Method for the site-specific mutagenesis of DNA and development of plasmid vectors.

p0001The invention relates to a method for site-specific mutagenesis of DNA, and the development of plasmid vectors.

p0002Corynebacterium glutamicum, Brevibacterium flavum and related strains or derived from these mutants are known organisms are by which L-amino acids such as lysine and threonine produced by fermentation.

p0003For the genetic strain improvement plasmid vectors are an essential prerequisite. The construction of plasmid vectors for Corynebacterium or Brevibacterium is generally based on cryptic plasmids, which can be found in this group of bacteria. Examples include the plasmid pCG1 from Corynebacterium glutamicum ATCC31808 (U.S. Patent 4,617,267), the plasmid pAM330 from Brevibacterium lactofermentum ATCC 13869 (EP-A-0 093 611) and the plasmid pHM1519 from Corynebacterium glutamicum ATCC 13058 (Miwa et al. 1984). The plasmid further contain at least one DNA region, which gives the host resistance to an antibiotic. Examples are the kanamycin resistance gene of the transposon Tn5 (Santa Maria et al. 1984), the kanamycin resistance gene of plasmid pUB110 from Staphylococcus aureus (EP-A-0 093 611), the hygromycin resistance gene from Streptomyces hygroscopicus (Santa Maria et al. 1987 ) and the chloramphenicol resistance gene from Streptomyces acrimycini (Santa Maria et al. 1987).

p0004Plasmid vectors for Corynebacterium and Brevibacterium can be used to clone genes in the biosynthesis of amino acids, to express the corresponding gene or enzyme in an increased amount and thereby to improve the amino acid excretion. Examples include improving the excretion of L-lysine by Corynebacterium glutamicum by cloning and overexpression of phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum (British patent application 8821319.4).

p0005Plasmid vectors are composed of several regions of DNA. A DNA region allows plasmid replication in the appropriate host organism. A second DNA region mediates the cell resistance to an antibiotic and may be used as a genetic marker for selection of plasmid-bearing cells in a population. A third region of DNA confers resistance to another antibiotic, and can be used as a genetic marker for insertional inactivation. In the presence of appropriate restriction sites in one of the two genetic marks these can each be used for insertional, while the second brand is used for selection. Examples are the known to the expert plasmid pBR322 (Bolivar et al. 1979) and pACYC177 (Chang et al. 1978) called for E.coli.

p0006Prerequisite for the construction of such plasmid vectors are genes that mediate the host resistance to antibiotics. Another requirement is the existence of singular restriction sites in the corresponding resistance genes of the plasmid, to use this to insertional can.

p0007In order to use restriction sites in the cloned antibiotic resistance genes for insertional inactivation, it is necessary to remove endogenous interfaces in the plasmid. This can be done by filling the interface with nucleoside triphosphates or by nuclease treatment z. B.. These methods, however, are only conditionally applicable when there is a location in a gene or other essential region interface, because it is a change in the reading frame or deletion of DNA is connected. The introduction of point mutations is not associated with these disadvantages. A method that uses point mutations, such as mutagenesis with hydroxylamine (Birch et al. 1985), in turn, carries the risk of unspecific more mutations distributed to produce a whole plasmid.

p0008Object of the invention is a method for site-directed mutagenesis of DNA, to view z. B. the restriction sites in the cloned antibiotic resistance genes for insertional can, and the construction of new plasmid vectors.

p0009The invention provides a method for site-specific mutagenesis vonDNA at the restriction cleavage sites, which is characterized in that one isolates the DNA, cleaving with an appropriate restriction enzyme, the so-treated DNA with a generally 0.5 to 2 mol / l, preferably 1 mol / l, hydroxylamine-containing mutagenesis and mixed for a sufficient time, min is generally 10 to 60, preferably 20 to 30 minutes at elevated temperature, preferably at 65 to 70 ^OIncubated with the proviso that the DNA double-strand or only in the area of ​​the interface "melts", then the DNA receives according to known methods, treated with a ligase, transformed a suitable microorganism with such mutated DNA and the transformants isolated ,

p0010The isolation of DNA from cells of microorganisms is carried out according to methods well-known, as are the cleavage at the restriction sites, treatment with ligase, and the following steps.

p0011The mutagenesis includes at Birch et al. described components.

p0012The invention is also the treated according to the method described, DNA which has one or more restriction sites inserted. Particularly suitable is a DNA which, in one or, if present, comprises one or more singular restriction sites more resistance genes and / or the replicon due to the mutagenesis.

p0013This coding for resistance DNA segments may also be derived from an inserted transposon, as they are generally known. Is preferable to use DNA in the form of plasmids, plasmid vectors or phage vectors that appear in the claims for convenience as plasmids.

p0014The invention provides also the mutated DNA bearing microorganisms, among which especially strains of the genus Corynebacterium, Brevibacterium or derived amino acids, secreting mutants are preferred.

p0015The invention further relates to mutant forms of DNA that are produced by the method according to the invention. , Eg, the plasmids pCV34 (Fig. 6a) and pCV36 (Fig. 6b) called, have the opposite the starting plasmid a mutated EcoRI site (pCV34) or a mutated EcoRI and a mutated PstI site (pCV36) and thus mutant forms of pHM1519 replicon (Miwa et al. 1984).

p0016These plasmids are represented by the corresponding restriction enzymes no longer be cleaved and, therefore, preferable to the Ausgangsplasmiden for the construction of plasmid vectors. Furthermore, DNA fragments can be exchanged in plasmid molecules against those carrying mutations which have been prepared using the described method. Such a procedure is shown in Example 5.4 in the construction of the plasmid vector pZ9 (Fig. 9). Hosts for the plasmids pCV34, pCV36 and pZ9 are strains of the genera Corynebacterium and Brevibacterium and especially derived from these, amino-secreting mutants. E.coli is also a host for plasmid pZ9. The plasmid stragenden strains of the genera Corynebacterium, Brevibacterium and Escherichia can be cultured by conventional methods as they are known in the art. The following strains were deposited at the German Collection of Microorganisms: Corynebacterium glutamicum ATCC 13032 / DSM 5025 as pCV34, Corynebacterium glutamicum ATCC 13032 / DSM 5026 as pCV36 and Escherichia coli DH5 / pZ9 as DSM 4938th

p0017Another component of the invention is the use of antibiotic resistance genes from Corynebacterium xerosis for the construction of plasmid vectors that replicate flavum and related species in Gram-negative and Gram-positive bacteria, particularly in amino acid-secreting strains of the species Corynebacterium glutamicum, Brevibacterium.

p0018One in the Kono et al. (1983) isolated strain of Corynebacterium xerosis M82B included, about 50 kilobase pairs (kb) comprehensive resistance plasmid was determined by a modified lysis by Birnboim and Doly (1982) isolated and designated pCxM82B. After digestion of plasmid DNA with restriction enzymes, the plasmid was characterized by comparing the values ​​obtained in single and double digestion DNA fragments. After cloning of overlapping DNA fragments the restriction maps shown in Figs. 1a and 1b have been found. From the plasmid pUC19 pCxM82B DNA fragments were isolated with the aid of the technique known E. coli vector, which carry genes t he give the host resistance to the antibiotics chloramphenicol, kanamycin and erythromycin. The chloramphenicol resistance gene is located on a 5 kb BglII DNA fragments (Fig. 2a). The kanamycin resistance gene located on a 1.7 kb Sal I DNA fragment (Fig. 2b). The erythromycin resistance is located on a 8.5 kb Sal I DNA fragment (Fig. 2c). A tetracycline resistance gene that is not in<u>E.coli</u>, but in <u>C. glutamicum</u> leading to the expression of resistance, could by fusion of the gene (Fig. 2d) containing the pUC19 plasmid are identified with a Corynebacteria replicon and transformation in C. glutamicum. The Corynebacterium glutamicum strain ATCC 13032 with the shuttle vector p2Hi4S (Tc^R) Was deposited with the German collection of microorganisms as DSM 5396th The described DNA fragment with the corresponding resistance genes can be isolated by any person skilled in the at the German collection of microorganisms DSM 5021 deposited strain of Corynebacterium xerosis M82B. vermittelden the DNA sequence of the chloramphenicol resistance DNA fragment was determined as part of the invention. The analysis of the coding region is shown in Fig. 3b and the DNA sequence in FIG. 4. The structural gene comprising 1173 base pairs with an ATG codon at the beginning and two TGA codons at the end of the structural gene.

p0019As part of the invention were further, starting from the above-described resistance genes from Corynebacterium xerosis, plasmid vectors konstruiiert that replicate in amino acid-secreting strains of the species Corynebacterium glutamicum and Brevibacterium flavum or related species. Thus, starting from the above-described plasmid pCV36, constructed using the chloramphenicol resistance gene of the plasmid shown in Fig. 7 pCVX4 carrying the kanamycin resistance gene of the transposon Tn5 next to the chloramphenicol resistance gene. The plasmid pCVX4 has a length of 6.5 kb and offers the possibility of insertional inactivation by the unique restriction cleavage sites EcoRI, PstI and MluI in the chloramphenicol resistance gene. Furthermore, the plasmid vector was pCVX10 (Fig. 8a) constructed which has a length of 7 kb and carries the kanamycin resistance gene of Corynebacterium xerosis with the unique restriction cleavage sites XhoI and ClaI and the above-described chloramphenicol resistance gene of Corynebacterium xerosis. Finally, the plasmid vector pCVX15 shown in Fig. 8b was constructed, having a length of 13.8 kb and carries the erythromycin resistance gene and the above beschriebne chloramphenicol resistance gene of Corynebacterium xerosis.

p0020Folgene strains were deposited at the German collection of microorganisms Corynebacterium glutamicum ATCC 13032 / pCVX4 as DSM 5022, Corynebacterium glutamicum ATCC 13032 / DSM 5023 as pCVX10, Corynebacterium glutamicum ATCC 13032 / DSM pCVX15 than 5024th

p0021Furthermore the advantageous expression vector pZ8-1, which is shown in Fig. 10, a part of the invention. Plasmid pZ8-1 has a length of 7.0 kb and carries the mutated form of the replicon pHM1519, which was introduced during the construction by replacing a DNA fragment into the plasmid produced according to the above-described new method of hydroxylamine mutagenesis. In particular, the plasmid carries pZ8-1 the tac promoter (De Boer et al. 1983), followed by a DNA fragment containing multiple Klonierschnittstellen and finally the T1T2 terminator of the rrnB gene of Escherichia coli (Brosius et al. 1981 ). By using the T1T2 terminator the stable replication of the plasmid pZ8-1 is saved in C. glutamicum. Escherichia coli DH5 / pZ8-1 was deposited with the German collection of microorganisms DSM 4939th The suitability of the plasmid pZ8-1 as an expression vector was demonstrated by the insertion of the phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum ATCC 13032 (British patent application 8821319.4) in the multiple Klonierschnittstelle. The resulting plasmid pDM7 characterized (Fig. 11) has a length of 10.4 kb and causes in Corynebacterium glutamicum an approximately 20-fold increased expression of the enzyme phosphoenolpyruvate carboxylase.

p0022The construction of plasmid pDM7 succeeds the skilled person by known methods using the plasmids deposited pZ8-1 (DSM 4939) and pDM6 (DSM 4242) containing the ppc gene (Fig. 12)

p0023The invention also microorganisms of the genera Escherichia, Corynebacterium, Brevibacterium, in particular the amino acids secreting mutants containing inserts according to the invention mutated DNA or plasmids and resistance genes of C. xerosis M82B.

Examples

1. Isolation and characterization of a Resistenzplasmids from

Corynebacterium xerosis

p0024Resistance plasmid-bearing strains of <i>C.xerosis</i> were first described in 1983 (Kono et al.). The authors examined clinical isolates and found in some strains of<i>C.xerosis</i> with a wide resistance range of large (> 40 kb) plasmids. The R-plasmid of the strain M82B (pCxM82B) carrying resistance to erythromycin, chloramphenicol, kanamycin and tetracycline has been characterized in detail in the context of the invention.

1.1 Modified lysis for the recovery of plasmid DNA

p0025The lysis method according to Birnboim and Doly (1982) with the modifications <i>C. glutamicum</i> (Thierbach et al. 1988) is on <i>C.xerosis</i> limited applicability. For this reason, the following pretreatment was that, on an acetone treatment according to Heath et al (1986) is based, for lysis of<i>C.xerosis</i> developed by Birnboim and Doly.

p002610 milliliters (ml) of an overnight culture of <i>C.xerosis</i> in LBG (Luria Broth with 2 g / l of glucose - Maniatis et al.1982) with added antibiotics are added to 200 ml of LBG with antibiotic and 10% glycine and grown for 20 hours at 30 ° C in a shaker (120 rev / min). The cells are harvested by centrifugation (10 minutes at 6000 rev / min) in the centrifuge Beckman J2-21 (JA14 rotor) and resuspended in 1 ml TES (50 mM Tris, 5 mM EDTA, 50 mM NaCl, pH 8.0) was added. After addition of 40 ml of acetone, the cells are incubated for 6 minutes in ice, while mixing at intervals of 30 seconds by shaking. The cells are then pelleted and washed twice with 40 ml of TES. After renewed centrifugation, the pellet was resuspended in 40 ml buffer FL (410mm sucrose, 10 mM MgCl, 50% MMYC medium - Katsumata et al.1984) was added with 20 mg / mL lysozyme and multiply by a 50ml- plastic syringe (Fresenius) pressed. The homogenate thus prepared is with shaking (100 rpm) at 37 ° C for 5 hours and then centrifuged. The steps of the procedure then in accordance with the lysis according to Birnboim and Doly (1982) without lysozyme. The precipitated with 96% ethanol plasmid DNA is then taken up in 150 ul TE and determined the DNA concentration at 20 nm in the photometer. In this way, from 200 ml stationary culture of<i>C.xerosis</i> about 20 ug pCxM82B DNA recovered.

1.2 Restriction mapping of the plasmid pCxM82B

p0027For mapping the out <i>C.xerosis</i> isolated plasmid pCxM82B restriction enzymes were used, the (kb) relatively rare cut this about 50 kilobase plasmid. The below mentioned enzymes (Table 1a) have between one and four recognition sites on the plasmid.<tables id="tabl0001" num="0001"><table frame="all"><title>Table 1a</title><tgroup cols="2" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col2" align="center">For a rough mapping of pCxM82B used enzymes</entry></row><row><entry namest="col1" nameend="col1" align="center">restriction enzyme</entry><entry namest="col2" nameend="col2" align="center">Number of interfaces</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><i>Cla</i>I</entry><entry namest="col2" nameend="col2" align="right">3</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>Eco</i>RV</entry><entry namest="col2" nameend="col2" align="right">3</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>hpa</i>I</entry><entry namest="col2" nameend="col2" align="right">1</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>Nsi</i>I</entry><entry namest="col2" nameend="col2" align="right">4</entry></row></tbody></tgroup></table></tables>

p0028All restriction digests were performed according to the manufacturer's instructions. By double digests the position of the interfaces could be determined relative to each other. A detailed mapping was carried out by cloning overlapping restriction fragments of the plasmid, with frequent cutting enzymes (Table 1b) first in the <i>E.coli</i> Plasmid pUC19 (Norrander et al.1983) cloned, then mapped and were later combined into a circular map of the plasmid. The coarse mapping provided the clues to create the exact map (Fig.1). The strain<i>Corynebacterium xerosis</i> M82B with the R plasmid pCxM82B been deposited under the designation DSM 5021 at the German collection of microorganisms. <tables id="tabl0002" num="0002"><table frame="all"><title>Table 1b</title><tgroup cols="2" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col2" align="center">For subcloning and fine mapping of pCxM82B used enzymes</entry></row><row><entry namest="col1" nameend="col1" align="center">restriction enzyme</entry><entry namest="col2" nameend="col2" align="center">Number of interfaces</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><i>Bam</i>HI</entry><entry namest="col2" nameend="col2" align="right">7</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>Bgl</i>II</entry><entry namest="col2" nameend="col2" align="right">8th</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>Eco</i>RI</entry><entry namest="col2" nameend="col2" align="right">10</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>Hind</i>III</entry><entry namest="col2" nameend="col2" align="right">8th</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>Kpn</i>I</entry><entry namest="col2" nameend="col2" align="right">5</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>shush</i>I</entry><entry namest="col2" nameend="col2" align="right">13</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>Sal</i>I</entry><entry namest="col2" nameend="col2" align="right">13</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>Xba</i>I</entry><entry namest="col2" nameend="col2" align="right">2</entry></row></tbody></tgroup></table></tables>

p0029All length determinations were performed by gel electrophoresis on agarose gels and compared with length standards (DNA of bacteriophage λ, digested with <i>Eco</i>RI and <i>down</i>dIII and <i>shush</i>I).

2. Cloning of antibiotic resistance genes from

C.xerosis

in

E.coli

p0030For the isolation of DNA fragments with antibiotic-resistance properties, the overlapping subclones were in the <i>E.coli</i>Plasmid pUC19 (see 1.2) used.

2.1 Cloning of the chloramphenicol resistance gene

p0031DNA of plasmid pCxM82B was made by the method described in 1.1 <i>C.xerosis</i> and isolated with the restriction enzyme <i>Bgl</i>II digested. Out<i>E.coli</i> according to known methods (Maniatis et al., 1982) recovered plasmid DNA of the vector pUC19 was treated with <i>Bam</i>HI digested and treated with alkaline phosphatase. In the case of plasmids were mixed and the mixture is treated with T4 DNA ligase. This mixture was then<i>E.coli</i> JM83 (Messing 1979) transformed. It was from the transformants, a plasmid can be isolated, which confers resistance to the antibiotic chloramphenicol (25 ug / ml onto agar plates of Antibiotic medium No.3-Oxoid) lend. This plasmid, called pBg12 is new and consists of the pUC19 vector with a 5 kb insert of pCxM82B DNA (Fig. 2a). Plasmid pBg12 was with the enzymes<i>Bam</i>HI and <i>shush</i>I treated. Digestion with<i>shush</i>I was partially carried out. The product isolated from E.coli JM83 plasmid pSVB21 (Arnold and washer, 1988) was also with<i>Bam</i>HI and <i>shush</i>I digested. Both were then mixed and the mixture is treated with T4 DNA ligase. After transformation of E. coli JM83 plasmid pCX10 was isolated. This plasmid is new and carries the vector pSVB21 a 1.9 kb<i>Bam</i>HI-<i>shush</i>I subfragment of the insert of pBg12 (Fig.2a).

2.2 Cloning of the kanamycin resistance gene

p0032Plasmid DNA from pCxM82B was with the enzyme <i>Eco</i>RV digested with <i>Sma</i>I-digested and treated with alkaline phosphatase pUC19 DNA mixed. The mixture was incubated with T4 DNA ligase, and by<i>E.coli</i> JM83 transformed. From a kanamycin-resistant transformant, plasmid was isolated pEVK1, the JM83 resistance to 25 ug / ml kanamycin and 10 ug / ml neomycin (Antibiotic medium No.3) gives the strain. pEVK1 is new and consists of the plasmid pUC19 with a 2.7 kb DNA fragment from pCxM82B (Fig. 2b).

2.3 Cloning of the erythromycin resistance gene

p0033Following the procedure described in 1.1 Method plasmid DNA isolated from pCxM82B was with the enzyme <i>Sal</i>I split and with the likewise <i>Sal</i>I-digested and treated with alkaline phosphatase plasmid pUC19 mixed. The mixture was treated with T4 DNA ligase, and by<i>E.coli</i> JM83 transformed. There was isolated a transformant that against 120 ug / ml erythromycin and after this primary selection having a resistance of more than 2 mg / ml to resistant Antibiotic medium No.3. This plasmid (pSalE2) is new and consists of a 8.5 kb insert in the vector pUC19 (Fig. 2c). The localized on this DNA fragment is erythromycin resistance by small amounts (10-100μg / ml) of erythromycin inducible.

2.4 Expression of the resistance genes in

E.coli

p0034The in <i>E.coli</i>-vector PUC19 cloned antibiotic resistance promoting DNA fragments of pCxM82B (see 2) were tested for minimum inhibitory concentration towards (Table 2). These were the in<i>E.coli</i>Strain JM83 inoculated clones present in LBG liquid medium from an antibiotic-containing pre-culture with a concentration of about 10⁶ cells per milliliter, was added various concentrations of the appropriate antibiotics and incubated at 37 ° C for about 16 hours.

3. DNA sequence analysis of the chloramphenicol resistance gene

p0035long The nucleotide sequence of the 1.9kb <i>Bam</i>HI-<i>shush</i>I DNA fragment was determined by the method of Maxam and Gilbert (1977) with the modifications of Arnold and Puhler (1988) and by the method of Sanger et al. (1978) is completely determined. Subcloning was carried out into the<i>E.coli</i>-Sequenziervektoren PSVB20, pSVB21, pSVB25 and pSVB27 (Arnold and-washer 1988). The sequencing strategy is shown in Fig.3a. The piece of DNA carrying restriction sites for the enzymes<i>Bam</i>HI, <i>Eco</i>RI, <i>Nru</i>I, <i>shush</i>I, <i>Sac</i>I and <i>Sma</i>I with those of the subclones were prepared. The sequence of both DNA strands was analyzed using the sequence analysis program package ANALYSEQ (Staden 1986). The coding region (Fig.3b) specifies a coding for a protein region between positions 520 and 1720th In the nucleotide sequence of the 1.9kb DNA fragment (Figure 4) is found there is a long open reading frame. It starts at position 545 to the ATG start codon and ends at position 1717 with 2 consecutive stop codons (TGA). Six nucleotides before the start codon is a ribosome binding site (GGAG). The molecular weight of the protein is 39.3 Mdals. It has no sequence homology with known chloramphenicol acetyl transferase genes from other organisms (as EMBL DATA Library Release 15).<tables id="tabl0003" num="0003"><table frame="sides"><title>Table 2</title><tgroup cols="5" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="31.50mm" /><colspec colnum="2" colname="col2" colwidth="31.50mm" /><colspec colnum="3" colname="col3" colwidth="31.50mm" /><colspec colnum="4" colname="col4" colwidth="31.50mm" /><colspec colnum="5" colname="col5" colwidth="31.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col5" align="center">Minimum inhibitory concentration (MIC) of the cloned resistance</entry></row><row><entry namest="col1" nameend="col1" align="center">strain</entry><entry namest="col2" nameend="col2" align="center">plasmid</entry><entry namest="col3" nameend="col3" align="center">kanamycin</entry><entry namest="col4" nameend="col4" align="center">chloramphenicol</entry><entry namest="col5" nameend="col5" align="center">erythromycin</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><i>C.xerosis</i> M82B</entry><entry namest="col2" nameend="col2" align="left">pCxM82B</entry><entry namest="col3" nameend="col3" align="right">> 1000</entry><entry namest="col4" nameend="col4" align="right">> 200</entry><entry namest="col5" nameend="col5" align="right">> 2000</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>E.coli</i> JM83</entry><entry namest="col2" nameend="col2" align="left">-</entry><entry namest="col3" nameend="col3" align="right">20</entry><entry namest="col4" nameend="col4" align="right">5</entry><entry namest="col5" nameend="col5" align="right">300</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>E.coli</i> JM83</entry><entry namest="col2" nameend="col2" align="left">pUC19</entry><entry namest="col3" nameend="col3" align="right">20</entry><entry namest="col4" nameend="col4" align="right">5</entry><entry namest="col5" nameend="col5" align="right">300</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>E.coli</i> JM83</entry><entry namest="col2" nameend="col2" align="left">pEVK1</entry><entry namest="col3" nameend="col3" align="right">> 1000</entry><entry namest="col4" nameend="col4" align="right">nt</entry><entry namest="col5" nameend="col5" align="right">nt</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>E.coli</i> JM83</entry><entry namest="col2" nameend="col2" align="left">pBg12</entry><entry namest="col3" nameend="col3" align="right">nt</entry><entry namest="col4" nameend="col4" align="right">> 100</entry><entry namest="col5" nameend="col5" align="right">nt</entry></row><row><entry namest="col1" nameend="col1" align="left"><i>E.coli</i> JM83</entry><entry namest="col2" nameend="col2" align="left">pSalE2</entry><entry namest="col3" nameend="col3" align="right">nt</entry><entry namest="col4" nameend="col4" align="right">nt</entry><entry namest="col5" nameend="col5" align="right">> 2000</entry></row><row><entry namest="col1" nameend="col5" align="justify">All values ​​are expressed in ug / ml.</entry></row></tbody></tgroup></table></tables>

4. Construction of plasmid vectors (pCV30, pCV33) for

Corynebacterium

-and

brevibacteria

and production of mutant forms of the replicon pHM1519 and of Tn

5

-Kanamycin Resistance gene

4.1 Construction of vector plasmids for

Corynebacterium

- and

brevibacteria

p0036The construction of the vectors is shown in Fig.5. The shuttle vector described in British Patent Application 8821319.4 pECS300 (Fig. 13) was prepared from<i>E.coli</i> JM 83 isolated and after <i>C. glutamicum</i> ATCC 13032 transformed (Thierbach et al. 1988). Plasmid pHM1519 (Miwa et al. 1984) was partially with<i>down</i>dIII restricted and completely with the <i>down</i>dIII-digested pECS300 ligated. The ligation mixture was after<i>C. glutamicum</i> transformed, from 24 of the transformants plasmid DNA was isolated and with <i>down</i>dIII digested. In the shortest of these plasmids was the kanamycin resistance carrying<i>down</i>dIII fragment in one of the <i>down</i>dIII interfaces of cryptic replicon pHM1519 cloned before (pCV30). The 4.5 kb plasmid was then treated with pCV30<i>Sma</i>I and <i>Sal</i>I split and about 25 bp <i>Sma</i>I-<i>Sal</i>I-piece from the <i>E.coli</i>-vector PUC19 (Norrander et al. 1983) inserted. The resulting plasmid pCV33 is 4.5kb long and carries individual interfaces for the enzymes<i>Sma</i>I, <i>Bam</i>HI, <i>Xba</i>I and <i>Sal</i>I.

4.2

In vitro

Mutagenesis of plasmids from

Corynebacterium glutamicum

p0037To use restriction sites in the cloned antibiotic resistance genes for insertional, it might be necessary to first remove endogenous interfaces in the plasmid. This can be done by filling the interface with nucleoside triphosphates or by nuclease treatment, for example. These methods, however, are only conditionally applicable when there is a location in a gene or other essential region interface, since a change in the reading frame or deletion of DNA is achieved. A method that uses point mutations, such as mutagenesis with hydroxylamine (Ashley et al. 1985) but carries the risk of non-specific more mutations distributed over a whole to generate plasmid. The developed modified hydroxylamine mutagenesis is characterized above all by a high site-specificity. This is achieved by linearization of the plasmids with the appropriate enzyme and by lower temperatures than described, so that the DNA double strand melts only in the interface area, or not. Both the<i>Eco</i>RI site in 4.2.1 (pHM1519 - Miwa et al.1984) and the <i>shush</i>I interface are the kanamycin resistance gene in essential areas, and could be removed either by filling or by S1-nuclease. A point mutation (cytosine -> thymine) in the kanamycin resistance gene, however, would receive both the reading frame and the amino acid sequence of the protein.

4.2.1 mutagenesis of

Eco

RI site in the replicon of pCV33 and generating pCV34

p0038Plasmid pCV33 was to Thierbach et al. (1988) isolated and with the enzyme<i>Eco</i>RI digested. The mutagenesis consists of approximately 2μg plasmid DNA in 60μl of TE buffer, 180μl 1.5M hydroxylamine HCl in 25 mM EDTA, 5μl 0.25M EDTA and 13μl 1M Tris-HCl, pH 8.0. The reaction was mixed and incubated at 68 ° C for 30 minutes. After phenol treatment and ethanol precipitation of the plasmid DNA (Maniatis et al. 1982) the DNA pellet was dissolved in 20 .mu.l TE and treated with T4 DNA ligase. After transformation of<i>C. glutamicum</i> ATCC 13032 with plasmid DNA transformants could be isolated by the enzyme <i>Eco</i>RI no longer be split. Plasmid pCV34 (Abb.6a) is new. The strain<i>Corynebacterium glutamicum</i> ATCC 13032 with plasmid pCV34 was deposited with the German collection of microorganisms DSM 5025th

4.2.2 mutagenesis of

shush

I site in the kanamycin resistance gene of pCV34 and generate plasmid pCV36

p0039was analog to the above procedure (4.2.1) pCV34 plasmid DNA with the enzyme <i>shush</i>I linearised and subjected to the hydroxylamine mutagenesis. The reaction time and temperature were here 20 minutes and 68 ° C. After ligation with T4 DNA ligase and transformation by<i>C. glutamicum</i> could pCV36 (Abb.6b) isolated the plasmid by restriction against <i>Eco</i>RI and <i>shush</i>I is resistant. pCV36 is new and has the same kanamycin resistance height as pCV33.<i>C. glutamicum</i> ATCC 13032 / pCV36 was deposited with the German collection of microorganisms DSM 5026th

5. Construction of plasmids with two antibiotic resistance genes and the possibility of insertional

5.1 Construction of plasmid pCVX4 using the chloramphenicol resistance gene from

C.xerosis

p0040From the <i>E.coli</i>Plasmid pCX10 (2.1), which is a 1.9 kb insert of pCxM82B DNA carries (Fig.2a), was a 1.9 kb <i>Bam</i>HI-<i>Sal</i>I fragment isolated. The<i>Sal</i>I interface not originates from the <i>C.xerosis</i>DNA fragment, but is immediately adjacent to the the DNA fragment limiting <i>shush</i>I site in the vector pSVB21. The isolated DNA piece was with the<i>Bam</i>HI and <i>Sal</i>I restricted and treated with alkaline phosphatase plasmid pCV36 mixed. This mixture was ligated and after<i>C. glutamicum</i> transformed. From a chloramphenicol-resistant transformant, plasmid was isolated pCVX2.1 (fig.7). The new plasmid was then treated with<i>Sal</i>I linearized and with the nuclease <i>Bal</i>31 treated. After treatment with T4 DNA polymerase and ligation with T4 DNA ligase<i>C. glutamicum</i> transformed with the plasmid mixture and plasmid pCVX4 isolated from a transformant. pCVX4 is new, 6.5 kb long and conveys the cell resistance to kanamycin and chloramphenicol. It has the interface for<i>Sal</i>I and one of the <i>shush</i>I recognition sites lost and provides Insertionsinaktivierungsmöglichkeiten by the enzymes <i>Eco</i>RI, <i>shush</i>I and <i>Mlu</i>I in the Cm gene (fig.7). The carrying plasmid pCVX4 strain<i>C. glutamicum</i> ATCC 13032 was deposited with the German collection of microorganisms DSM 5022nd

5.2 Preparation of the plasmid pCVX10 using the kanamycin resistance gene from

C.xerosis

p0041Plasmid pCVX2.1 (see 5.1) was prepared from <i>C. glutamicum</i> isolated, with <i>Bam</i>HI and <i>Bgl</i>II digested, treated with T4 DNA ligase, and by <i>C. glutamicum</i> transformed. From a kanamycin-sensitive and chloramphenicol-resistant transformant, plasmid was isolated pCVX2.1ΔBB (Fig.8). This has a deletion of 1.4 kb compared to pCVX2.1 on which the whole kanamycin resistance gene from Tn<i>5</i> includes. was pCVX2.1ΔBB with<i>Sal</i>I linearized, treated with alkaline phosphatase and with the likewise with <i>Sal</i>I-digested plasmid pCxM82B mixed. After ligation and transformation, the plasmid was pCVX10 from a kanamycin-resistant transformant isolated (Fig.8a). pCVX10 is new, 7 kb in size and carries resistance to kanamycin and chloramphenicol. The plasmid also provides the Cm gene new Insertionsinaktivierungsmöglichkeiten by the enzymes<i>Xho</i>I and <i>Cla</i>I in the kanamycin resistance gene. The plasmid pCVX10 strain carrying<i>C. glutamicum</i> ATCC 13032 / pCVX10 was deposited with the German collection of microorganisms DSM 5023rd

5.3 Preparation of Plasmid pCVX15 using the erythromycin resistance gene from

C.xerosis

p0042Plasmid pCVX2.1ΔBB (5.2) was treated with the restriction enzyme <i>Sal</i>I digested and treated with alkaline phosphatase. pSalE2 (see 2.3) was charged with<i>Sal</i>I restricted and mixed both plasmids together. The mixture was treated with T4 DNA ligase, and by<i>C. glutamicum</i> transformed. From a to 10 ug / ml erythromycin resistant transformant, plasmid was isolated pCVX15 (Fig.8b), which comprises 8.5 kb, in<i>E.coli</i> also Em-conferring resistance, DNA fragment carries. Plasmid pCVX15 is new and carries unique cleavage sites for the enzymes<i>Bgl</i>II, <i>Bam</i>HI and <i>Xba</i>I on which the erythromycin resistance-carrying DNA fragment and for <i>Eco</i>RI and <i>shush</i>I in the Cm gene. The plasmid pCVX15 strain carrying<i>C. glutamicum</i> ATCC 13032 / PCVX15 was deposited with the German collection of microorganisms DSM 5024th

5.4. preparation of

E. coli C. glutamicum

Shuttle vector p2Hi4S and identification of the tetracycline resistance gene from

C.xerosis

in

C. glutamicum

p0043Of the <u>E.coli</u>-vector PUC19 was with the enzyme <u>down</u>dIII linearized and with the also <u>down</u>dIII digested plasmid pCxM82B ligated. It was isolated a pUC19 derivative containing a 10.7 kb DNA piece of pCxM82B. Plasmid p2Hi4 is new and carries on the cloned fragment contains the erythromycin resistance. was p2Hi4 with <u>Xba</u>I split. The Corynebacteria vector pCV33 (s. 4.1.) Was also with<u>Xba</u>I digested and treated with alkaline phosphatase. After ligation of both plasmids using T4 DNA ligase and transformation into the<u>E.coli</u>Strain DH5a was plasmid p2Hi4S (Fig. 14) can be isolated, which in <u>E.coli</u> Resistance to ampicillin, kanamycin and erythromycin mediated. p2Hi4S is new and can be used as shuttle vector in <u>E.coli</u> and <u>C. glutamicum</u> replicate. After transformation of<u>C. glutamicum</u> by p2Hi4S kanamycin and erythromycin-resistant colonies were isolated, which were additionally tetracycline-resistant. By plasmid analysis and retransformation could be demonstrated that the tetracycline resistance by the<u>C.xerosis</u>DNA fragment is mediated.

5.4 Replacement of mutant DNA fragments in plasmid vectors - Construction of plasmid pZ9

p0044Plasmid pZ1, which is described in German patent application 3737729.9, was from <i>E.coli</i> by known methods (Maniatis et al. 1982) was isolated. After linearization with the restriction endonuclease<i>shush</i>I plasmid using the nuclease was <i>Bal</i>31 is reduced by about one kilobase (kb). After treatment with T4 DNA ligase and transformation of<i>E.coli</i> DH5 (Hanahan, 1985) was a process called pZ2-1 plasmid isolated. After digestion of the plasmid with pCV34<i>down</i>cII and <i>shush</i>I, the 2.4 kb long <i>down</i>cII DNA fragment was isolated by electroelution. The fragment thus obtained was transformed with the plasmid pZ2-1, with the<i>down</i>cII and alkaline phosphatase treated, mixed, and the mixture is treated with T4 DNA ligase. After transformation of<i>E.coli</i> DH5 was isolated from a kanamycin-resistant transformant, plasmid pZ3-4 represented by the restriction endonuclease <i>Eco</i>RI is no longer cleaved. The plasmid pCVX2.1 which the chloramphenicol resistance gene from <i>C.xerosis</i> bears (see 5.1), was charged with <i>Bam</i>HI and <i>Sal</i>I treated. The 1.9 kb DNA fragment was then by treatment with<i>Bal</i>31 is reduced by about 0.1 kb. After treatment with T4 DNA polymerase, the described DNA fragment was inserted into the ScaI cleavage site of pZ3-4, wherein the vector was designated as pZ9 (Fig.9).<i>E.coli</i> DH5a / pZ9 was deposited with the German Collection of Microorganisms under DSM 4938th Plasmid pZ9 is new and carries unique restriction sites for<i>Xho</i>I and <i>Cla</i>I in the kanamycin resistance gene and <i>shush</i>I, <i>Mlu</i>I and <i>Eco</i>RI in chloramphenicol resistance gene that can be used for insertional inactivation.

5.6. Expression of the genes for kanamycin, chloramphenicol, erythromycin and tetracycline resistance from

C.xerosis

in

C. glutamicum

p0045The previously constructed plasmid vectors were after transformation (Thierbach et al. 1988) in <u>C. glutamicum</u> ATCC 13032 tested for their minimum inhibitory concentrations (Table 3). <tables id="tabl0004" num="0004"><table frame="all"><title>Table 3</title><tgroup cols="6" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="26.25mm" /><colspec colnum="2" colname="col2" colwidth="26.25mm" /><colspec colnum="3" colname="col3" colwidth="26.25mm" /><colspec colnum="4" colname="col4" colwidth="26.25mm" /><colspec colnum="5" colname="col5" colwidth="26.25mm" /><colspec colnum="6" colname="col6" colwidth="26.25mm" /><thead valign="top"><row><entry namest="col1" nameend="col6" align="center">Minimum inhibitory concentration (MIC) of the cloned resistance</entry></row><row><entry namest="col1" nameend="col1" align="center">strain</entry><entry namest="col2" nameend="col2" align="center">plasmid</entry><entry namest="col3" nameend="col3" align="center">kanamycin</entry><entry namest="col4" nameend="col4" align="center">chloramphenicol</entry><entry namest="col5" nameend="col5" align="center">erythromycin</entry><entry namest="col6" nameend="col6" align="center">tetracycline</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">C_x</entry><entry namest="col2" nameend="col2" align="left">pCxM82B</entry><entry namest="col3" nameend="col3" align="right">> 1000</entry><entry namest="col4" nameend="col4" align="right">> 200</entry><entry namest="col5" nameend="col5" align="right">> 2000</entry><entry namest="col6" nameend="col6" align="right">40</entry></row><row><entry namest="col1" nameend="col1" align="left">C_G</entry><entry namest="col2" nameend="col2" align="left">-</entry><entry namest="col3" nameend="col3" align="right"><10</entry><entry namest="col4" nameend="col4" align="right"><3</entry><entry namest="col5" nameend="col5" align="right"><5</entry><entry namest="col6" nameend="col6" align="right"><3</entry></row><row><entry namest="col1" nameend="col1" align="left">C_G</entry><entry namest="col2" nameend="col2" align="left">pCV34</entry><entry namest="col3" nameend="col3" align="right">> 500^*1</entry><entry namest="col4" nameend="col4" align="right">nt</entry><entry namest="col5" nameend="col5" align="right">nt</entry><entry namest="col6" nameend="col6" align="right">nt</entry></row><row><entry namest="col1" nameend="col1" align="left">C_G</entry><entry namest="col2" nameend="col2" align="left">pCVX10</entry><entry namest="col3" nameend="col3" align="right">> 1000</entry><entry namest="col4" nameend="col4" align="right">> 80</entry><entry namest="col5" nameend="col5" align="right">nt</entry><entry namest="col6" nameend="col6" align="right">nt</entry></row><row><entry namest="col1" nameend="col1" align="left">C_G</entry><entry namest="col2" nameend="col2" align="left">pCVX4</entry><entry namest="col3" nameend="col3" align="right">> 500^*1</entry><entry namest="col4" nameend="col4" align="right">> 80</entry><entry namest="col5" nameend="col5" align="right">nt</entry><entry namest="col6" nameend="col6" align="right">nt</entry></row><row><entry namest="col1" nameend="col1" align="left">C_G</entry><entry namest="col2" nameend="col2" align="left">pCVX15</entry><entry namest="col3" nameend="col3" align="right">nt</entry><entry namest="col4" nameend="col4" align="right">> 80</entry><entry namest="col5" nameend="col5" align="right">> 1000</entry><entry namest="col6" nameend="col6" align="right">nt</entry></row><row><entry namest="col1" nameend="col1" align="left">C_G</entry><entry namest="col2" nameend="col2" align="left">pZ9</entry><entry namest="col3" nameend="col3" align="right">> 500^{* 2}</entry><entry namest="col4" nameend="col4" align="right">> 80</entry><entry namest="col5" nameend="col5" align="right">nt</entry><entry namest="col6" nameend="col6" align="right">nt</entry></row><row><entry namest="col1" nameend="col1" align="left">C_G</entry><entry namest="col2" nameend="col2" align="left">p2Hi4S</entry><entry namest="col3" nameend="col3" align="right">> 500^*1</entry><entry namest="col4" nameend="col4" align="right">nt</entry><entry namest="col5" nameend="col5" align="right">> 1000</entry><entry namest="col6" nameend="col6" align="right">> 20</entry></row></tbody></tgroup></table></tables> The minimum inhibitory concentration was determined in LBG medium analogous to 2.4. Cx means<u>C.xerosis</u> M82B and Cg <u>C. glutamicum</u> ATCC 13032. The kanamycin resistance and the plasmids pCV34 pCVX4 (* ¹) determined by Tn<u>5</u> and the plasmid pZ9 of Tn<u>903</u> (* ²). All values ​​are expressed in ug / ml.

6. Construction of expression vectors for Corynebacterium and Brevibacterium using the mutant replicons pHM1519

6.1 Construction of the expression with the pZ8-1

tac

-Promoter And the

rrn

B-T1T2 terminator

p0046The expression vector pKK223-3 (Brosius, 1984) was purchased from Pharmacia and with the enzymes <i>Sca</i>I and <i>Bam</i>HI treated. Treatment with<i>Bam</i>HI was carried out here as partial digestion. The resulting 1.1 kb<i>Sca</i>I-<i>Bam</i>HI fragment containing the <i>tac</i>Promoter, a DNA sequence with recognition sites for the enzymes <i>shush</i>I, <i>Sal</i>I, <i>Bam</i>HI and <i>Eco</i>RI and the T1T2 terminator of <i>rrn</i>bears B gene was isolated by electroelution and treated with T4 DNA polymerase. The fragment described was transformed with plasmid pZ3-4 that with<i>Sca</i>I had been linearized, mixed, treated with T4 DNA ligase and <i>E.coli</i> DH5 transformed with the ligation mixture. From a kanamycin-resistant transformant, the plasmid pZ8-1 was isolated, which is shown in Fig.10.<i>E.coli</i> DH5 / pZ8-1 was deposited with the German collection of microorganisms under the number DSM 4939th After transformation of<i>C. glutamicum</i> ATCC 13032 was replicability, be detected by pZ8-1. Plasmid pZ8-1 proliferates under non-selective culture conditions for at least 70 generations in stable<i>Corynebacterium glutamicum</i>,

6.2 overexpression of PEP carboxylase gene (

ppc

) from

C. glutamicum

using the vector pZ8-1

p0047Plasmid pDM2, which is described in British patent application 8821319.4, was charged with <i>Sal</i>I and <i>Sma</i>I treated and mixed with the vector pZ8-1, with the <i>Sal</i>I had been linearized. The DNA mixture was treated with T4 DNA ligase and the ligation mixture used to transform<i>E.coli</i> XH11 (Mountain et al. 1984) used from a kanamycin-resistant and succinate-prototrophic transformant the plasmid shown in Fig.11 pDM7 was isolated. <i>C. glutamicum</i> ATCC 13032 was transformed with the plasmid pDM7. After culturing in MMYE medium (Katsumata et al. 1984) the specific PEP carboxylase content in the transformant ATCC 13032 / pDM7 and the control strain ATCC was prepared as described in British Patent Application 8821319.4, 13032 / pZ8-1 determined. The specific content of PEP carboxylase was 13032 / pDM7 3:00 U / mg protein in strain ATCC 13032 / pZ8-1 12:16 U / mg protein and strain ATCC.

literature

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