Microbial degradation of trichloroethylene.

PATENT OF INVENTION

No. 90,196

NAME: AMGEN INC., American, established in 1900 Oak Terrace Lane, Thousand Oaks, California 91320, United States of America,

EPÍGRAFE ('Process for microbial degradation of trichlorethylene

INVENTORS: Robert 3. Winter,

Kwang-Mu Yen,

Burt D. Ensley,

Claim of priority right under Article 4? of the Paris Union Convention of 20 March 1883.

USA, on April 5, 1988, under the N? 177,640, and 10/1983, under No. 235,354AMGEN INC.

Process for microbial degradation of tric1oroeti1eη o

BACKGROUND OF THE INVENTION

I Field of the Invention

The present invention relates to an improved process for microbial degradation of trichlorethylene by Pseudomonas mendocin KR-1 (PmKR1) cells or Pseudomonas putida KT2440 cells containing PmKR1 pAUTI plasmid (Pp Y2101) or genetically engineered microorganisms contain the PmKR1 genes of the 1ueno-moioo oxygenase. It has now been unexpectedly found that the toluene monooxygenase enzymatic system of PmKR1 is usable for degradation of the trichloromethane. 0 Isolation and cloning of gene segments encoding the PmKR1 toluene monooxygenase enzyme system is described in U.S. Patent Application No. 9. No. 77771, filed April 5, 1988, which is incorporated herein by reference.

In one aspect, the present invention relates to a process for microbial degradation of trichlorethylene by treating trichlorethylene with a microorganism host cell. which contains a recombinant plasmid, containing the recombinant plasmid toluene monooxygenase genes PmKR1. The host cell of the microorganism containing the recombinant plasmid should be treated with a toluene monooxygenase gene inducer to degrade trichlorethylene. The present invention provides a novel process for the degradation of trichloroethylene by providing genetically engineered microorganisms that exhibit levels of toluene monooxygenase enzyme activity under certain cell culture and assay conditions, which exceed levels expressed in wild type PmKR1 cells.

The present invention thus provides a more efficient means of effecting certain biodegradations related to this enzyme system, in particular the degradation of trichlorethylene. The invention is applicable to the degradation of existing trichloride in the form of a pollutant or contaminant in open or closed environmental systems.

tric 1 oroethylene (TCE) is a widely used industrial solvent that often exists as a contaminant of groundwater, drinking water and wastewater. Groundwater is the main reservoir of improperly treated hazardous waste. Over 200 organic and inorganic chemicals have been identified in various groundwater supplies, however, of all these identified contaminant chemicals, the EPA has found TCE to be the most frequently observed chemical contaminant at National Priority List (NPL) sites in the United States. -United.

The scale of the problem of groundwater contamination is further exemplified by the fact that groundwater supplies 25% of all water used in the United States. Calculations performed by The Conservation Eoundation (Groundwater-5th Edition of the Unseen Resource, November 1985, p. 5) show that in the United States groundwater is the source of: (1) 35% of all shrimp distribution water, (2) 50% of all drinking water (97% in rural areas), (3) 40% of all water used for irrigation in agriculture and (4) 26% of all water used in industry (excluding power stations). Thus, it is never too much to stress the importance of developing effective environmental techniques for the degradation of TBI in harmless products.

The development of genetically engineered microorganisms that have high capacities to degrade specific chemical contaminants such as TCE in innocuous products is an important strategy in the development of economically and environmentally acceptable processes for the removal of hazardous chemicals. 0 Development and use of microorganism host cells with recombinant plasmids containing PmKR1 toluene monooxygenase genes in the present invention is the first process using such genetically engineered microorganisms usable for the treatment of TBI chemical residues. 0 The development and use of host cells of microorganisms containing the recombinant plamids described herein are particularly advantageous because the specific and well characterized gene segments encoding the PmKR1 toluene monooxygenase genes have been cloned and used to construct the recombinant plamids. as described in US Pat. 177 631, cited above. It is such gene segments placed under the regulation of certain promoters that specifically confer high capacities to degrade TCE to certain host cells of microorganisms used in the present invention. This system easily allows the manipulation of isolated genes by cloning into a variety of cloning and expression vectors across a variety of different promoter systems to increase and optimize metabolic activity to degrade TCE. In addition, this system easily allows the study and manipulation of enzymes and specific proteins involved in TBI degradation. As a consequence of the preparation of a DNA segment containing PmKR1 toluene monooxygenase genes, the incorporation of these DNA segments into appropriate plasmid vectors, and the transformation of the host cells of the microorganism, the enzyme products of each other can be isolated and isolated. toluene monooxygenase from PmKR1. Therefore, a different pathway for TBI degradation can be followed using isolated and purified enzyme products instead of host cells of transformed microorganisms. Must understand5

<img file="PT90196B_D0001.tif" />

whereas PmKR1 toluene monooxygenase enzyme products can be used directly to degrade TCE. Such enzymatic products may be released to or applied to TCE chemical waste sites and may be usable for pollution control, for example for treating TCE-contaminated industrial wastewater.

II. Description of the technique

Many different processes have been proposed to render toxic waste safe. These include incineration, chemical transformation and microbial degradation. Since it does not involve the use of chemical reagents that may themselves be toxic and does not result in the production of large amounts of harmful fumes, such as those produced in the incineration of toxic waste, microbial degradation of toxic waste has become a preferred process. for the rejection of toxic waste.

Most of the microbial degradation of toxic products is based on the discovery of a particular microorganism that metabolizes the toxic product into innocuous metabolic products, usually in the case of toxic organic compounds, converting these compounds into carbon dioxide, water and salts. . Discovering microorganisms, in particular genetically engineered microorganisms, that can safely and effectively convert toxic waste into harmless metabolic products is a highly complex process that involves many woodpages if a significant amount of time is required. Most of this effort has focused on the discovery of indigenous microorganisms and their isolation from contaminated soil or water.

One way is to obtain a soil or water sample and enrich the sample with a mixture of microorganisms or isolate from a mixture a purified culture of a microorganism capable of degrading one or more of the toxic compounds. Several studies using mixtures of microorganisms containing methane-using bacteria obtained by methane enrichment from a soil sample have shown that these mixtures have the ability to degrade TCE and other chlorinated ethenes:

Fogel et al., Appl. Environ. Microbiol. 51, 1986, 720-724; Wilson and Wilson, Appl. Environ. Microbiol. 49, 1985, 242-243. Although these cultures using methane contain more than one type of bacteria, it has been proposed that methanotrophs are responsible for the degradation of TBI. Fogel et al. (supra) report a TBI degradation rate of 0.03 nanomoles TCE per minute per milligram of cellular protein.

Other studies did not use mixtures of microorganisms but purified strains isolated from soil or water. One such pathway is described in U.S. Patent No. 4,581,581.

493 895, in which a process for microbial degradation of contaminated halogenated aromatic compounds is claimed.

Ί

<img file="PT90196B_D0002.tif" />

to obtain harmless products. This process comprises the steps of (1) taking a product sample from a place contaminated with harmful chemicals; (2) enrichment of living microorganisms contained in the sample; (3) separation of strains of microorganisms capable of different metabolism for the various chemicals in the sample from one location to another; (4) purification of strains which are capable of biodegrading the chemicals to be rejected; (5) applying the strain to the place where the contaminants are deposited; and (6) controlling the removal of contaminants at the application site. U.S. Patent No. 2. No. 4,477,570 discloses and claims the microorganisms used in the process referred to above and claimed in U.S. Patent No. 2,478,507. 4,493,895.

Another route is described in U.S. Pat. 4 664 805 in which a process for decontaminating environments by halogenated organic compounds is claimed using (1) indigenous microorganisms from decontaminated environments which may metabolize but not develop into the contaminant; (2) an inoculum of non-indigenous environmental microorganisms that metabolize the contaminant faster than, but cannot develop into, the indigenous microorganism; and (3) a non-toxic contaminant analogue that serves as a substrate for the development of indigenous and non-indigenous microorganisms. Trust is placed on existing microorganisms in the environment, the so-called indigenous microorganisms, to carry out

χ is the degradation. Degradation is enhanced by non-indigenous microorganisms.

Still another route is described in U.S. Patent No. 5,481,379. No. 4,551 1,657 comprises a process for the treatment of earthwaste leachate containing chemical residues with activated sludge containing bacteria capable of metabolizing harmful organic products present in the leachate. All the techniques described above involve the use of microorganisms that are indigenous or isolated from contaminated soil or leachate.

The degrading enzymes required for microorganisms to degrade halogenated organic compounds can be encoded by plasmid genes. An individual plasmid generally contains genes encoding enzymes according to a single cycle of degradation. Plasmids have been used in methods for the biodegradation of certain chlorinated aromatic organic compounds as illustrated in US Pat.

535 061 (which describes plasmid-assisted reproduction processes for generating pure and mixed cultures of microorganisms capable of dissimulating persistent chemical compounds in the environment) and U.S. Patent No. 5,461,640.

664 805 discussed earlier.

European patent application NS. 8511008.1 describes the preparation of hybrid and transconjugated plasmids containing these plasmids, which come from microorganisms that degrade halogenated aromatic organic compounds

<img file="PT90196B_D0003.tif" />

(ATCC 31 939-31 945) (These microorganisms have been described in U.S. Patent Nos. 4,477,570 and U.S. 4,493,895). 8511008.1 describes a process for producing a microorganism that has specificity for biodegrading halogenated organic compounds comprises the steps of: (1) cultivating separately and maintaining (a) a broad spectrum of microorganisms chosen from the group of ATCC 31945, ATCC 31941, ATCC 31942 , ATCC 31940, ATCC 31943, ATCC 31944, ATCC 31939, and mutants thereof and (b) a vector from a wide range of hosts; (2) separately isolating plasmid DNA from (a) and (b) above; (3) separately purifying the plasmid DNA from (a) and (b) above; (4) separately performing the enzymatic restriction of purified DNA from (a) and (b) above; (5) combining the products of step (4) and enzymatically linking the combined products; (6) transforming the binding products into a recipient microorganism such as E. coli. coli or Pseudomonas species; (7) choosing transformants that have the desired plasmid DNA inserted into the vector; (8) conjugation of plasmid DNA to a recipient host selected from the group of Pseudomonas, Klebsiella, Rhizobium, Agrobacterium, Escherichia with the aid of an auxiliary plasmid; and (9) choosing transconjugants that have the desired plasmid DNA.

Eleven transconjugates have been described and, unexpectedly, most transconjugates have been found to use as their sole carbon source certain aliphatic halogenated organic compounds, specifically tetrachloromethylene, ethylene dichloride, methylchloroform and TCE, while parent microorganisms ATCC 31939- 31945 use only a broad spectrum of aromatic organic compounds as described in US Pat. 4,493,895. The only attempt for the use of these aliphatic halogenated organic compounds was to grow in medium containing the growth having been 50% greater than the average growth. Except for this developmental test, transconjugates are completely uncharacterized. In particular, nothing is described about the extent of degradation of these aliphatic halogenated organic compounds by these microorganisms or about the nature and toxicity of metabolic products. Nothing is said or described with respect to the genes, gene segments, enzymes, proteins or protein products involved in the ability of transconjugates to metabolize these aliphatic halogenated organic compounds, including TCE. In particular, the teachings of European patent application NS. 851008.1 are limited to the use of aliphatic halogenated organic compounds, including TCE, by plasmids selected from the group of microorganisms designated as ATCC 31939-31945.

Regarding TBI metabolism specifically, partial degradation of TBI by anaerobic organisms has been reported, but metabolism of the degradation process includes vinyl chloride and dichloroethylene which are also undesirable contaminants.

<img file="PT90196B_D0004.tif" />

groundwater sources. Kleopfer et al., Environ. Know.

T ech 1. 1. 19, 1 985, 277-280; Parsons et al., J. Am. Water Works Assoe. , 76, 1984, 56-59; Vogel &amp; McCarty, Appl. Environ Microbiol. , 49, 1985, 1080-1083.

Recent work describes a naturally occurring bacterial isolate that is capable of degrading TBI under aerobic conditions. Nelson et al., Appl. Environ Microbiol., 52, 1986, 383-384; Nelson et al., Appl. Environ. Microbiol., 53, 1986, 949-954. The microorganism designated Strain G4 requires phenol, toluene, N-crosses or n-cresol for TCE degradation. As characterized, Strain G4 (1) does not use the TOL scheme for toluene degradation; (2) Does not appear to have the toluene dioxigenase enzyme, the first enzyme in the TOD scheme for toluene degradation and (3) does not use the TMO scheme for toluene degradation. These three toluene degradation schemes (TOL, TOD, TMO) are summarized in U.S. patent application no. Serial No. 177,631. Nelson et al. does not explain or describe which genes, gene segments, enzymes, proteins or protein products are involved in the ability of Strain G4 to degrade TCE, or whether the genes involved are plasmid encoded or chromosomally encoded. Genetic engineering of Strain G4 has not been reported.

More recently, Nelson et al. Appl. Environ. Microbiol.

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54, 1988, 604-606, tested the degradability of TCE of 6 strains of microorganisms capable of degrading naphthalene, biphenyl, phenol and toluene. Only two of the strains tested,

PpE1 from Pseudomonas puti da (PpF1) and 85 from Pseudomonas putida (B5) degraded TCF. PpF1 and B5 are toluene degrading strains; however, a third strain that degrades toluene, Pseudomonas putida mt-2 (Pp mt-2), does not degrade TCE. Therefore it appears that not all toluene degrading strains are capable of degrading TCE.

Pp mt-2, which Nelson et al., Supra, found not to degrade TCE contains plasmid pWWO, which codes for the enzymes in the toluene degradation scheme known as TOL. The PpE1 that Nelson et al., Supra, found not to degrade TCE, is known to contain enzymes from the toluene degradation scheme designated by TOD. Wackett and Gibson, Appl.Environ. Io ίο robot 1. , 54, 1988, 1703-1 708 have also recently shown that PpE1 cells have the ability to degrade TCE. Under these culture and assay conditions, Wackett and Gibson, supra, found that approximately 50% to 60% of the initial TBI (20 µM) was degraded. In contrast, for high TCE concentrations (32 O ^ /^4, M) no degradation of TBI was detected. The highest rate of TBI degradation observed by these authors was 1.8 nanomoles per minute per milligram of cellular protein.

The genes for the PpE1 TOD scheme are encoded by

3 chromosomal pathway, in contrast to the Pp mt-2 genes of the TOL scheme which are encoded by plasmid. Therefore, it is not possible to predict whether genes involved in TBI degradation are encoded chromosomally or by plasmid. Studies by Nelson et al., Supra, and Wackett and Gibson, supra, with multi-component defective PpF1 mutants in the TOD toluene degradation scheme suggest that the ability of PpF 1 to degrade TCE is associated with the enzymatic activity of all. 1uene dioxigenase, which is the first chromosomal coding enzyme in the TOD scheme. Studies by Nelson et al., Supra, with Pp mt-2 showed that the degradation capacity of TBI is not associated with any enzymes of the plasmid-encoding TOL scheme.

None of the microorganisms tested so far for TBI degradability use the plasmid-coding THO scheme for toluene degradation. Furthermore, no microorganism has so far been genetically engineered to increase the enzymatic activity and degradability of TCE. Also, the use of any of the above described microorganism systems to degrade TCE presents several associated problems. A first problem is that in order to degrade TCE an enzymatic degradation scheme (eg the TOD scheme) must be induced in the microorganisms and the inducers to be added to the TCE contaminated sample are hydrocarbons. A second problem is that since TCE (a substrate for induced degradation enzymes) itself cannot be used by these microorganisms as a carbon source for cell development, cells from another substrate must be supplied ( a co-substrate) for development. These co-substrates added to the TCE contaminated sample are hydrocarbons such as toluene. These two problems are related to the practical problem, in view of the degradation of TCE in a contaminated sample such as an aquifer, it is not desirable to add hydrocarbons such as toluene (as inducer and / or carbon source) because hydrocarbons such as Toluene are themselves environmentally toxic compounds. A third problem closely related to the first two is that in the systems described above, where a hydrocarbon such as toluene acts as the inducer and substrate for the degradation scheme enzymes (where these enzymes metabolize the hydrocarbon and degrade the TCE), there is a competition between hydrocarbon and TCE for the same enzyme system. Under conditions where the hydrocarbon concentration is in excess of the TCE concentration, the hydrocarbon should compete more effectively for the enzyme system and retard the degradation of the TCE. These three problems illustrate various aspects of what is called the co-substrate problem that occurs in the inducible enzyme systems described above for TCE degradation.

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SUMMARY OF THE INVENTION

It has now unexpectedly been found that PmKR1 and genetically engineered microorganisms containing PmKRI toluene monooxygenase genes have the ability to degrade TCE. Accordingly, PmKRI and the host cells of microorganisms containing PmKRI toluene monooxygenase genes are usable in a process for the degradation of TCE.

The present invention thus encompasses a novel process for the degradation of TCE using these microorganisms that contain PmKRI toluene nonooxygenase genes. In particular, the present invention comprises a process for the degradation of TCE using genetically engineered microorganisms that have been developed and which have high capacities to degrade a specific chemical contaminant such as TCE with formation of innocuous products.

Thus, it is an object of the present invention to provide a process for the degradation of TBI wherever it appears as a contaminant or pollutant.

Another object of the present invention is to provide a process for microbial degradation of TCE where necessary, for example as a means for cleaning container and outdoor water, industrial effluent discharges, commercial or industrial government installations or factories or various laboratory operations and in other situations where the ECA is

6th .X may have accumulated. In particular, the present invention provides an improved process for the degradation of TCE using genetically engineered TCE-degrading microorganisms according to the present invention after TCE is removed from contaminated water (e.g. by entrainment). by air).

Another object of the present invention is to provide a process for the easy, efficient and relatively economical degradation of TCE.

It is a further object of the invention to provide microorganisms containing recombinant plasmids with toluene monooxygenase genes derived from enzymes encoding PmKR1 capable of degrading TCE, resulting in a non-toxic cell mass, which microorganisms are non-pathogenic to humans, animals. or marine fauna and flora.

Still another object of the present invention is to provide a process for TCE degradation wherein the host cell of the microorganism containing the recombinant plasmid with the PmKR1 to 1uene monooxygenase genes and capable of degrading TCE can be applied directly to the site. contamination of the ECA.

Yet another object of the invention is to provide a process for the degradation of TCE using genetically engineered microorganisms with PmKR1 to 1ueno-mooxyoxygenase genes

7th ί

in which the genes are placed under the control of various promoters and result in enhanced expression of toluene monooxygenase activity and enhanced degradation of TCE under certain cell culture and assay conditions.

Another object of the invention is to provide a process for the degradation of TBI by using genetically engineered microorganisms with PmKR1 toluene monooxygenase genes in which the genes are placed are under the control of non-hydrocarbon-induced promoter systems such as toluene, while solving the co-substrate problem presented by the inducible enzyme systems described above for TBI degradation. Advantages of using genetically engineered microorganisms according to the present invention therefore include: the elimination of a hydrocarbon such as toluene as an inducer; the elimination of a hydrocarbon such as toluene as co-substrate for the enzymatic degradation system; and the elimination of competitive inhibition of TCE degradation by the co-substrate / inducer hydrocarbon.

The invention provides a process for microbial degradation of TCE comprising treating TCE with PmKR1 cells or PpY21O1 cells containing a PmKR1 pAUTI plasmid or a microorganism host cell containing a recombinant plasmid. The recombinant plasmid contains toluene monooxy genes.

<img file="PT90196B_D0005.tif" />

genase isolated from PmKR1. The genes encode the protein enzymes of the TMO scheme for toluene degradation. According to the procedure, PmKR1 cells or ΡρY2101 cells or microorganism host cells with a recombinant plasmid are treated with a toluene monooxygenase gene inducer.

An advantage of the present invention in one aspect is the use of genetically engineered microorganisms with well characterized cloned genes, the expression of which is carried out under the control of well characterized and easily regulated promoters such as microorganisms such that these microorganisms are constructed. genetically have the ability to effectively degrade TBI. In addition, these genetically engineered microorganisms can be rapidly and economically developed to very high cell densities with the modern fermentation technology developed for the E. strains. coli, in contrast to the difficulty in developing natural isolates such as PmKR1 cells. Also, these genetically engineered microorganisms can maintain, in the presence of low glucose concentrations, the degradation of TCE for periods longer than 12 hours. Another advantage of using these recombinant microorganisms is that they are able to degrade TCE to extremely low levels, because metabolism no longer requires the presence of aromatic hydrocarbons (such as toluene) in the medium such as was required by PmKR1 cells.

Genetically engineered microorganisms can be applied directly to the TCE-containing environment to be decontaminated. Alternatively, cloned gene enzyme products may be used to degrade TCE instead of genetically engineered microorganisms at TCE chemical waste sites. The acceleration of the decontamination rate results from the use of these genetically engineered microorganisms with inducible genes for TBI degradation.

These and other objects and advantages of the present invention will become apparent to those skilled in the art from the following description and claims.

BRIEF DESCRIPTION OF DRAWINGS

Referring to the accompanying drawings, there are 8 figures, which will be described in detail, and illustrating the present invention:

FIG. 1 is a graph depicting the disappearance of 10yz, M (1.3 ppm) of TCE added to cells tested for TCE degradation capacity due to TCE metabolism by cells in non-volatile product as a function of time. The cells tested were Pseudomonas mendocina KR-1 (PmKR1), Pseudomonas putida E1 (PpF1) and Pseudomonas putida Y2101 (Pp21O1)

<img file="PT90196B_D0006.tif" />

FIG. 2 is a graph depicting the effect of adding toluene simultaneously with the addition of TCE on the disappearance of 20 μ · Μ (2.6 ppm) TCE added to cells tested for TBI degradation capacity due to TBI metabolism in non-volatile product as a function of time. The cells tested were: (1) PmKR1 with and without toluene addition at the same time as TCE addition; and (2) PmYAOOI.

FIG. 3 is a graph depicting the disappearance of 50 (6.5 ppm) TCE added to cells tested for TCE degradation capacity due to TCE metabolism in non-volatile product as a function of time. The cells tested were: (1) E. coli HB101. coli containing recombinant plasmid pMYA02 (HB101 / pMY402) under conditions such that cells were induced with IPTG before and during the assay (control cells were not induced with IPTG); and (2) EA coli FM5 cells containing the recombinant plasmid pKY287 (FM5 / pKY287) in which the cells were induced by a temperature increase (the control cells were FM5 cells containing the plasmid vector pCFM1146).

FIG. A is a graph depicting the disappearance of TCE added to cells at various concentrations due to TCE metabolism in non-volatile product as a function of time by HB101 cells containing the recombinant plasmid pMYA02.

FIG. 5 is a graph depicting the high pressure liquid chromatography (HPLC) elution profile of ^ C-labeled metabolites present in cell media after degradation of ^ C-TCE by PmKR1 cells (control cells were PmY4001 cells).

FIG. 6 is a graph similar to FIG. 5 but wherein the degradation of C-TCE was effected by EM5 / pKY287 cells (control cells were FM5 / pCEM 1146 cells).

FIG. 7 is a graph plotting the disappearance of 20 M (2.6 ppm) TCE added to cells due to TCE metabolism in non-volatile product as a function of time to compare the rate and extent of TCE degradation by wild-type PmKRI cells and by HB101 cells containing the recombinant plasmid pMY402 (HB101 / pMY402).

FIG. 8 is a graph similar to FIG. 7 but using 50 µl / 4M (6.5 ppm) TCE instead of 20 M (2.6 ppm). In addition, this figure includes the results obtained with? coli containing recombinant plasmid pKY287 (EM5 / pKY287) to compare the rate and extent of TCE degradation by wild-type PmKRI, HB101 / pMY402 and EM5 / pKY287 cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As noted above, TCE is an important industrial solvent and has been found to be widely distributed in the aquatic environment. Approximately 234 000 tonnes of TCE are produced annually worldwide (US Environmenta 1 Protection Agency, 1 980, EPA 440 / 5-80-077). TBI is very persistent and can be extremely difficult to remove once present in the environment. Little is currently known about the microbial metabolism of TBI. Isolated and cloned genes such as PmKR1 toluene monooxygenase genes used in the present invention should allow elucidation of a detailed understanding of a single type of TBI microbial metabolism. In addition, cloned genes can be engineered, for example, by combining genes with different promoters to increase expression of enzyme products to increase the rate and extent of TBI degradation. As illustrated in the present invention, the use of certain promoter systems under certain cell culture and assay conditions may increase the expression of toluene monooxygenase genetic products and may result in acceleration of the rate of TBI degradation by the microorganisms containing such antibodies. cloned genes.

The development of genetically engineered microorganisms that have high capacities to degrade specific chemical contaminants such as TCE to produce harmless products is an important strategy in the development of economically and environmentally appropriate methods for the disposal of hazardous chemical waste. .

<img file="PT90196B_D0007.tif" />

The development and use of microorganism host cells with recombinant plasmids containing PmKRI toluene monooxygenase genes according to the present invention is a novel and useful process that can be applied to the disposal of TCE chemical residues. In particular, these genetically engineered microorganism host cells are usable in an improved process for degrading TCE after it is removed from contaminated waters (e.g. by air entrainment). These host cells of genetically engineered microorganisms with PmKRI toluene monooxygenase genes have been fully described in US Patent Application NS. No. 177,631 which is incorporated herein by reference in its entirety.

It should also be understood that toluene monooxygenase gene enzyme products can be used to degrade TCE, rather than using host cells of microorganisms containing recombinant plasmids with toluene monooxygenase genes to degrade TCE. Enzyme products can be applied directly to TBI chemical waste sites.

The present invention provides a process that can be used to degrade TCE anywhere it is a contaminant or pollutant. Therefore, with the process, it becomes possible to clean and degrade TCE in wasteful places.

TCE chemical sts located in water or soil.

The following examples are given by way of illustration of the present invention and are not limitative.

EXAMPLE 1

Bacterial Strains and Development

The following bacterial strains are described in U.S. Patent Application No. 9. No. 177,631, which is incorporated herein by reference. In particular, the construction and characteristics of host cells of microorganisms including plasmids, vectors, genes and gene segments are described in said patent application. The following bacterial strains were grown overnight at 30 ° C in PA5 medium (Chakabarty et al., Proc. Natl. Acad. Scl. USA 70, 1973, 1137-1140) enriched with the following developmental substrates and inducers: Pm 'R1, toluene (supplied as vapor) for the development and induction of toluene degrading enzymes or 0.2% glucose for development; Pseudomonas puida F1 (PpF1), toluene (supplied in vapor state) for development and induction of 0.2% toluene or glucose degrading enzymes for development; Pseudomonas putida KT2440 (KT2440), 0.2% L-glutamate for development; Pseudomonas putida KT2440 containing plasmid pND50 (KT2440 / pND50), 2.5 mM p-cresol (diluted ί '

1000 times from a concentrated dimethylformamide stock solution) for development and induction of p-cresol degrading enzymes; Pseudomonas KT2440 crude containing plasmid pAUT 1 (KT2440 / pAUT1), toluene (supplied as a vapor) for development and induction of toluene degrading enzymes; Pseudomonas mendocin Y4001 (PmY4001), p-cresol

2.5 mM for development and induction of p-cresol degrading enzymes; the HB101 strain of E. .. coli containing the recombinant plasmids pKY277, pMY402 or the plasmid vector pMMB66EH (HB101 / pKY277, HB101 / pMY402, HB101 / pMMB66EH, respectively), 0.2% L-glutamate or 0.2% glucose, casaminic acids a 0.2% and 2yxg / ml vitamin B1 for development, 250) Ug / ml ampicillin for plasmid maintenance, and 1 mM isopropyl-1 (¼-D-1ioga1actoside (IPTG) for induction of toluene monooxygenase synthesis; KT2440 containing recombinant plasmid pMY402 (KT2440 / pMY402), 0.2% L-glutamate for development, 1 mg / ml ampicillin for plasmid maintenance and 5 mM IPTG for induction of toluene monooxygenase synthesis; £ 5 EM5. coli containing recombinant plasmid pKY287 or plasmid vector pCEM1146 (EM5 / pKY287 and FM5 / pCEM1146, respectively), 0.2% glucose and 0.05% yeast extract for development, 50yxg / m 1 kanamycin for maintenance of plasmid and for EM5 / pKY287 an increase in culture temperature to 42 ° C for two hours, followed by return to 30 ° C for two hours to induce synthesis of toluene monooxygenase. Cells were grown to at least one equal to 0.5, which corresponds to about 5 x 10 CFU / ml.

In addition to the above strains, several isolated strains designated E1, E2, E4, E5, E7, and E9 using ethylbenzene or toluene as carbon source for development have been developed and tested for TBI degradability. Strains using ethylbenzene were isolated as follows. Seven samples (numbered 1 to 7) were obtained from LaBrea Tarpit and nine samples (numbered 1 to 9) from Thousand Oaks Sewage Treatment Plant. An aliquot of 1 ml of each sample was inoculated into 50 ml of PAS medium supplemented with steam-supplied ethylbenzene from 0.2 ml of ethylbenzene and cultures were grown at 25 ° C for 5 days. The crops with the numbers 1, 4, 5_and 9 Thousand Oaks

Sewage Treatment Plant and LaBrea Tarpit number 2 and 7 cultures were grown to saturation (no development from other cultures). An aliquot of 1 ml of cultures 1, 2, 4, 5, 7, and 9 was taken and each was inoculated with 50 ml of steam-supplied ethylbenzene enriched PAS medium as described above. These 50 ml cultures were grown overnight at 25 ° C. A sample of each culture was spread over a PAS-ethylbenzene plate twice for individual colonies. These natural bacterial isolates using ethylbenzene have also been found to grow in toluene and develop during {

overnight at 30 ° C in PAS medium enriched with the following development substrates and inducers: 0.2% L-glutamate for development, 1 mM toluene for induction of toluene degrading enzymes. The cells were developed as described for the other strains indicated above until an OD of at least 0.5 was obtained.

EXAMPLE 2

Radioactivity Assay for TBI Degradation

This example describes a radioactivity assay in which the appearance of non-volatile TCE metabolites is determined.

The bacterial strains used in this assay were developed as described in Example 1. If necessary, cells were diluted with PAS medium to an OD of 0.5 µg for the assay. One ml of cell suspension in a 50 ml serum vial was added.

TCE (1.2 ° C, 72mCi per mmole, New England Nuclear, Boston, Massachusetts) until a final concentration of 5 µm. The solution of C-TCE was prepared by mixing radioactive and non-radioactive TCE to give a 5 mM solution in dimethylformamide at about 1 x 10 4 counts per minute and per minute. 4yttl of solution was added to each 4 ml of cell suspension, the serum vial was rolled with a Teflon-coated rubber stopper and a flip-down metal cap, shaken and incubated, shaking at room temperature. 30 ° C. At time zero and then at 30 minute intervals about 100 µl of cell suspension was collected using a needle and syringe and 20 µl of this sample was spread on a Whatman silica gel thin layer chromatography plate (Whatman, Clifton, New Jersey), air dried for 15 minutes and counted in a Beckman LS-100 scintillation counter (Beckman Instruments, Inc., Palo Alto, California) using a Biofluor liquid scintillation fluid (New England Nuclear). For Table 1, data obtained after air drying was converted to nanomoles of TCE metabolized to nonvolatile product, using 200 counts per minute per picomole as specific input activity.¹⁴C-TEC.

TABLE 1

ECA Degradation

^ C-TCE nanomoles converted to non-volatile product within 2 hours

Strain

Inductor

<td>PpE1</td><td>none</td><td> 0,4</td>

<td></td><td>toluene</td><td> 1,1-2,0</td>

<td>PmKR1</td><td>none</td><td> 0,4</td>

<td></td><td>toluene</td><td> 5,1-6,5</td>

<td>PmY4001</td><td>p-cresol</td><td> 0,4</td>

<td>Ρ ρ Y 2 1 0 1</td><td>none</td><td> 0,4</td>

<td></td><td>toluene</td><td> 3,8-5,1</td>

<td>PpY21 19</td><td>p-cresol</td><td> 0,4</td>

<td>PpY21 18</td><td>none</td><td> 0,4</td>

<td></td><td>IPTG</td><td> 0,7-1,1</td>

<img file="PT90196B_D0008.tif" />

^ C-TCE nanomomas converted to non-volatile product

<td>Strain</td><td>Inductor</td><td>labile in 2 hours</td>

<td>HB101 / pKY277</td><td>none</td><td> 0,4</td>

<td></td><td>IPTG</td><td> 1,6</td>

<td>HB101 / pMY402</td><td>none</td><td>0.3, 0.5 after 6 hours</td>

<td></td><td>IPTG</td><td>3.3-3.5, 4.7 after 6</td>

ho r as

Table 1 shows the results of extending the conversion of C-TCE to non-volatile product for 2 hours as a function of the inducer present during one night development. In these experiments, about 0.4 nanomoles of C-TCE remained volatilized; This represents a base of about 2% of the input C-TCE.

EXAMPLE 3

Gas chromatography assay for TBI degradation.

This example describes a gas chromatography assay in which the disappearance of volatile TCE is determined. The bacterial strains used in this assay were developed as described in Example 1. When necessary, overnight cultures were diluted to an OD of 0.5 g in PAS medium for the assay and 4 ml of PBS added. cell culture to serum vials. TCE (Aldrich, Mi 1 waukee, Wisconsin, spectrophotometer grade) was diluted to N, N'-dimethylformamide (DMF) (Aldrich, spectrum spectrum 30

<img file="PT90196B_D0009.tif" />

(photometric) at concentrations of 10 mM or 20 mM and 4jA was added to the cell suspension to give a final TCE concentration of 10 µM (1.3 ppm) (Figure 1) or 20 µM (2.6 µM). ppm) (Figure 2). The vials were rolled, shaken and 10 µl of gas phase collected using a gas syringe at the times shown in the figures. Gas phase samples were analyzed on a Hewlett-Packard 5890A gas phase chromatograph equipped with a 25-meter 5% phenylmethyl silicon column (Hewlett-Packard, Palo Alto, California) and an electron capture detector.

6 Ni. Injector, kiln and detector temperatures were 120 ° C, 100 ° C and 300 ° C respectively. The vector gas was helium and a mixture of 95% argon and 5% methane was used as the addition gas. The maximum areas were calculated using a Hewlett-Packard 3392A integrator. Data are shown in Figures 1, 2 and 3 as the percentage of TBI remaining after various times after addition to the cell suspension. The amount of TBI present at time zero was considered as 100%

Figure 1 represents the rate of TCE degradation at 10 M (1.3 ppm) TCE for PmKRI, KT2440 / pAUT1 and PpF1. Degradation is rapid 1 to 2 hours after TBI addition and becomes slower thereafter. PmKRI exhibits the highest activity of the three strains tested.

Figure 2 depicts the stimulation of degradation of pre-developed PmKRI cells in PAS medium containing toluene is present at the time of addition of TCE from TCE by toluene when

PmKRI should

<img file="PT90196B_D0010.tif" />

degrade more than 90% of the TCE initially present at 20 µL (2.6 ppm) when toluene is present as a vapor; Only approximately 50% of TCE is degraded by PmKR1 when toluene is not present at the time of TCE addition.

Fig. 3 represents the rate of TCE degradation to 20 µM (2.6 ppm) by HB101 / pMY402 cells and EM5 / pKY287 cells. More than 95% of the TBI is degraded after 4 hours.

EXAMPLE 4

TBI degradation by natural bacterial isolates

Strains using ethylbenzene (and toluene) isolated and developed as described in Example 1 were tested for their ability to degrade TCE. TCE concentrations were assayed by gas chromatography according to the technique described in Example 3. TCE at 20yx, M (2.6 ppm) was added. The results are presented in Table 2. In comparison with wild-type PmKR1 cells, the percentage of TBI remaining after an 18-hour degradation period was 6 to 14 times higher than for wild-type PmKR1 cells, indicating that these strains were substantially less effective than PmKR1 cells. wild type in its ability to degrade TBI.

ί

X__

Table 2

TBI Degradation by Natural Bacterial Isolates

<td></td><td>% TBI remaining</td>

<td>Strain</td><td>after 18 hours</td>

<td>PmKR1</td><td> 4</td>

<td>PmY4001</td><td> 84</td>

<td>PpE1</td><td> 49</td>

<td>E1</td><td> 48</td>

<td>E2</td><td> 39</td>

<td>E4</td><td> 56</td>

<td>E5</td><td>Not rehearsed</td>

<td>E7</td><td> 38</td>

<td>E9</td><td> 24</td>

TCE concentrations were assayed by gas chromatography according to example 3. TCE was added at a concentration of 20yUM (2.6 ppm). In this experiment, cells E1, E2, E4, E5, E7 and E9 were developed in toluene as a carbon source although as described in Example 1, these cells were originally chosen for development in ethylbenzene.

EXAMPLE 5

Effect of higher TBI concentrations

This example describes the degradation of increasing concentrations.

<img file="PT90196B_D0011.tif" />

<img file="PT90196B_D0012.tif" />

of TCE by recombinant cells according to the present invention. In this assay bacterial cells were developed as described in Example 1, except that 0.5% yeast extract (Difco, Detroit, Michigan) was used in place of casaminic acids and vitamin B1 in the development medium.

The gas chromatography assay for TBI degradation was performed as described in Example 3. Figure 4 shows the disappearance of TCE added to cells at varying concentrations of a gas phase due to TBI metabolism to of nonvolatile product, as a function of time, by HB101 cells containing recombinant plasmid pMY402 (vector of plasmid pMMB66EH with PmXR1 toluene monooxygenase genes). Blank circles represent 20 µM TCE metabolism (2.6 ppm); solid squares represent the 50yjM TCE metabolism (6.5 ppm); blank triangles represent the metabolism of 125 µM TCE (6 ppm); solid triangles represent TCE 310 µM metabolism (41 ppm); and the blank squares represent the metabolism of 780 µM TCE (100 ppm). Controls were performed for each TCE concentration using HB101 cells containing plasmid pMY402 in the absence of IPTG inducer or HB101 cells containing plasmid vector PMMB66EH

Figure 4 shows that HB101 cells containing the IPTG-induced plasmid pMY402 metabolize almost 100% TCE at TCE concentrations to about 20 ppm within 6 hours or less.

For 48 ppm TBI, 75% is metabolized within 8 hours. In addition, Figure 4 shows that increasing rates of TBI degradation occur as TCE concentration increases.

EXAMPLE 6

TBI degradation kinetics

In order to study the kinetics of TBI degradation by PmR1 to 1uene monooxygenase genetic products, PmKR1 and PmY4001 cells were developed according to the technique described in Example 1. In addition, for these experiments EM5 / pKY287 cells and EM5 / pCEM1146 cells were developed as follows: a cell inoculum was added to a L broth and the culture incubated at 30 ° C until the cells reached OD 0.5 after which the temperature was raised to 42 ° C for

1.5 hours to allow enzyme induction and synthesis, and then the temperature was lowered to 30 ° C for 4 to 6 hours to continue development. Cell cultures were centrifuged and resuspended in PAS medium or PAS medium containing 0.2 glucose.

In order to determine the degradation kinetics of TBI, cell cultures were diluted with 0.1 M KPO ^ pH 7.5 to a θ ^ 550 θ shown in Table 3 below. A portion of the diluted cells were retained for protein assays as follows. TBI degradation was assayed by gas chromatography substantially as indicated in Example 3, except that cell reactions were performed in 10 ml (instead of 4 ml) volumes in the serum vials and added to the vials. 5 to 101 TCE in dimethylformamide at the final concentrations indicated in Table 3 below.

Cell reactions were incubated with shaking at 30 ° C and at time zero and several times after TCE addition, 10 µl gas phase samples were taken and TCE concentration was analyzed as follows. shown in Example 3.

The initial rates of TBI degradation were calculated from the amount of TBI degraded during the initial 20 to 30 minutes of the reaction and were expressed as nanomoles per minute per mg of protein in Table 3 below. Most kinetic experiments were performed using EM5 / pKY287 cells, however several experiments were performed using PmKRI cells in order to compare rates when using recombinant cells with rates when using wild type cells. EM5 / pCEM1146 cells were used as control cells for EM5 / pKY287 cells, and PmY4001 cells were used as control cells for PmKRI cells to determine any TBI losses due to vial spillage or due to adsorption. in the cells. Experiments showed that losses in control vials were usually less than 5% after 1 hour incubation at 30 ° C.

Total cellular protein can be assayed by a variety of techniques including the Bradford, Anal method. Biochem.,

2, 1 976, 248-254, commercially available as Bio-Rad Protein Assay (Bio-Rad, Richmond, California, Catalog No. 500-0006). To lyse the cells and expose the cell protein to the reaction in the protein assay technique, sodium hydroxide was added to the cell suspension to a final concentration of 0.1 N, after which it was incubated at 100 ° C for 30 minutes before the test technique. Heated, sodium hydroxide-treated bovine plasma albumin was used as described as a protein standard in the assay technique.

The rates of TCE degradation by EM5 / pKY287 cells for various cell densities (ϋΟ ^ θ 0.05 to 1.00) and for various TCE concentrations (1 to 400.13 to 5.2 ppm) are indicated in Table 3

Table 3

Initial Cell Density

TBI Degradation Kinetics by EM5 / pKY287 cells (nmo1es / min / mg protein)

TBI concentration (RM)

<img file="PT90196B_D0013.tif" />

2,5

5,0

10,0

20,0

40,0

0,05 0,10 0,20 0,50 1 ,00

0,4 0,5

1,5

1,3

0,9

1,2 1,0 1,4 1 ,4 0,2

0,1

In experiments with toluene-induced PmKR1 cells, the rates of TCE degradation for an OD 50 of 0.50 and for TCE concentrations of 5.0 µM and 20.0 µM were 1.3 and 2 µM. , 4 nons / min / mg protein, respectively. At an OD50-g of 1.0 and a TBI concentration of 5 µM, the rate of TCE degradation by PmKR1 cells was 2.7 nmoles / min / mg protein. These results indicate that the rate of TCE degradation by wild-type PmR1 cells is at least comparable and is generally slightly higher than the rate of recombinant cells. The results presented in Table 3 also demonstrate that recombinant EM5 / pKY287 cells can effectively degrade TCE to a low cell density (OD50-q from 0.05 to 0.10) and to low TCE concentrations (1 µM at 2.5 µM). Therefore, even at low cell densities, these cells can be used in an effective process for TBI degradation.

EXAMPLE 7

4

C-TEC metabolism

In order to study the metabolism of ^ C-TCE by the PmKR1 toluene monooxygenase genetic products, PmKR1 cells, PmY4001 cells and HB101 cells containing pMY402 were developed as described in Example 1 and incubated as follows. described in Example 1, paragraph 1. Incubation was performed for 16 to 18 hours at 30 ° C.

1414 14 in COg, C in cell mass and C in development medium

<img file="PT90196B_D0014.tif" />

ί was determined as described by Nelson et al., Appl. Environ. Micro 53, 1 987, 949-954 and Spain and Nishino, Appl. Environ. Micro

, 1 987, 1010-101 9.

Shortly after the incubation period, the medium was acidified with 100 Π / l 2N sulfuric acid. The

The aqueous phase was introduced by introducing an air line into the flask and bubbling the air for 1 to 2 hours, while the free air containing it passed through another line into a tube containing 5 ml of 1N sodium hydroxide. . Radioactivity was determined in 0.5 ml of sodium hydroxide solution and the percentage C as carbonic anhydride in a 5 ml sample was calculated. After removal of the O 2 as described above, a 1 ml aliquot of the cell suspension was collected and the radioactivity of the cell suspension was determined. The remaining suspension of medium and cells was removed from the centrifuged flask until a cell pellet was obtained and the supernatant was passed through a 0 ° C filter.²Z The radioactivity of a 50 µl aliquot of the filtrate was determined and the percentage of C in the medium was calculated. The difference between the radioactivity in the cell suspension and the filtrate was taken as the radioactivity of present 14 in cell mass. Alternatively, the C in the cell mass could be determined directly by resuspending the cell aggregate and determining an aliquot of the suspended cells. Results from a representative experiment using PmKR1, PmY4001 and HB101 / pMY402 cells are shown in

9

Table 4

TABLE 4

4

C-TCE metabolism

Strain, 1 4 „o, 1 4„% As% Creates

C0 „cell mass%¹⁴c in the middle

P. mendocina KR-1

P. mendocina Y4001

HB101 / pMY402 (+ IPTG)

HB101 / pMY402 (-IPTG)

In other experiments, PmKR1 cells were developed,

PmY4001, EM5 / pKY287 and EM5 / pCEM1146 as described in the Example and then incubated with ^ C-TCE. The reaction was carried out with 14 C-TCE as described above with the difference: (i) purged 14.

the reaction of 1 N sodium hydroxide; and (ii) 1.2 ml of

CO2 from the aqueous phase and taken up in 20 ml of a barium chloride solution to 5 ml of the 20 ml solution of sodium hydroxide to precipitate CO2. The resulting precipitate was analyzed over an aliquot of the supernatant. . The results are shown in Table 5. Almost all present in the 1 N sodium hydroxide solution was precipitated by barium chloride, confirming that the present was in the form

<img file="PT90196B_D0015.tif" />

in ¹ ^ CO ^ ·

TABLE 5

4

C-TCE metabolism

<td>Strain</td><td>oJ4 / 0 L as C0_Q</td><td>oJ4_ç '° ^{L ra} 11 cell mass</td><td>o / 1 4 p .0 L Middle</td>

<td>P. mendocina KR-1</td><td> 32</td><td> 31</td><td> 31</td>

<td>P. mendocina Y4001</td><td> &lt; 1</td><td> 1</td><td> 2</td>

<td>EM5 / pKY287</td><td> 45</td><td> 1 8</td><td> 39</td>

<td>FM5 / pCEM1146</td><td> &lt; 1</td><td> 1</td><td> 2</td>

In order to further analyze the metabolites that were present in the cell medium each fraction of the cell medium indicated in Table 5 was treated as follows. Three drops of 45% potassium hydroxide were added to 10 ml of cell medium containing water-soluble metabolites to bring the pH of the solution to 11 to 12. This solution was lyophilized for 18 hours to 1 hour. 0.4 to 0.6 ml final volume with 60 to 70% recovery of C-labeled material.

Concentrated material was analyzed by high pressure liquid chromatography (HPLC) using an Aminex ion exclusion column (Bio-Rad) and using H2 SO4 as eluent.

0.01 N. 0.6 ml fractions were collected and the C was determined in 0.2 ml of each fraction as shown in Figures 5 and 6.

The C-labeled products were identified by comparing HPLC elution times with the elution times of unlabeled standards as shown in Figures 5 and 6. The standards used were: monochloric acid, oroacetic acid, glyoxylic acid and formic acid.

Table 6 summarizes the C-labeled metabolic products from TCE degradation by PmKR1 cells (Figure 5) and EM5 / pKY287 cells (Figure 6).

TABLE 6

Cell Media Analysis for TBI Degradation Products

<td rowspan="2">Compound</td><td colspan="2">1 4 % C in Cellular Medium</td>

<td>PmKR1</td><td>FM5 / pKY287</td>

<td>Dichloroacetic Acid</td><td> 9</td><td> 5</td>

<td>Glyoxylic acid</td><td> 64</td><td> 71</td>

<td>Formic acid</td><td> 1 6</td><td> 1 5</td>

<td>Unidentified compound</td><td> 1 1</td><td> 1 0</td>

<td>Of the total ^ c-TCE added</td><td colspan="2">to cells P m KR 1 or EM5 / pKY287 (d</td>

<td>note that approximately 30</td><td>at 40%</td><td>1 4 of total C was detected</td>

<td>in the cell fraction),</td><td>fence</td><td>3 to 5% of ¹⁴C total was</td>

identified under the roacetic. The remaining part was a chlorinated compound, the acidic acid of the acidic acid was dichloromethane.

<img file="PT90196B_D0016.tif" />

The compound is made up of COg (-30 to 40%), cell mass-innocuous cellular constituents (-18 to 35%) or predominantly non-chlorinated water-soluble components.

EXAMPLE 8

ECA dechlorination

This example describes an assay wherein the release of TCE chloride ions by PmKRI cells or host cells of microorganisms containing a recombinant plasmid with PmKRI toluene monooxygenase genes is determined.

PmKRI and EM5 / pKY287 cells were grown as described in Example 1. After cell growth the bacterial cultures were centrifuged at 5,000 rpm for 5 minutes, the growth medium was discarded and resuspended. cell aggregate or aggregated cells in 15 ml of 0.1 M potassium phosphate, pH 7.0, were centrifuged again and resuspended in 15 ml in 0.1 M potassium phosphate, pH 7. 0 The cells were diluted in the same buffer to an OD of 0.8 g and a final volume of 10 ml. The PmKRI cell suspension was added to toluene to a final concentration of 1 mM. The EM5 / pKY287 cell suspension was added glucose to a final concentration of 0.2% and kanamycin to a final concentration of 50y / µg / ml. The vials were rolled and TCE was added by syringe to a final concentration of 40yU.M (5.2 ppm). Incubated at 30 ° C for 5 to 18 hours

3 and the extent of TBI degradation was determined by gas chromatography.

After TCE degradation the cells were centrifuged for 5 minutes and the supernatants passed through a 0.2 µl filter. Chloride ion concentrations in the supernatants were determined by means of an EA 920 Orion ion meter using a Model 94-17B chloride electrode and a Model 90-02 reference electrode, both from Orion. A calibration curve with 20uM potassium chloride at 200 µM in 0.1 M potassium phosphate, pH 7.0 was determined by adding aliquots of potassium chloride to a background control sample (PmKR1 or EM5 / pKY287 cells in 0.1 M KP without any addition of TCE). Chloride ion concentrations in samples containing TCE were determined from this curve.

The results showed that 2.5 moles of chloride ion per mole of TCE were released using induced PmKRI cells and 2.7 moles of chloride ion per mole of TCE were released using induced EM5 cells containing pKY287.

EXAMPLE 9

TBI degradation capacity and to 1uene monooxygenase activity

A. T-1uene monooxygene assay for high cell densities and correlation with TCE degradation.

4th

<img file="PT90196B_D0017.tif" />

ί

For these assays, cells were grown in PAS medium containing 0.4% glutamate or L broth until saturation. However, FM5 cells were grown as described in Example 6 except that the cells were induced at 42 ° C for 3 hours and then the temperature was lowered to 30 ° C for 2 hours. The cells were resuspended in an appropriate volume of the same medium to an OD._no of 3.0. An aliquot of cells was used to determine total cellular protein as described in Example 6. An aliquot of 0.5 ml of cells was mixed with 4 itmoles of p-cresol in 10 µl and 15 µg.

I Ji-Í

<td>les de</td><td>toluene</td><td>radioactive</td><td>(toluene-</td><td>C-ring,</td><td>Sigma chemical</td><td>Co.</td>

<td colspan="2">56.3 mCi / nmole</td><td>) at 5 ^ tl and</td><td>incubated</td><td>the mix</td><td>the temperature</td><td>both</td>

<td>environment</td><td>during</td><td>20 minutes,</td><td>I acted</td><td>from time to</td><td>When. After</td><td>gives</td>

incubation, 20 µl of the mixture was spread on a small portion of a thin layer chromatography plate and the plate was air dried for 20 minutes. The remaining non-volatile radioactivide was determined by means of a liquid phase scintillation counter and this value was used to calculate the amount of toluene degradation product in the plate and the specific activity of toluene monooxygenase.

Table 7 presents the results of the assay described above for high cell density and a variety of strains that were also tested for TCE degradability. Table 7 also shows the correlation of the degradation capacity of TCE with toluene activity.

Α 5

-monοοχigeηase (BMT). In particular, Table 7 shows that under high cell density conditions pMYA02-containing HB1O1 cells and pKY287-containing FM5 cells exhibit approximately 2 to A times higher levels of PmKR1 toluene monooxygenase enzymatic activity than cells Wild type PmKR1. These two same genetically engineered host cells have high capacities to degrade TCE as evidenced by a longer sustained degradation rate and a greater amount of degradation to higher TCE concentrations relative to wild type PmKR1 cells. This is illustrated in Figures 7 and 8 for HB1O1 cells containing IPTG-induced pMYA02 (blank circles) compared to wild-type PmKR1 cells (blank triangles) and also shown in Figure 8 for EM5 / pKY287 cells (square to full). Increased enzymatic activity under the conditions described above therefore correlates with the increased ability to degrade TCE as illustrated in Figure 8 and Table 7.

<img file="PT90196B_D0018.tif" />

/

TABLE 7

TBI degradation and To 1uene monooxygenase activity at high cell densities

<td>PLASMIDEO</td><td>INDUCER</td><td>HOST</td><td>BMT activity *</td><td>Vector</td><td>tcE θ °</td>

<td>pAUTI</td><td>Toluene</td><td>PmKR1</td><td> 0,130</td><td> -</td><td> +</td>

<td>pAUTI</td><td>none</td><td>PmKR1</td><td> 0,010</td><td> -</td><td> -</td>

<td>pMY4O2</td><td>none</td><td>E. coli HB1O1</td><td> 0,005</td><td>pMMB66EH</td><td> -</td>

<td>pMY402</td><td>IPTG</td><td>E. coli</td><td> 0,200</td><td>pMMB66EH</td><td>-H</td>

<td></td><td></td><td>HB101</td><td></td><td></td><td></td>

<td>pKY287</td><td>heat</td><td>E. coli</td><td> 0,500</td><td>pCFM1146</td><td> +</td>

<td></td><td></td><td>FM5</td><td></td><td></td><td></td>

<td>pCEM1146</td><td>heat</td><td>E. coli</td><td> 0,005</td><td> -</td><td> -</td>

<td></td><td></td><td>EM5</td><td></td><td></td><td></td>

<td>pMMB66EH</td><td>IPTG</td><td>E. coli</td><td> 0,005</td><td> -</td><td> -</td>

<td></td><td></td><td>HB101</td><td></td><td></td><td></td>

<td></td><td>* An</td><td colspan="2">business unit of</td><td>BMT</td><td></td>

is expressed as 1 nmole of I 4 labeled toluene

C converted to non-volatile product per minute per milligram of total cellular protein

In addition to the plasmids listed in Table 7, which contain PmKR1 to 1αη-moηooxygenase genes, other plasmids containing these genes, including pKY277, pKY280, pKY281, pKY282, pMY401, pMY404, are suitable for degradation of TCE.

Ν9.

U.S. Patent Application Serial No. 177,631 (filed April 5, 1988 and incorporated herein by reference).

B. Toluene monooxygenase assay for lower cell densities and correlation with TCE degradation.

Cells were grown as described in Example 6.

Cell cultures were centrifuged, resuspended in PAS medium containing 0.2% glucose to a DG g of 0.5. The toluene monooxygenase assay was performed as described above in part A, except that the incubation time with 1 4

C-toluene is 5 minutes and not 20 minutes.

Table 8 summarizes the results of the assay described above for lower cell densities for strains that were also tested for TCE degradability. Table 8 also indicates the correlation of the degradation capacity of TCE with toluene monooxygenase (TMO) activity. In contrast to the results obtained for higher cell densities as shown in Table 7, Table 8 shows that under conditions of lower cell density, HB101 cells containing pMY402 and FM5 cells containing p? Y287 show levels of PmKR1 toluene monooxygenase enzymatic activity smaller than wild-type PmKR1 cells.

/

-/

TABLE 8

ECA Degradation and

To 1u-monooxygenase at low cell densities

<td>PLASMIDEO</td><td>INDUCER</td><td>HOST</td><td>Activity Unit * from BMT</td><td>Vector</td><td>Degradation from TCE</td>

<td>pAUTI</td><td>Toluene</td><td>PmKR1</td><td> 8.35</td><td></td><td> +</td>

<td>pAUTI</td><td>none</td><td>PmKR1</td><td> 0.08</td><td> -</td><td> -</td>

<td>pMY402</td><td>none</td><td>AND . coli HB1 01</td><td> 0.01</td><td>pMMB66EH</td><td> -</td>

<td>pMY402</td><td>IPTG</td><td>AND . coli HB101</td><td> 0.72</td><td>pMMB66EH</td><td> +</td>

<td>pKY287</td><td>heat</td><td>AND . coli FM 5</td><td> 1 .29</td><td>pCEM1146</td><td></td>

<td>pKY287</td><td>none</td><td>AND . coli EM5</td><td> 0.12</td><td>pCEM1146</td><td> -</td>

<td>pCEM1146</td><td>Heat</td><td>AND . coli EM5</td><td> 0.05</td><td> -</td><td> -</td>

<td>pMMB66EH</td><td>IPTG</td><td>AND . coli HB 1 01</td><td> &lt;0.01</td><td> —</td><td> -</td>

<td>* An</td><td>unity of</td><td>activity</td><td colspan="2">of BMT is expressed</td><td>how</td>

nmole of C-labeled toluene converted to nonvolatile product per minute per milligram of total cellular protein.

Genetically constructing the PmKR1 to 1ueno-monooxygase nase genes to place them under the control of several

promoters, increasing levels of expression of PmKR1 to 1uene monooxygenase gene products were achieved with a concomitant increase in TBI degradability.

Under conditions where PmKR1 cells were incubated with at least 40-fold excess toluene over TCE, a time lag of several hours was observed before PmKR1 cells began to degrade TCE (Figure 7, triangles in white). This is because toluene and TCE are co-substrates for PmKR1 toluene monooxygenase and both compete with the available PmKR1 to 1uene monooxygenase enzymes. Thus, when toluene and TCE are simultaneously present and toluene is present at concentrations in excess of TCE, the PmKR1 cells initiate TCE degradation only after the toluene concentration is considerably reduced. This same time lapse was not observed when PmKR1 cells were first induced with toluene and then toluene was eliminated prior to the addition of TCE (Figure 2). By cloning the PmKR1 allo-monooxygenase genes and placing them under the control of the non-toluene-induced promoters, the co-substrate problem described above was eliminated.

Many other promoter systems than the toluene-inducible, IPTG-inducible and heat-inducible promoters described herein are expected to be suitable for

<img file="PT90196B_D0019.tif" />

increase the expression of toluene monooxygenase and thus increase the degradation of TCE. In addition, many other types of plasmid vectors and host cells of microorganisms are expected to be suitable for PmKRI toluene monooxygenase expression and TCE degradation. Therefore, the present invention should not be limited by the exemplary embodiments described above. The invention will be defined by the following claims.