). Expression of reverse degradation pathway genes is expressed using methods well known in the art for determining polypeptide expression or enzymatic activity, such as Northern blots, PCR amplification of mRNA, immunoblotting and the like. support. The enzymatic activity of the expressed enzyme is confirmed using an assay specific to the individual activity. The ability of the genetically engineered E. coli strain to produce adipate is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and / or liquid chromatography-mass spectrometry (LCMS). The number of microbial strains genetically engineered to have a functional adipate synthetic pathway is further increased by optimizing the efficient use of that pathway. Simply put, the genetically engineered strain is evaluated to determine if any of the exogenous genes are expressed at rate-determining levels. For example, the introduction of additional gene copy numbers increases expression for any enzyme expressed at low levels that can limit the flux through that pathway. Metabolic modeling is used to optimize growth conditions for better product production. Modeling is also used to design gene knockouts that further optimize the use of that pathway (eg, US Patent Publication US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003). / 0059792, US 2002/0168654 and US See 2004/0009466 and US Pat. No. 7,127,379). Modeling analysis allows reliable prediction of effects on cell proliferation that shift metabolism to more efficient production of adipate. One modeling method is the two-layer optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84: 647-657 (2003)), a gene that results in jointly producing better production of adipate. Used to select knockout. Adaptive evolution can also be used to produce better products of, for example, acetyl-CoA and succinyl-CoA intermediates or adipate products. Perform adaptive evolution to improve both proliferative and production properties (Fong and Palsson, Nat.Genet. 36: 1056-1058 (2004); Alper et al., Science 314: 1565-1568 (2006)). Based on these results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the adipate producer to further increase production. For large-scale production of adipate, the reverse degradation pathway-containing organism is cultured in a fermenter under anaerobic conditions using a medium known in the art to support the growth of the organism. Fermentation is carried out in either batch, fed-batch or continuous mode. Anaerobic conditions may be maintained by first spraying the medium with nitrogen and then sealing the culture vessel, for example, the flask may be sealed with a septum and crimp cap. Microaerobic conditions can also be used by providing a small hole in the septum for restricted ventilation. The pH of the medium is H2SO4Maintain at a pH of about 7 by adding acids such as. The growth rate is determined by measuring the optical density using a spectrophotometer (600 nm), and the glucose uptake rate is determined by monitoring carbon source depletion over time. By-products such as unwanted alcohols, organic acids and residual glucose can be obtained using a refractometer detector for glucose and alcohol, and a UV detector for organic acids, eg, the Aminex® series HPLC. It can be quantified by HPLC using a column (eg HPX-87 series) (BioRad, Hercules, CA) (Shimadzu, Columbia, Maryland) (Lin et al., Biotechnol. Bioeng., 775-779 (2005)). )). This example illustrates the production of adipate-producing microorganisms using the reverse degradation pathway. Example III Adipate synthesis via 3-oxoadipate This example illustrates an exemplary adipate synthesis pathway via 3-oxoadipate. An additional pathway from those described in Examples I and II, using acetyl-CoA and succinyl-CoA as precursors for adipate formation, and passing through the metabolic intermediate, 3-oxoadipate, is shown in FIG. The first two transformations in this pathway are the two terminal steps of the degradation pathway for aromatic and chloroaromatic compounds acting in the opposite direction (Kaschabek et al., J. Bacteriol. 184: 207-215 (2002)). Nogales et al., Microbiol.153: 357-365 (2007); Ismail et al., Eur.J.Biochem. 270: 3047-3054 (2003)). Specifically, the first step is to form 3-oxoadipyl CoA by condensation of succinyl-CoA and acetyl-CoA. The second step is to form 3-oxoadipate and has been reported to be reversible in Pseudomonas strain B13 (Kaschabek et al., J. Bacteriol. 184: 207-215 (2002)). Subsequent steps include reduction of 3-oxoadipate to 3-hydroxyadipate (conversion of keto group to hydroxyl group), dehydration of 3-hydroxyadipate to produce hexa-2-endioate, and formation of adipate. Includes reduction of hexa-2-engioate for. These steps of this pathway are similar to the conversion of oxaloacetate to succinate by the reducing TCA cycle (see Figure 4). This supports that these steps of this pathway are thermodynamically suitable, subject to the presence of appropriate metabolite concentrations. The final reduction step can be performed biochemically or by utilizing a chemical catalyst to convert hexa-2-engioate to adipate. Chemical hydrogenation can be carried out using a Pt catalyst supported on activated carbon, as described in (Niu et al., Biotechnol. Prog. 18: 201-211 (2002)). The maximum theoretical yield of adipate using this pathway is 0.92 mol per mole of glucose consumed and does not require oxygen to obtain these yields (see Table 2). The coenergy properties are the same as those of the inverse adipate pathway. Theoretically, through this pathway, ATP formation is observed up to 1.55 moles per mole of glucose utilized. This ATP yield increases to approximately 2.47 mol, assuming that phosphoenolpyruvate kinase (PPCK) acts in the direction of ATP production. Interestingly, using chemical hydrogenation in the final step and assuming 100% catalytic efficiency, the product yield can be further increased to 1 mole of adipate per mole of glucose consumed. In this scenario, theoretically up to 1.95 mol of ATP is formed without considering the reverse functionality of PPCK.<tables num="2"><img id="000016" he="33" wi="125" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> To successfully reshape this pathway in genetic engineering, it is necessary to identify the appropriate set of enzymes with sufficient activity and specificity. This requires identification of the appropriate set of enzymes, cloning of their corresponding genes into the production host, optimization of fermentation conditions, and assays for post-fermentation product formation. One or more exogenous DNA sequences can be expressed in the appropriate host microorganism to genetically engineer the production host for the production of adipate. In addition, the host microorganism may carry a functionally deleted endogenous gene (s). These modifications allow the production of adipates using renewable feedstocks. A number of biochemically characterized candidate genes that can encode enzymes that catalyze each step of the 3-oxoadipate pathway for adipate synthesis are described below. Although this method is described for E. coli, one of ordinary skill in the art can apply these teachings to any other suitable host organism. Specifically, the genes inherent in E. coli as well as genes in other organisms that can be used to catalyze proper conversion when properly cloned and expressed are listed below. With reference to FIG. 3, step 1 comprises succinyl-CoA: acetyl-CoA acyltransferase (β-ketothiolase). First stage in this route floor, to form a 3-Okisoajipiru-CoA were combined acetyl-CoA and succinyl-CoA. PcaF in Pseudomonas strain B13 (Kaschabek et al., J. Bacteriol. 184: 207-215 (2002)), phaD in Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci. USA 95: 6419-6424 (1998)) , PaaE (Di) in Pseudomonas fluorescens ST The gene products encoded by Gennaro et al., Arch.Microbiol.188: 117-125 (2007)) and paaJ from E. coli (Nogales et al., Microbiol.153: 357-365 (2007)) are phenylacetates. Alternatively, it catalyzes the conversion of 3-oxoadipyl-CoA to succinyl-CoA and acetyl-CoA during the decomposition of aromatic compounds such as styrene. Since β-ketothiolase enzymes catalyze reversible conversion, these enzymes can be utilized for the first step of adipate synthesis shown in FIG. For example, ketothiolase phaA from R. eutropha combines two acetyl-CoA molecules to form acetoacetyl-CoA (Sato et al., J. Biosci. Bioengineer. 103: 38-44 (2007)). Similarly, β-ketothiolase (bktB) has been reported to catalyze the condensation of acetyl-CoA and propionyl-CoA to form β-ketovaleryl-CoA in R. eutropha (Slater et al., J. .Bacteriol. 180: 1979-1987 (1998)). Protein sequences for the above gene products are well known in the art and are available in public databases such as GenBank using the accession numbers below.<maths num="14"><img id="000017" he="33" wi="111" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> These sequences can be used to identify homologous proteins by sequence similarity search, eg, BLASTp, in GenBank or other databases. The resulting homologous proteins and their corresponding gene sequences provide additional exogenous DNA sequences for transformation into E. coli or other microorganisms to produce the production host. For example, the paaJ ortholog from Escherichia coli K12 can be found using the GenBank accession number below:<maths num="15"><img id="000018" he="19" wi="69" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> An example of a pcaF ortholog from Pseudomonas knackmussii can be found using the GenBank accession number below:<maths num="16"><img id="000019" he="19" wi="70" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> Additional natural candidate genes for the ketothiolase step include atoB (Sato et al., J. Biosci. Bioengineer. 103: 38-44 (2007)), which can catalyze the reversible condensation of two acetyl-CoA molecules. The homolog yqeF can be mentioned. Non-natural gene candidates include phaA from R. eutropha (Sato et al., Supra, 2007) and bktB (Slater et al., J. Bacteriol. 180: 1979-1987 (1998)), and two ketothiolase from Clostridium acetobutylicum. , ThiA and thiB (Winzer et al., J. Mol. Microbiol. Biotechnol. 2: 531-541 (2000)). Protein sequences for each of these exemplary gene products can be found using the GenBank accession numbers below:<maths num="17"><img id="000020" he="36" wi="84" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> In this exemplary pathway, it is less desirable to use the thiolase-encoding genes fadA and fadB in the fatty acid degradation pathway in E. coli. These genes form a complex that encodes a number of activities, most of which are undesirable in this pathway. With reference to FIG. 3, step 2 comprises 3-oxoadipyl-CoA transferase. At this stage, 3-oxoadipate is formed by the transfer of the CoA group from 3-oxoadipyl-CoA to succinate. This activity has been reported in a two-unit enzyme encoded by pcaI and pcaJ in Pseudomonas (Kaschabek et al., J. Bacteriol. 184: 207-215 (2002)). This enzyme catalyzes reversible conversion. The protein sequence of the exemplary gene product for subunit A of this complex can be found using the following GenBank accession numbers:<maths num="18"><img id="000021" he="19" wi="79" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> The protein sequence of the exemplary gene product for subunit B of this complex can be found using the following GenBank accession numbers:<maths num="19"><img id="000022" he="20" wi="77" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> With reference to FIG. 3, step 3 comprises 3-oxoadipate reductase. E. coli has several candidate alcohol dehydrogenases; two with similar functions are malate dehydrogenase (mdh) and lactate dehydrogenase (ldhA). Although these two enzymes have not been shown to have broad substrate specificity in E. coli, lactate dehydrogenase from Ralstonia eutropha can be used for substrates of various chain lengths, such as 2-oxobutyrate and 2-oxopentano. It has been shown to demonstrate high activity against ate and 2-oxoglutarate (Steinbuchel and). Schlegel, Eur.J. Biochem. 130: 329-334 (1983)). An additional non-natural enzyme candidate for this stage is mitochondrial 3-hydroxybutyric acid dehydrogenase (bdh) from the human heart, which has been cloned and characterized (Marks et al., J. Biol. Chem. 267). : 15459-15463 (1992)). This enzyme is of particular interest in that it is a dehydrogenase that acts on 3-hydroxy acids. Given that dehydrogenases are generally reversible, this gene product, or homologs thereof, reduces 3-oxo acids, such as 3-oxo adipates, to the corresponding 3-hydroxy acids, such as 3-hydroxy adipates. It is expected that it can be done. Protein sequences for each of these exemplary gene products can be found using the GenBank accession numbers below:<maths num="20"><img id="000023" he="26" wi="75" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> With reference to FIG. 3, step 4 comprises 3-hydroxyadipate dehydratase. In this reaction, 3-hydroxyadipate is dehydrated to hexa-2-engioate. Although no direct evidence has been identified for this enzymatic conversion, most dehydrogenases catalyze α, β-elimination of water. This involves activation of α-hydrogen by electron-withdrawing carbonyl, carboxylate, or CoA-thiol ester groups, and removal of hydroxyl groups from the β-position (Martins et al., Proc. Natl. Acad. Sci. USA 101). : 15645-15649 (2004); Buckel and Golding, .FEMS Microbiol. Rev. 22: 523-541 (1998)). Protein sequences for exemplary gene products can be found using the GenBank accession numbers below:<maths num="21"><img id="000024" he="19" wi="71" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> Another good candidate for performing this function is serine dehydratase. These enzymes catalyze conversions that are very similar in removing ammonia from serine as needed during this dehydration step. Protein sequences for exemplary gene products can be found using the GenBank accession numbers below:<maths num="22"><img id="000025" he="7" wi="71" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> Non-natural gene candidates for this conversion have also been identified. For example, the multi-subunit L-serine dehydratase from Peptostreptococcus asaccharolyticus has been shown to supplement E. coli strains lacking L-serine dehydratase activity (Hofmeister et al., J. Bacteriol. 179: 4937-4941). 1997)). In addition, the putative 2- (hydroxymethyl) glutarate dehydratase encoded by the gene hmd in Eubacterium barkeri provides similarities to both the α-subunit and β-subunit of the [4Fe-4S] -containing bacterial serine dehydratase. (Alhapel et al., Proc.Natl.Acad.Sci.USA 103: 12341-12346 (2006)). Protein sequences for exemplary gene products can be found using the GenBank accession numbers below:<maths num="23"><img id="000026" he="5" wi="77" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> With reference to FIG. 3, step 5 comprises 2-enoate reductase. The final step in the 3-oxoadipate pathway is the reduction of the double bond in the hexa-3-engioate to form the adipate. Biochemically, this conversion is known to catalyze the NADH-dependent reduction of a wide variety of α, β-unsaturated carboxylic acids and aldehydes 2-enoate reductase (EC 1.3.1.31) (Rohdich et al.) , J. Biol. Chem. 276: 5779-5787 (2001)). This enzyme is part of Clostridia, including C. tyrobutyricum and C. thermoaceticum (now called Moorella thermoaceticum) (Rohdich et al., J. Biol. Chem. 276: 5779-5787 (2001)). Coded by enr in species of (Giesel and Simon, Arch. Microbiol. 135: 51-57 (1983)). Among the recently published genomic sequences of C. kluyveri, nine coding sequences for enoic acid reductase have been reported, one of which is characterized (Seedorf et al., Proc. Natl. Acad. Sci). .USA 105: 2128-2133 (2008)). The enr genes from both C. tyrobutyricum and C. thermoaceticum have been cloned and sequenced, showing 59% identity to each other. The earlier gene has also been found to have approximately 75% similarity to the gene characterized in C. kluyveri (Giesel and). Simon, Arch. Microbiol. 135: 51-57 (1983)). Based on these sequences, enr has been reported to be very similar to dienoyl CoA reductase (fadH) in E. coli (Rohdich et al., J. Biol. Chem. 276: 5779-5787 (2001)). ). Thus, there are several gene candidates for catalyzing this final step in the 3-oxoadipate pathway, which are listed below. The C. thermoaceticum enr gene is also expressed in an enzymatically active form in E. coli (Rohdich et al., Supra, 2001). Protein sequences for exemplary gene products can be found using the GenBank accession numbers below:<maths num="24"><img id="000027" he="24" wi="85" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> The above description provides an exemplary adipate synthesis pathway by the 3-oxoadipate pathway. Example IV Production of adipate-producing microorganisms having a 3-oxoadipate pathway This example illustrates the production of microorganisms capable of producing adipate using the 3-oxoadipate pathway. Escherichia coli is used as the target organism to genetically engineer the 3-oxoadipate pathway as shown in Figure 3. E. coli is a good host for producing non-naturally occurring microorganisms capable of producing adipate. E. coli is easy to genetically engineered, and it is known that E. coli can produce various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid effectively under anaerobic or microaerobic conditions. Is. Nucleic acids encoding enzymes utilized in the 3-oxoadipate pathway to produce E. coli strains genetically engineered to produce adipate have been developed into well-known molecular biological techniques (eg, Sambrook). , 2001; Ausubel, supra, 1999), and expressed in E. coli. Specifically, succinyl-CoA: acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase and 3-oxoadipate reductase activity encoding paaJ (NP_415915.1), pcaIJ (AAN69545.1 and NP_746082.1), and The bdh (AAA58352.1) gene is cloned into the pZE13 vector (Expressys in Ruelzheim, Germany) under the PA1 / lacO promoter. In addition, the acnA (P25516.3) and enr (ACA54153.1) genes encoding 3-hydroxyadipate dehydratase and 2-enoate reductase activity were added to the pZA33 vector (Ruelzheim, Germany) under the PA1 / lacO promoter. Clone to each of Expressys). These two sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for adipate synthesis by the 3-oxoadipate pathway. The resulting genetically engineered organism is cultured in glucose-containing medium according to procedures well known in the art (see, eg, Sambrook et al., Supra, 2001). Using methods well known in the art for determining polypeptide expression or enzymatic activity, such as Northern blots, PCR amplification of mRNA, immunoblotting and the like, for 3-oxoadipate pathway genes. Support expression. The enzymatic activity of the expressed enzyme is confirmed using an assay specific to the individual activity. The ability of the genetically engineered E. coli strain to produce adipate is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and / or liquid chromatography-mass spectrometry (LCMS). The number of microbial strains genetically engineered to have a functional adipate synthetic pathway is further increased by optimizing the efficient use of that pathway. Simply put, the genetically engineered strain is evaluated to determine if any of the exogenous genes are expressed at rate-determining levels. For example, the introduction of additional gene copy numbers increases expression for any enzyme expressed at low levels that can limit the flux through that pathway. Metabolic modeling is used to optimize growth conditions for better product production. Modeling is also used to design gene knockouts that further optimize the use of that pathway (eg, US Patent Publication US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003). / 0059792, US 2002/0168654 and US See 2004/0009466 and US Pat. No. 7,127,379). Modeling analysis allows reliable prediction of effects on cell proliferation that shift metabolism to more efficient production of adipate. One modeling method is the two-layer optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84: 647-657 (2003)), a gene that results in jointly producing better production of adipate. Used to select knockout. Adaptive evolution can also be used to produce better products of, for example, acetyl-CoA and succinyl-CoA intermediates or adipate products. Perform adaptive evolution to improve both proliferative and production properties (Fong and Palsson, Nat.Genet. 36: 1056-1058 (2004); Alper et al., Science 314: 1565-1568 (2006)). Based on these results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the adipate producer to further increase production. For large-scale production of adipate, 3-oxoadipate pathway-containing organisms are cultured in a fermenter under anaerobic conditions using a medium known in the art to support the growth of the organism. Fermentation is carried out in either batch, fed-batch or continuous mode. Anaerobic conditions may be maintained by first spraying the medium with nitrogen and then sealing the culture vessel, for example, the flask may be sealed with a septum and crimp cap. Microaerobic conditions can also be used by providing a small hole in the septum for restricted ventilation. The pH of the medium is H2SO4Maintain at a pH of about 7 by adding acids such as. The growth rate is determined by measuring the optical density using a spectrophotometer (600 nm), and the glucose uptake rate is determined by monitoring carbon source depletion over time. By-products such as unwanted alcohols, organic acids and residual glucose can be obtained using a refractometer detector for glucose and alcohol, and a UV detector for organic acids, eg, the Aminex® series HPLC. It can be quantified by HPLC (Shimadzu) using a column (eg HPX-87 series) (BioRad) (Lin et al., Biotechnol. Bioeng., 775-779 (2005)). This example illustrates the production of adipate-producing microorganisms containing a 3-oxidoadipate pathway. Example V cis, cis-Adipate synthesis via muconic acid This example illustrates a previously described adipate synthetic pathway (Niu et al., Biotechnol.Prog.18 (2): p.201-11.2002; Frost et al., USA, January 30, 1996). See Pat. No. 5,487,987). Adipate synthesis by a combined biological and chemical transformation process has been previously described (Niu et al., Biotechnol. Prog. 18 (2): 201-211 (2002)), which is shown in Figure 5. This method is further described in US Pat. No. 5,487,987. Adipate synthesis by this route requires the introduction of three heterologous genes into E. coli, which can convert dehydroshikimates to cis, cis-muconic acid (Niu et al., Supra, 2002). The final chemical hydrogenation step results in the formation of adipic acid. At this stage, pretreated fermented broth containing 150 mM cis, cis-muconate was mixed with 10% platinum (Pt) supported on activated carbon. The hydrogenation reaction was carried out at 250 ° C. for two and a half hours at a hydrogen pressure of 3400 KPa with stirring. Table 3 shows the adipate yield calculated assuming that cis, cis-muconate is converted to adipate using either the enzymatic or chemical catalytic step. Under aerobic conditions, an 85% molar yield of adipate can be obtained when a chemical reaction is used for hydrogenation, and a 75% molar yield can be obtained when using a NADH-based hydrogenase.<tables num="3"><img id="000028" he="27" wi="128" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> Although this is an exemplary method, it has drawbacks compared to others, such as those described in Examples I-IV. For example, the first limitation of this method is the low theoretical yield compared to the inverse adipate degradation and the 3-oxoadipate pathway. The second limitation is that the ATP yield of this pathway is negligible. A third limitation of this pathway is the need for dioxygenase, which forces the bioreactor to supply oxygen and hampers the choice of anaerobic fermentation. The above description provides exemplary adipate synthesis by the cis, cis-muconic acid pathway. Example VI Alpha-Adipate synthesis via ketoadipate This example illustrates an exemplary adipate synthesis pathway by the alpha-keto adipate pathway. Alpha-ketoadipate is a known intermediate in lysine biosynthesis at S. cerevisiae, and this information was used to identify additional pathways for adipic acid biosynthesis (see Figure 6). The conversion of alpha-ketoglutarate to alpha-ketoadipate is catalyzed by homocitrate synthase, homoaconitase and homoisocitrate dehydrogenase, as indicated by the dashed arrow in FIG. The conversion of alpha-ketoadipate to alpha-hydroxyadipate is 2-ketoadipate reductase (an enzyme reported to be found in rat and human placenta) (Suda et al., Arch.Biochem.Biophys.176: 610-620 ( 1976); Suda et al., Can be catalyzed by Biochem.Biophys.Res.Commun.77: 586-591 (1977)). Subsequent steps include dehydratase for the conversion of alpha-hydroxyadipate to hexa-2-endioate, which is then reduced to adipic acid. This final step may be catalyzed by an enzyme or it may be caused by a chemical reaction as described in Example II. The gene encoding the enzyme for the alpha-ketoadipate pathway is identified as described in Examples I-IV. Table 4 shows the adipate yield associated with this pathway. Two COs during conversion of acetyl-CoA to adipate2Due to the loss of molecules, only 67% of glucose can be converted to adipate. This is reflected in the molar yield for this route under aerobic conditions. This yield is further reduced in the absence of oxygen uptake. Also, the maximum ATP yield under anaerobic conditions is so small that genetically engineered organisms can form energy for cell proliferation and maintenance under such conditions. Additional substrates must be utilized.<tables num="4"><img id="000029" he="36" wi="126" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> The above description provides an exemplary adipate synthetic pathway by the alpha-keto adipate pathway. Example VII Adipate synthesis by lysine degradation This example illustrates an exemplary adipate synthesis pathway by the lysine degradation pathway. Two additional pathways for adipate synthesis rely on lysine degradation to form adipate. One pathway begins with alpha-ketoglutarate to form lysine (a pathway found in S. cerevisiae that is not inherent in E. coli) and the other uses aspartate as a starting point for lysine biosynthesis. (E.coli's inherent route). FIG. 7 shows the formation of adipate from lysine. E. coli stoichiometric model is used to show the maximum theoretical yield for adipate, both in the presence and absence of oxygen, using alpha-ketoglutarate and aspartate as their respective starting points for lysine. Shown in 5 and 6. The maximum ATP yields associated with these theoretical yields have also been calculated and shown in the same table. These yields are low compared to the other routes described in Examples I-IV. The gene encoding the enzyme for the alpha-ketoadipate pathway is identified as described in Examples I-IV.<tables num="5"><img id="000030" he="28" wi="110" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></tables><tables num="6"><img id="000031" he="27" wi="111" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> The above description provides an exemplary adipate synthesis pathway by the lysine degradation pathway. Example VIII Production of caprolactam and 6-aminocaproic acid via adipyl-CoA This example illustrates an exemplary caprolactam and / or 6-aminocaproic acid synthesis pathway by the adipyl-CoA pathway. An exemplary pathway for forming caprolactam and / or 6-aminocaproic acid using adipyl-CoA as a precursor is shown in FIG. This pathway includes a CoA-dependent aldehyde dehydrogenase capable of reducing adipyl-CoA to adipic acid semialdehyde and a transaminase or 6-aminocaproic acid dehydrogenase capable of converting this molecule to 6-aminocaproic acid. The final step in converting 6-aminocaproate to caprolactam may be performed by amide hydrolase or by chemical conversion (Guit and Buijs, USA, published March 7, 2002). Patent No. 6,353,100; US Pat. No. 5,700,934 by Wolters et al. Issued December 23, 1997; US Pat. No. 6,660,857 by Agterberg et al. Issued December 9, 2003). Assuming that the reaction scheme shown in FIG. 8 complements the reverse adipate degradation pathway, the maximum theoretical yield of caprolactam was calculated to be 0.8 mol per mol of glucose consumed. This pathway is energetically suitable as it forms up to 0.78 moles of ATP per mole of glucose consumed at the maximum theoretical yield of caprolactam. Assuming that phosphoenolpyruvate carboxylase (PPCK) functions in the direction of ATP production towards oxaloacetate formation, the ATP yield can be further improved to 1.63 moles of ATP produced per mole of glucose. The final amide hydrolase step is energetically and redoxically neutral, so the product and ATP molar yields associated with 6-aminocaproic acid production are equivalent to those associated with caprolactam production. Thus, microorganisms and related fermentation processes that form 6-aminocaproic acid rather than caprolactam, followed by additional unit operations to dehydrate / cyclize 6-aminocaproic acid to caprolactam can be considered as alternatives.<tables num="7"><img id="000032" he="34" wi="115" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> To successfully reshape this pathway in genetic engineering, it is necessary to identify the appropriate set of enzymes with sufficient activity and specificity. This requires identification of the appropriate set of enzymes, cloning of their corresponding genes into the production host, optimization of fermentation conditions, and assays for post-fermentation product formation. One or more exogenous DNA sequences can be expressed in the host microorganism to genetically engineer the production host for the production of 6-aminocaproic acid or caprolactam. In addition, the microorganism may carry a functionally deleted endogenous gene (s). These modifications allow the production of 6-aminocaproic acid or caprolactam using renewable feedstocks. A number of biochemically characterized candidate genes that can encode the enzymes that catalyze each step of the caprolactam formation pathway described in FIG. 8 are described below. Although E. coli is described, one of ordinary skill in the art can apply these teachings to any other suitable host organism. Specifically, the genes listed are those inherent in E. coli or genes in other organisms that can be utilized to catalyze proper conversion when correctly cloned and expressed. With reference to FIG. 8, step 1 comprises a CoA-dependent aldehyde dehydrogenase. An exemplary gene encoding an enzyme for catalyzing the reduction of its corresponding aldehyde to acyl-CoA is Acinetobacter calcoaceticus acr1 (Reiser and Somerville, .J. Bacteriol. 179: 2969), which encodes a fatty acid acyl-CoA reductase. -2975 (1997)), Acinetobacter species M-1 fatty acyl-CoA reductase (Ishige et al., Appl.Environ.Microbiol .. 68: 1192-1195 (2002)) and sucD gene from Clostridium kluyveri (Sohling and Gottschalk, J) .Bacteriol. 178: 871-880 (1996)), which can convert succinyl-CoA to semialdehyde succinate.<maths num="25"><img id="000033" he="27" wi="106" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> With reference to FIG. 8, step 2 comprises a transaminase. The second step in this pathway is the conversion of 6-aldehyde to amines. This conversion is encoded by gamma-aminobutyric acid transaminase (GABA transaminase), a natural enzyme encoded by gabT that transfers an amino group from glutamate to the terminal aldehyde of succinic semialdehyde (Bartsch et al., J. Bacteriol. 172: 7035-7042). 1990)), which is likely to be accomplished by. GABA transaminase in Mus musculus, Pseudomonas fluorescens, and Sus scrofa has been shown to react with 6-aminocaproic acid (Cooper, Methods Enzymol. 113: 80-82 (1985); Scott and Jakoby, J. Biol. Chem. .234: 932-936 (1959)). Protein sequences for exemplary gene products can be found using the GenBank accession numbers below:<maths num="26"><img id="000034" he="24" wi="101" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> With reference to FIG. 8, or step 2 may include 6-aminocaproic acid dehydrogenase, which constitutes the reductive amination of adipic acid semialdehyde to form 6-aminocaproate. This conversion can be accomplished by lysine-6-dehydrogenase, which naturally converts L-lysine to 2-aminoadipic acid-6-semialdehyde. Illustrative enzymes are Geobacillus stearothermophilus (Heydari et al., Appl.Environ.Microbiol.70 (2): 937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., J.Biochem. (Tokyo), 106 (1):76). -80 (1989); found in Misono et al., J. Biochem. (Tokyo), 105 (6): 1002-1008 (1989)), and Agrobacterium denitrificans (Ruldeekulthamrong et al., BMB Reports 790-795 (2008)). it can.<maths num="27"><img id="000035" he="19" wi="111" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> With reference to FIG. 8, step 3 comprises an amide hydrolase. The final step in caprolactam synthesis is the cyclization of 6-aminocaproic acid. This conversion is not enzymatically characterized, but lysine cyclization by D-lysine lactamase (EC 3.5.2.11) from Cryptococcus laurentii (Fukumura et al., FEBS Lett. 89: 298-300 (1978)). Very similar to. However, the protein and nucleotide sequences of this enzyme are currently unknown, and so far lysine-lactamase activity has not been demonstrated in other organisms. It has been demonstrated that plasmids contained in several strains of Pseudomonas species isolated from soil confer the ability to grow using caprolactam as a single carbon source (Boronin et al., FEMS Microbiol. Lett). .22: 167-170 (1984)), but to date, related gene or protein sequences have not been associated with this function. The most closely related candidate enzyme with available sequence information is 6-aminohexanoate cyclic dimer hydrolase, which is characterized in Pseudomonas and Flavobacterium species. The nylB gene product from Pseudomonas NK87 was cloned and expressed in E. coli ((Kanagawa et al., J.Gen. Microbiol. 139: 787-795 (1993)). The substrate specificity of this enzyme is Flavobacterium species. Tested in K172, it was shown to react with higher-order oligomers of 6-aminohexanoate but not caprolactam (Kinoshita et al., Eur. J. Biochem. 116: 547-551 (1981)). The ability of 6-aminohexanoate dimer hydrolase in other organisms to react with the desired substrate in the sex and direction of interest can be further tested. The protein sequence for the exemplary gene product is as follows: Can be found using the GenBank accession number:<maths num="28"><img id="000036" he="19" wi="106" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> The above description provides an exemplary pathway for producing caprolactam and / or 6-aminocaproic acid by the adipyl-CoA pathway. Example IX Production of 6-aminocaproate or caprolactam-producing microorganisms with 3-oxoadipate pathway This example illustrates the production of microorganisms that can use the reverse degradation pathway and convert intracellular adipates to 6-aminocaproate and / or caprolactam. Escherichia coli is used as a target organism for genetically engineering the genes required for adipate, 6-aminocaproate and / or caprolactam synthesis (see Figures 2 and 8). E. coli is a good host for producing non-naturally occurring microorganisms capable of producing adipate, 6-aminocaproate and / or caprolactam. E. coli is easy to genetically engineered, and it is known that E. coli can produce various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid effectively under anaerobic or microaerobic conditions. Is. Used in the reverse adipate degradation pathway and the 6-aminocaproate or caprolactam synthetic pathway to produce E. coli strains genetically engineered to produce 6-aminocaproate and / or caprolactam. Nucleic acids encoding the enzymes are expressed in E. coli using well-known molecular biological techniques (see, eg, Sambrook, supra, 2001; Ausubel, supra, 1999). Specifically, succinyl-CoA: acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyadipyl-CoA dehydratase activity encoding paaJ (NP_415915.1), paaH (NP_415913.1), and maoC. The (NP_415905.1) gene is cloned into the pZE13 vector (Expressys in Ruelzheim, Germany) under the PA1 / lacO promoter, respectively. In addition, the bcd (NP_349317.1), etfAB (349315.1 and 349316.1) and sucCD (NP_415256.1 and AAC73823.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthesizer activity, PA1 Under the / lacO promoter, clone into the pZA33 vector (Expressys in Ruelzheim, Germany), respectively. Finally, acr1 (YP_047869.1), gabT (NP_417148.1) and nylB (AAA24929.), Which encode CoA-dependent aldehyde dehydrogenase, transaminase and amide hydrolase activities. 1) The gene is cloned into a third compatible plasmid, pZS23, under the PA1 / lacO promoter. pZS23 is obtained by substituting the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module using well-known molecular biology techniques. These three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for 6-aminocaproate and / or caprolactam synthesis. The resulting genetically engineered organism is cultured in glucose-containing medium according to procedures well known in the art (see, eg, Sambrook et al., Supra, 2001). 6-Aminocaproate and 6-aminocaproate and the like using methods well known in the art for determining polypeptide expression or enzymatic activity, such as Northern blots, PCR amplification of mRNA, immunoblotting and the like. Supports the expression of caprolactam. The enzymatic activity of the expressed enzyme is confirmed using an assay specific to the individual activity. The ability of genetically engineered E. coli strains to produce 6-aminocaproate and / or caprolactam, HPLC, gas chromatography-mass spectrometry (GCMS) and / or liquid chromatography-mass spectrometry (LCMS) Confirm using. Microbial strains genetically engineered to have functional synthetic pathways for 6-aminocaproate and / or caprolactam are further increased by optimization for efficient utilization of those pathways. Simply put, the genetically engineered strain is evaluated to determine if any of the exogenous genes are expressed at rate-determining levels. For example, the introduction of additional gene copy numbers increases expression for any enzyme expressed at low levels that can limit the flux through that pathway. Metabolic modeling is used to optimize growth conditions for better product production. Modeling is also used to design gene knockouts that further optimize the use of that pathway (eg, US Patent Publication US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003). / 0059792, US 2002/0168654 and US See 2004/0009466 and US Pat. No. 7,127,379). Modeling analysis allows reliable prediction of effects on cell proliferation that shift metabolism to more efficient production of 6-aminocaproate and / or caprolactam. One modeling method is the two-layer optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84: 647-657 (2003)), which produces better production of 6-aminocaproate and / or caprolactam. Is used to select gene knockouts that result in co-occurrence. Adaptive evolution can also be used to produce better products, for example the products acetyl-CoA and succinyl-CoA intermediates. Perform adaptive evolution to improve both proliferative and production properties (Fong and Palsson, Nat.Genet. 36: 1056-1058 (2004); Alper et al., Science 314: 1565-1568 (2006)). Based on these results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the 6-aminocaproate and / or caprolactam producers to further increase production. For large-scale production of 6-aminocaproate and / or caprolactam, the above organisms are used in a fermenter using a medium known in the art to support the growth of the organism under anaerobic conditions. Incubate in. Fermentation is carried out in either batch, fed-batch or continuous mode. Anaerobic conditions may be maintained by first spraying the medium with nitrogen and then sealing the culture vessel, for example, the flask may be sealed with a septum and crimp cap. Microaerobic conditions can also be used by providing a small hole in the septum for restricted ventilation. The pH of the medium is H2SO4Maintain at a pH of about 7 by adding acids such as. The growth rate is determined by measuring the optical density using a spectrophotometer (600 nm), and the glucose uptake rate is determined by monitoring carbon source depletion over time. By-products such as unwanted alcohols, organic acids and residual glucose can be obtained using a refractometer detector for glucose and alcohol, and a UV detector for organic acids, eg, the Aminex® series HPLC. It can be quantified by HPLC (Shimadzu) using a column (eg HPX-87 series) (BioRad) (Lin et al., Biotechnol. Bioeng. 775-779 (2005)). Example X 2-Hydroxyadipyl-Adipate synthesis via CoA This example illustrates two exemplary adipate synthetic pathways starting with alpha-ketoadipate and passing through a 2-hydroxyadipyl-CoA intermediate. As described in Example VI, alpha-ketoadipate is a known intermediate in lysine biosynthesis that can be formed from alpha-ketoglutarate by homocitrate synthase, homoaconitase and homoisocitrate dehydrogenase. The two routes illustrated in Figure 9 can convert alpha-ketoadipate to 2-hydroxyadipyl-CoA. The 2-hydroxyadipyl-CoA can then be dehydrated and reduced to adipyl-CoA, and then the adipyl-CoA can be converted to adipate, as shown in FIG. The maximum yield of adipate from glucose by these pathways is 0.67 mol / mol. The conversion of alpha-ketoadipate to 2-hydroxyadipate is 2-ketoadipate reductase (an enzyme reported to be found in rat and human placenta) (Suda et al., Arch.Biochem.Biophys.176: 610-620 ( 1976); Suda et al., Can be catalyzed by Biochem.Biophys.Res.Commun.77: 586-591 (1977)). Alternatively, an enzyme capable of reducing alpha-ketoglutarate to 2-hydroxyglutarate may also be active against alpha-ketoadipate, which is one carbon atom longer. One such enzyme with alpha-ketoglutaric acid reductase activity is serA in Escherichia coli (Zhao and Winkler, J. Bacteriol. 178 (1): 232-9 (1996)). Additional exemplary enzymes can be found in Arabidopsis thaliana (Ho et al., J. Biol. Chem. 274 (1): 397-402 (1999)) and Haemophilus influenzae.<maths num="29"><img id="000037" he="19" wi="98" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> With reference to FIG. 9, it is likely that 2-hydroxyadipate can be converted to 2-hydroxyadipyl-CoA by the synthesizers, transferases, phosphotransadipylases and kinases described in Example I. Alternatively, an enzyme with 2-hydroxyglutarate CoA-transferase or glutaconate CoA-transferase activity is likely to be suitable for transferring the CoA moiety to 2-hydroxyadipate. An example of such an enzyme is Acidaminococcus. By the gctA and gctB genes of fermentans (Buckel et al., Eur.J.Biochem.118 (2): 315-321 (1981); Mack et al., Eur.J.Biochem.226 (1): 41-51 (1994)) It is coded. Similarly, as illustrated in FIG. 9, a synthesizer, transferase, or phosphotransadipylase and kinase would be required to convert alpha-ketoadipate to alpha-ketoadipyl-CoA. Conversion of alpha-ketoadipyl-CoA to 2-hydroxyadipyl-CoA can be performed by the alpha-hydroxyacyl-CoA dehydrogenase enzyme. Similar activity has been reported in propionate-adapted E. coli cells, and their extracts catalyzed the oxidation of lactyl-CoA to form pyrvir-CoA (Megraw et al., J. Bacteriol. 90 (4). ): 984-988 (1965)). Additional hydroxyacyl-CoA dehydrogenases have been described in Example I.<maths num="30"><img id="000038" he="13" wi="108" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> Dehydration of 2-hydroxyadipyl-CoA to form 5-carboxy-2-pentenoyl-CoA can be performed by 2-hydroxyacyl-CoA dehydratase. The 2-hydroxyglutaryl-CoA dehydratase system is characterized in Acidaminococcus fermentans and requires both the hgdA and hgdB subunits and the activator protein, hgdC, for optimal activity (Dutscho et al., Euro). .J.Biochem.181 (3): 741-746 (1989); Locher et al., J.Mol.Biol.307 (1): 297-308; Muller and Buckel, Euro.J.Biochem.230 (2): 698-704 (2001); Schweiger et al., Eur.J.Biochem.169 (2): 441-448 (1987)). This enzymatic system is mechanically similar to lactoyl-CoA dehydratase from Clostridium propionicum (Hofmeister and). Buckel, Eur.J.Biochem.206 (2): 547-552 (1992); Kuchta and Abeles, J.Biol.Chem.260 (24): 13181-13189 (1985)). HgdA, hgdB and hgdC homologs are present in some organisms.<maths num="31"><img id="000039" he="56" wi="121" file="JP5951990B2_D0001.tif" img-format="tif" img-content="drawing" /></maths> Conversion of 5-carboxy-2-pentenoyl-CoA to adipate is performed by the enzyme described in Example I. The above description provides an exemplary adipate synthetic pathway by the 2-hydroxyadipyl-CoA pathway. Example XI Production of adipate-producing microorganisms with 2-hydroxyadipyl-CoA pathway This example illustrates the production of microorganisms capable of producing adipate using the 2-hydroxyadipyl-CoA pathway. Escherichia coli is used as a target organism for genetically engineering the genes required for adipate synthesis (see Figure 9). E. coli is a good host for producing non-naturally occurring microorganisms capable of producing adipate. E. coli is easy to genetically engineered, and it is known that E. coli can produce various products such as ethanol, acetic acid, formic acid, lactic acid and succinic acid effectively under anaerobic or microaerobic conditions. Is. Well-known molecular biology of nucleic acids encoding enzymes utilized in the 2-hydroxyadipyl-CoA pathway to adipate to produce E. coli strains genetically engineered to produce adipate. It is expressed in E. coli using a technique (see, eg, Sambrook, supra, 2001; Ausubel, supra, 1999). Specifically, the serA (NP_417388.1), gctA (Q59111), and gctB (Q59112) genes encoding 2-hydroxyadipate dehydrogenase and 2-hydroxyadipyl-CoA: acetyl-CoA transferase activity were presented in PA1 / lacO. Under the promoter, clone into pZE13 vector (Expressys in Ruelzheim, Germany) respectively. In addition, the hgdA (P11569), hgdB (P11570), and hgdC (P11568) genes encoding 2-hydroxyadipyl-CoA dehydrogenase activity were added to the pZA33 vector under the PA1 / lacO promoter (Expressys in Ruelzheim, Germany). ), Respectively. In addition, the bcd (NP_349317.1), etfAB (349315.1 and 349316.1) and sucCD (NP_415256.1 and AAC73823.1) genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthesizer activities were added to PA1 /. Under the lacO promoter, clone into pZS23, a third compatibility plasmid. pZS23 is obtained by substituting the ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module using well-known molecular biology techniques. These three sets of plasmids are transformed into E. coli strain MG1655 to express the proteins and enzymes required for adipate synthesis. The resulting genetically engineered organism is cultured in glucose-containing medium according to procedures well known in the art (see, eg, Sambrook et al., Supra, 2001). 2-for adipate synthesis using methods well known in the art for determining polypeptide expression or enzymatic activity, such as Northern blots, PCR amplification of mRNA, immunoblotting and the like. Supports expression of the hydroxyadipyl-CoA pathway. The enzymatic activity of the expressed enzyme is confirmed using an assay specific to the individual activity. The ability of the genetically engineered E. coli strain to produce adipate is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) and / or liquid chromatography-mass spectrometry (LCMS). The number of microbial strains genetically engineered to have a functional adipate synthetic pathway is further increased by optimizing the efficient use of that pathway. Simply put, the genetically engineered strain is evaluated to determine if any of the exogenous genes are expressed at rate-determining levels. For example, the introduction of additional gene copy numbers increases expression for any enzyme expressed at low levels that can limit the flux through that pathway. Metabolic modeling is used to optimize growth conditions for better product production. Modeling is also used to design gene knockouts that further optimize the use of that pathway (eg, US Patent Publication US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003). / 0059792, US 2002/0168654 and US See 2004/0009466 and US Pat. No. 7,127,379). Modeling analysis allows reliable prediction of effects on cell proliferation that shift metabolism to more efficient production of adipate. One modeling method is the two-layer optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84: 647-657 (2003)), a gene that results in jointly producing better production of adipate. Used to select knockout. Adaptive evolution can also be used to produce better products, for example alpha-ketoadipate intermediates or adipate products. Perform adaptive evolution to improve both proliferative and production properties (Fong and Palsson, Nat.Genet. 36: 1056-1058 (2004); Alper et al., Science 314: 1565-1568 (2006)). Based on these results, subsequent rounds of modeling, genetic engineering and adaptive evolution can be applied to the adipate producer to further increase production. For large-scale production of adipate, 2-hydroxyadipyl-CoA pathway-containing organisms are used in fermenters under anaerobic conditions using media known in the art to support their growth. Incubate. Fermentation is carried out in either batch, fed-batch or continuous mode. Anaerobic conditions may be maintained by first spraying the medium with nitrogen and then sealing the culture vessel, for example, the flask may be sealed with a septum and crimp cap. Microaerobic conditions can also be used by providing a small hole in the septum for restricted ventilation. The pH of the medium is H2SO4Maintain at a pH of about 7 by adding acids such as. The growth rate is determined by measuring the optical density using a spectrophotometer (600 nm), and the glucose uptake rate is determined by monitoring carbon source depletion over time. By-products such as unwanted alcohols, organic acids and residual glucose can be obtained using a refractometer detector for glucose and alcohol, and a UV detector for organic acids, eg, the Aminex® series HPLC. It can be quantified by HPLC (Shimadzu) using a column (eg HPX-87 series) (BioRad) (Lin et al., Biotechnol. Bioeng. 775-779 (2005)). This example illustrates the production of adipate-producing microorganisms with a 2-hydroxyadipyl-CoA pathway. We have referred to various publications throughout this application. To better explain the state of the art to which this application belongs, the disclosures of these publications are incorporated herein by reference in their entirety. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.

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Document	Relation	Office	Cited during
KR20230159611A	Cited by	Republic of Korea	Applicant
WO2022210708A1	Cited by	World Intellectual Property Organization (WIPO)	Applicant
JP9505463A	Cites	Japan	–
US20030113886A1	Cites	United States of America	–
Metab Eng.,2002年,Vol.4,p151-158	Non-patent	–	–
Eur J Biochem.,1981年,Vol.116,p547-551	Non-patent	–	–

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US7799545B2	United States of America	B2	2010-09-21
EP2265709A2	European Patent Office (EPO)	A2	2010-12-29
US2010330626A1	United States of America	A1	2010-12-30
CN102066551A	China	A	2011-05-18
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US8216814B2	United States of America	B2	2012-07-10
EP2265709A4	European Patent Office (EPO)	A4	2012-08-29
US2012264179A1	United States of America	A1	2012-10-18
US2012309062A1	United States of America	A1	2012-12-06
US2013095540A1	United States of America	A1	2013-04-18
CN102066551B	China	B	2013-10-09
US8592189B2	United States of America	B2	2013-11-26
CN103555643A	China	A	2014-02-05
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DK2265709T3	Denmark	T3	2018-02-05
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JP2018102317A	Japan	A	2018-07-05
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BRPI0910928A2	Brazil	A2	2018-12-18
US10415042B2	United States of America	B2	2019-09-17
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