IE83308B1 - Bioconversions catalyzed by the toluene monooxygenase of pseudomonas mendocina KR-1 - Google Patents

Abstract

Abstract, University of Geneva EMBO Workshop, August 31-September 4, 1986), the art has not been provided with information regarding the genes encoding the enzymes and proteins of the toluene monooxygenase system in PmKRl or the usefulness of such genes and gene products in certain microbial bioconversions. The art has also not been provided with microorganism host cells harboring novel recombinant plasmids containing PmKRl toluene monooxygenase genes, in which induction of the toluene monooxygenase genes does not involve use of tonic compounds or simultaneous induction of other undesirable genes and in which some of the microorganism host cells harboring such recombinant plasmids under certain conditions express toluene monooxygenase enzyme activity at levels that equal, or under certain assay conditions, exceed the activity of wildtype PmKRl cells.

Description

BIOCONVERSIONS CATALYZED BY THE TOLUENE MONOOXYGENASE OF PSEUDOMONAS MENDOCINA KR-l AMGEN, INC.

The present invention is directed to the use of recombinant DNA techniques to confer upon microorganism host cells the capacity for selected bioconversions.

More specifically, the invention is directed to the cloning of toluene monooxygenase genes from a newly isolated and characterized Pseudomonas strain, Pseudomonas mendocina KR-1. The present invention provides genetically engineered plasmids that allow production of toluene monooxygenase enzymes and proteins in a variety of Gram-negative bacteria in the absence of a toxic inducer, and provides more efficient means of conducting bioconversions dependent on this enzyme system.

A bacterial strain identified as Bgeudomogas mendocina KR—l (PmKRl) was isolated by Richardson and Gibson from an algal-mat taken front a fresh water lake. Whited, Ph.D. Dissertation, The University of Texas at Austin, Library Reference No. W586 (1986).

PmKRl utilizes toluene as a sole carbon and energy source. Other bacterial strains have been isolated and described which metabolize or degrade toluene, including Pseudomonas putida mt-2 (Pp mt—2) (Williams and Murray, J. Bacteriol. 120: -423 (1974) and Pseudomonas putida PpFl (PpFl) (Gibson, et al. Biochemistry 221626- (l970)). In. addition, a bacterial strain designated. G4, isolated frmn a waste treatment lagoon, can metabolize toluene However, the genes, the enzymes and the pathways for toluene metabolism in these various bacterial strains are distinct and non- overlapping. particular, detailed studies of the organization and regulation of brief summary of the TOL pathway is as follows: initial attack of toluene is at the methyl group which undergoes successive oxidations to form benzoic acid, which is further oxidized by formation of a gig-carboxylic acid diol, which is oxidized to form catechol, which is then degraded by enzymes of a mgga cleavage pathway to acetaldehyde and pyruvate.

A second catabolic pathway for the degradation of toluene by In contrast to the PpF1 has been established and designated TOD.

TOL pathway, the genes for the TOD pathway are located on the bacterial chromosome and are not plasmid-encoded. Finette, et al., The genetics and biochemistry of the TOD pathway has been studied by Finette, et al. (supra); Finette (supra); Gibson, et al.

Biochemistry 2:1626-1630 (1970); Kobal, et al., J. Am. Chem. Soc.

Biochemistry 1:2653-2662 (1968). A brief summary of the TOD pathway is as follows: the initial attack of toluene is by a dioxygenase methylcatechol which is further degraded by enzymes of a meta cleavage pathway. Zylstra and Gibson, J Biol. Chem. gggz 14940- (1989) and Mccombie, Abstr. Annu. Meet. Am. Soc. Microbiol. gggz 155 (1984) have reported the cloning and sequencing of the tod genes which encode the first three enzymes in the TOD pathway. by a unique enzyme complex, toluene monooxygenase. The biochemistry of the partially purified enzymes and proteins of this pathway has been recently studied by Whited, Ph.D. Dissertation, The University of Texas at Austin, Library Reference No. W586 (1986). recently, a toluene catabolic apparently More pathway, distinct from the three above-described pathways, has been described in the trichloroethylene-degrading bacterium C4 by Shields et al., App. Environ. Microbiol. 5;: 1624-1629 (1989). The bacterial strain designated G4 was isolated from a waste treatment lagoon. Strain G4 is uncharacterized with respect to genus and species. The toluene pathway of G4 appears to involve dihydroxylations of the aromatic ring by two monooxygenations, first ortho and then meta. The enzymes involved in these reactions have not been isolated and studied, and therefore remain completely uncharacterized.

The steps of the TMO pathway as outlined by Whited (ggpra) are In the initial step toluene is oxidized to diagrammed in Figure l. p-cresol, followed by methyl group oxidation to form p- hydroxybenzoate, followed by hydroxylation to protocatechuate and subsequent ortho ring cleavage. In the first step of the TMO pathway, toluene is converted by toluene monooxygenase to p-cresol.

PmKRl elaborates a unique multicomponent enzyme system which catalyzes this first step monooxygenase reaction. The implications of the teachings of Whited, (ggpga), suggest that at least three protein components may be involved: component a (possibly NADH oxidoreductase, molecular weight unknown), component b (possibly an oxygenase, at least 2 subunits) and component c (red-brown, probably ferredoxin, 23,000d.).

Despite beginning biochemical studies of the enzymes and proteins of the TMO pathway (Whited, supra) and beginning genetic studies (Yen et al. Abstract, University of Geneva EMBO Workshop, August 31-September 4, 1986), the art has not been provided with information regarding the genes encoding the enzymes and proteins of the toluene monooxygenase system in PmKRl or the usefulness of such genes and gene products in certain microbial bioconversions. The art has also not been provided with microorganism host cells harboring novel recombinant plasmids containing PmKRl toluene monooxygenase genes, in which induction of the toluene monooxygenase genes does not involve use of tonic compounds or simultaneous induction of other undesirable genes and in which some of the microorganism host cells harboring such recombinant plasmids under certain conditions express toluene monooxygenase enzyme activity at levels that equal, or under certain assay conditions, exceed the activity of wildtype PmKRl cells.

SUMMARY OF THE INVENTION According to the first aspect of the present invention there is provided an isolated nucleotide sequence encoding the tmoABCDE gene product, said product having the sequence of Figures 5A to E, as well as an isolated nucleotide sequence encoding the tmoABCDEF gene product, said product having the sequence of Figures 5A to F, and a recombinant plasmid comprising such a nucleotide sequence.

‘The present invention also provides a DNA sequence encoding for proteins having substantially the same amino acid sequences and having substantially the same activity as the tmoABCDEF gene products of Figures 5A to F, as well as isolated proteins having an amino acid sequence of the TmoA protein of Figure 5, the TmoB protein of Figure 5, the TmoC protein of Figure 5, the TmoD protein of Figure 5, the TmoE protein of Figure 5 and the TmoF protein of Figure 5.

The present invention further provides an improved method for the microbial degradation of TCE comprising reacting a TCE-containing substance with microorganism host cells, the microorganism host cells having been treated with an inducer of toluene monooxygenase genes. sguggr OF THE INVENTION The present invention provides novel gene segments, biologically functional plasmids and recombinant plasmids, and microorganism host cells, all of which contain the PmKRl toluene monooxygenase genes. The present invention further provides a microorganism host cell harboring a novel recombinant plasmid containing PmKRl toluene monooxygenase genes, in which synthesis of only toluene monooxygenase but not other undesirable enzymes can be induced specifically with an innocuous and inexpensive inducer and exceed the at levels that equal or, under certain conditions, activity of wildtype PmKRl cells. In addition, the present invention provides a method for using transformed microorganism host cells containing the PmKRl toluene monooxygenase genes in microbial bioconversions. Thus, the present invention provides microorganisms genetically engineered to produce toluene monooxygenase enzymes and proteins specifically and under innocuous conditions and therefore provides a more efficient means of conducting bioconversions with this enzyme system.

The present invention encompasses a biologically functional plasmid derived from PmKRl containing toluene monooxygenase genes.

This plasmid (designated pAUTl) can be transferred by conjugation to a microorganism host cell lacking the toluene monooxygenase gene system and thus unable to convert toluene to p-cresol. In a particularly preferred embodiment of the present invention, the microorganism host cell for the pAUTl plasmid is Pseudomonas putida KTZAAO.

The present invention also encompasses the toluene monooxygenase genes which have been isolated as various DNA gene segments from PmKRl and cloned into a suitable, autonomously- replicating plasmid vector, resulting in a series of recombinant plasmids each of which contains a toluene monooxygenase gene segment. Each such recombinant plasmid is biologically functional and can be used to transform a microorganism host cell, conferring on the microorganism host cell the ability to convert toluene to p- cresol.

The present invention further encompasses a series of such transformed microorganism host cells. In a preferred embodiment of the present invention, the microorganism host cell is E. coli H8101, the recombinant plasmid is pMY402 and the inducer is isopropyl— thiogalactoside (IPTG). The pMY402 recombinant plasmid is the pMMB66EH plasmid into which a 4.7 kb _X_h_gI fragment encoding the PmKRl toluene monooxygenase genes has been inserted. In another preferred embodiment of the present invention, the microorganism host cell is E. coli FMS, the recombinant plasmid is pKMY287 and the inducer is heat (42°C). The pKMY287 recombinant plasmid is the pCFM1l46 plasmid into which a 4.7 kb @I fragment encoding the PmKRl toluene monooxygenase genes has been inserted. Under certain assay conditions, these resulting recombinant host cells express toluene monooxygenase enzyme activity at levels exceeding the activity PmKR1 of wildtype cells from which the toluene monooxygenase genes were isolated.

Other preferred embodiments of the present invention include the recombinant plasmids pKMY336 and pKMY3lLO in E. coli FMS cells and a particularly preferred embodiment is plasmid pKMY342 in PpY25OO cells (PpY251l). These cells synthesized the highest levels of TMO enzyme observed for recombinant microorganisms described here in.

Under alternative assay conditions, the levels of TMO activity detected in the recombinant host cells equal but do not exceed the activity detected in wildtype PmKRl cells. However, it is advantageous to use the recombinant host cells for certain microbial bioconversions. Advantages of using recombinant host cells with cloned TMO genes according to the present invention versus PmKRl cells in these bioconversions include: (i) the ability to use innocuous inducers (e.g., IPTG, heat, salicylate) instead of toluene for TMO enzyme induction, and (ii) the ability to prevent further conversion of product by enzymes of subsequent steps in the TMO pathway that are present in PmKR1 but not in the recombinant host cells. Thus, it is particularly advantageous to use the cloned TMO gene cluster to generate recombinant plasmids and recombinant host cells for certain bioconversions according to the present invention because the need for toluene, which is volatile and toxic, is eliminated as inducer of TMO enzyme activity. For large-scale bioconversions using fermentors or otherwise, the disadvantages of toluene vapors in the bioconversion process are clearly evident.

For safety reasons, some laboratories simply do not permit the use of toluene vapors in their fermentation processes. Additionally, it is particularly advantageous to use the isolated and cloned TMO gene because it can be manipulated and cluster in bioconversions introduced into certain host cells that will not further convert the -10. desired product obtained in the bioconversion using the TMO genes.

In contrast, since wildtype PmKRl cells contain the genes for the entire TMO pathway and not just the TMO genes for the first step of the pathway, further conversion of the product of the first step monooxygenase reaction by the PmKRl cells is likely.

The present invention is directed to the characterization and nucleotide sequence analysis of an isolated gene cluster of five TMO genes (tmoA, B, C, D, E), localized on the 4.7 kb xhgl fragment from PmKRl. The five-gene cluster, when expressed in E. ggli, gave significant TMO activity. Expression of this gene cluster carrying mutations in the individual genes demonstrated that each of the five genes is essential for TMO activity. The present invention is further directed to the characterization and nucleotide sequence analysis of a sixth TMO gene (tmoF), isolated on an ~l.3 kb fl;ndIII- final fragment downstream of the five essential TMO genes. The product of the tmoF gene is useful to enhance the activity of the gene cluster gave significantly higher TMO enzyme activity than expression of the five—gene cluster.

The present invention is also directed to an improved method for the degradative bioconversion of trichloroethylene using transformed microorganism host cells containing the PmKRl tmoABCDEF gene cluster. Further aspects and advantages of the present invention will become apparent to those skilled in the art upon consideration of the following detailed description. -12.

BEIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the steps of the PmKR1 toluene monooxygenase (TMO) pathway.

Figure 2 shows a map of the pKMY235 plasmid vector.

Figure 3 illustrates a summary of recombinant plasmids, plasmid vectors and restriction maps of the PmKRl DNA segments containing toluene monooxygenase genes.

Figure 4 illustrates a summary of recombinant plasmids and restriction maps of the PmKRl DNA segments containing toluene monooxygenase genes. Arrows indicate transcriptional direction and sizes of the TMO genes. Heavy lines underneath the map denote the different inserts in the plasmids indicated.

Figure 5 shows the nucleotide sequence of an.-h.7 kb PmKR1 DNA region carrying the tmoABCDEF genes. A region of dyad symmetry is underlined.

Figure 6 shows the sequence homology of the TmoC protein with the ferredoxin (NahAg) of naphthalene dioxygenase and the ferredoxin (BdoB) of benzene dioxygenase. Vertical lines indicate identical amino acid residues. Double dots indicate evolutionarily related amino acid residues based on the Gap program in UWGCG software (Devereux et al., Nucleic Acids Res. lg: 387-395 (1984)) with the similarity threshold set at 0.5. The amino acid sequences of the TmoC protein and the naphthalene dioxygenase ferredoxin protein are deduced from nucleotide sequences of the tmoC gene and the NahAh gene of the NAH7 plasmid (see Yen and Serdar, ggpga), respectively. The amino acid sequence of the benzene dioxygenase ferredoxin protein was determined by Morrice et al., FEBS Lett. gfilz 336-340 (1988) .13- from the protein B of E. putida ML2 (NCIB 12190). A methionine residue is inserted at the N—terminus of this protein to reflect the nucleotide sequence.

The methods and materials that provide an illustration of the practice of the invention and that comprise the presently preferred embodiments relate specifically to plasmid-borne DNA gene segments of PmKRl origin encoding the genes for the toluene monooxygenase enzyme system.

After cloning into a plasmid, these plasmid-borne DNA gene segments can be introduced and expressed in certain microorganism host cells, for example, by conjugation or transformation.

Microorganism host cells containing PmKR1 toluene monooxygenase genes are useful in certain bioconversions. For example, many phenyl compounds, including toluene, methylphenylacetic acid, ethylphenylacetic acid, acetanilide, 2- phenylethanol, fluorobenzene and ethylbenzene may serve as substrates and be converted to phenolic compounds by the TMO system as described herein. In addition, the broad substrate specificity of the TMO system makes it potentially useful in biodegradation of toxic compounds. Methods for the complete degradation of trichloroethylene (TCE) by the TMO system have been described in co- pending and co-assigned U.S. Patent Application Ser. No. 177,640, filed August 26, 1988, and hereby incorporated by reference in its entirety, and by Winter et al., Bio(Technology 1: 282-285 (1989).

Improved methods for the complete degradation of TCE utilizing an isolated tmoABCDEF gene complex are described herein.

The 4.7 kb Xhgl fragment identified from PmKRl that encodes TMO protein components has now been further characterized. This Xhgl fragment originally designated as 4.6 kb by restriction enzyme The potential exists, using recombinant DNA technology, to prepare variants, mutants or derivatives of one or more of the six TMO genes, which would encode a variant, mutant or derivative TMO protein. The complete gene sequence for each of the six TMO genes Various modifications might result in is disclosed in Figure 5. single or multiple amino acid deletions, substitutions, insertions or inversions, for example, by means of in vitro mutagenesis of the underlying DNA by methods well-known in the art. In addition, various fragments of one or more of the proteins encode by the TMO genes, whether produced in vivo or in vitro, may possess the requisite useful TMO activity. Experiments have shown, for example, that changes in the N-terminal sequences of TmoF do not substantially change its functional activity. All such variations, mutant or modifications or fragments resulting in a variant, derivative of one or more of the six TMO genes are included within the scope of this invention so long as they encode the functional segment(s) of TMO protein(s) and the characteristic functional TMO activity remains substantially the same, e.g. unaffected in kind.

Functional TMO activity results from a functional five—gene or six- gene TMO gene complex, as measured by the assays described herein, for example, in Example ll. From the disclosure of the TMO DNA sequences herein and the amino acid sequence of each of the six TMO genes, a TMO variant, mutant or derivative may be prepared and identified by those skilled in the art.

The invention is now illustrated by the following Examples, with reference to the accompanying drawings. Plasmids previously designated with the prefix "pKY" have been redesignated herein as "pKMY". The examples do not include detailed descriptions for conventional methods employed in the isolation of DNA, the cleavage of DNA with restriction enzymes, the construction of vectors, the insertion of DNA gene segments encoding polypeptides of interest into such vectors (e.g. plasmids) or the introduction. of the resulting recombinant plasmids into microorganism host cells. Such methods are well-known to those skilled. in the art of genetic engineering and are described in numerous publications including the following laboratory manuals: Maniatis et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory (1982); et al., Davis Basic Methods in Molecular Biology, Elsevier Science Publishing Co. (1986); Current Protocols in Molecular Biology, edited by Ausubel et al., Greene Publishing Associates and Wiley Interscience (1987); Sambrook et al., Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press (1989). In addition to using published methods, methods for enzymatic cleavage, modification, and ligation of DNA may be achieved according to manufacturer's instructions distributed by various commercial suppliers of restriction enzymes, including New (NEB), MA 01915 and Boehringer England Biolabs, Inc. Beverly, Mannheim Biochemicals, Indianapolis, IN 46250.

EXAMPLE 1 Growth of PmKR1 Cells Pseudomonas mendocina KR-1 was grown overnight at 30° in PAS medium or on a PAS agar plate (Chakrabarty, et al., Proc. Natl.

Acad. Sci. U.S.A., 1921137-1140 1973) with toluene (supplied as vapor) for growth and for induction of the toluene monooxygenase genes. (13-17 hours). After incubation, growth phase were collected by centrifugation. The cells were lysed and total PmKR1 DNA was then extracted and purified as described by Dhaese et al., Nucleic Acid Res. 1: 1837-1849 (1979).

B. Preparation of Plasmid DNA Plasmid DNA may be isolated according to the method of Johnston and Gunsalus, Biochem. Bionhvs. Res. Comm. 1;: 13-19 (1977). E. ggli HBl0l containing the pRK290 plasmid (Ditta, et al., Proc. Natl. Acad, Sci. U.S.A. 11: 7347-7351 (1980)) was inoculated into L broth and incubated with shaking at 37°C overnight. The bacterial cells were collected by centrifugation, lysed and the bulk of chromosomal DNA and cellular debris was removed by centrifugation. The pRK290 plasmid DNA was then purified by conventional techniques using cesium chloride/ethidium bromide density gradients.

. Preparation of Recombinant Plasmid Total PmKRl DNA obtained in Part A above and pRK29O plasmid DNA obtained in Part B above were separately treated with the restriction endonuclease figlll, under conditions of complete digestion. The figlll digested PmKR1 DNA was mixed with figlll digested pRK29O plasmid DNA and the mixture then incubated with DNA ligase.

D. Transformation with Recombinant Plasmid Transformation of E. 99;; with plasmid DNA may be achieved by the calcium chloride procedure originally described by Mandel and Higa, J. M01. Biol. fig: 159-162 (1970). The ligated DNA obtained in Part C above was used to transform E. ggli HB10l and the transformed cells were plated on selection plates of L-agar containing 10 pg/ml tetracycline. Only those cells which are successfully transformed and which contain the pRK290 plasmid or a recombinant pRK29O plasmid with PmKRl DNA can grow on the selection plates. Colonies which grew on the selection plates were tested for the presence of recombinant plasmids containing PmKRl toluene monooxygenase genes by the conjugation and complementation screening assay of Example 3.

Elililléi Conjugation and Complementation Screening Assay Colonies were removed from the selection plates of Example 2 or Example 8 by gentle scraping in L-broth with a slide. The resulting bacterial cell suspension was washed to remove any tetracycline and suspended in L-broth for the mating. Suspensions of donor cells, helper cells (if necessary) and recipient cells in logarithmic phase were mixed in equal volumes. Small aliquots of the mixture were placed on L-agar plates thus allowing all cell After overnight incubation at 30°C, the cells were types to grow. replated on a PAS agar selection plate containing 50 pg/ml tetracycline. Toluene was provided as sole carbon source for growth. Toluene vapor was supplied to the selection plate by taping a cotton-stoppered toluene containing tube to the lid of the plate.

This selection plate permits only the desired trans-conjugates to grow. In all experiments performed, the donor cells were from an E. to be transferred in the mating. The helper cells used were E. coli HBl0l cells carrying the helper plasmid pRK20l3 which provided the transferring functions for those transferring plasmids which do not carry the Egg genes. Alternatively, the helper plasmid pRK20l3 was introduced directly into the donor cells to provide its transferring function. The recipient strain was one of several mutant strains of Pseudomonas mendocina KR-1 (Pm Y400l, Pm Y4002, Pm YAOO7) prepared as described in Example 4. Each of the mutant strains has a defective toluene monooxygenase gene and is unable to convert toluene to p-cresol. When a recombinant plasmid containing the specific PmKR1 toluene monooxygenase gene which is defective in the recipient strain has been successfully transferred. during conjugation, the resulting transconjugate will be able to grow as a colony on the selection.plates containing toluene as the sole carbon source for growth.

The colonies which grew on the selection plates were purified by restreaking each colony once or twice on a selection plate.

These transconjugates are further manipulated according to Example EXAMPLE 4 Preparation of Pseudomonas mendocina KR—1 Mutant Strains PmKR1 cells were mutagenized and the toluene monooxygenase defective mutants were isolated according to the following protocol.

Cells were grown in 5 ml of L broth to 0.D.5m of approximately 0.7 and resuspended into 2 ml of 50 mM citrate buffer pH 6.0 containing N-methyl-N’-nitro-N-nitrosoguanidine (nitrosoguanidine) at a concentration of 0.1 mg per ml. After incubation at room temperature for 20 minutes, the cells were washed twice with 2 ml of l M phosphate buffer pH 7.0 and resuspended into 50 ml of L broth.

After growth overnight, the cells were streaked on L agar plates for single colonies. The individual colonies were picked and streaked onto PAS plates containing toluene or p-cresol (2.5 mM) as sole carbon source. The toluene monooxygenase defective mutants, PmYhOOl, PmY4002 and PmY4007 were isolated as strains which grew on p-cresol but not on toluene. The toluene monooxygenase assay as described in Example 11 further confirmed that these mutants have a defective toluene monooxygenase enzyme system.

Similar mutagenesis techniques may be used to obtain mutants in the p-cresol hydroxylase or p~ defective enzyme hydroxybenzaldehyde dehydrogenase of the TMO pathway. After nitrosoguanidine treatment of PmKRl cells as described above, p- cresol hydroxylase defective mutants can be isolated as strains which grow on p-hydroxybenzyl alcohol but not on p-cresol and p- hydroxybenzaldehyde dehydrogenase defective mutants can be isolated which grow on p-hydroxybenzoate but not p- as strains hydroxybenzylalcohol or p-hydroxybenzaldehyde.

Example 3 were further characterized as follows. Each colony was grown and plasmid DNA was isolated by conventional methods. The plasmid DNA from each isolate was used to transform E. coli HBl0l cells. The plasmid in each transformant was transferred to PmY400l by conjugation according to Example 3 except that the selection plates contained tetracycline and glucose (2 mg/ml). Each transconjugate was tested for growth on toluene by plating the cells on PAS agar supplemented with 50 pg/ml tetracycline and toluene vapor. After the toluene monooxygenase complementing activity of the plasmid was confirmed in the transconjugates each such HBlOl transformant was grown and plasmid DNA was isolated by conventional methods.

The DNA was digested with flglll and a 9.4 kb fragment was isolated from each transconjugate colony which complemented each PmKRl mutant strain of Example 4 for toluene utilization. This result indicated that the 9.4 kb gglll fragment from PmKRl contained one or more toluene monooxygenase genes. Two Sacl sites were mapped could be detected from the pRK290 plasmid (Example 2) carrying this .4 kb gglll insert in either E. coli HBl0l or E. putida KTZAAO insert into E. coli expression vectors pUCl8 and pUCl9 (Yanisch- Gene 33: not appear to contain the entire TMO gene cluster required for activity.

EXAMPLE 6 Construction of pKHY235 Plasmid Vector The starting material for the construction of the pKMY235 plasmid was the pKY2l7 plasmid described by Yen and Gunsalus, J‘ Bacteriol. lfigz 1008-13 (1985). The pKMY23S plasmid was constructed In the first step, two according to the following series of steps. adjoining flindlll fragments (~l.l and ~3.2 kb) from pKY2l7 containing the nahR and nahG genes was cloned into the fligdlll site (1983). The resulting plasmid from this first step was was designated pKMY223. In the next step, an ~6 kb gsgl fragment from pKMY223 containing the nah; gene, -200 base pairs of the gahg gene and the pKT23l gene conferring kanamycin resistance was cloned into the Eggl site of the pUC19 plasmid described by Yanisch—Perron 103-119 (1985). The resulting plasmid was et al., Gene 3;: designated pKMY256. The orientation of the -6 kb Esgl fragment in pKMY256 placed the multi-cloning site of pUC19 from the fiall to the EcoRI site immediately downstream to the Estl site in the nahG gene. inserted into the pRK29O plasmid described by Ditta et al., Proc.

Natl. Acad. Sci. U.S.A. 11; 7347-7351 (1980) to replace the ~l kb pKMY235 and a map of pKMY235 is shown in Figure 2.

EXAMPLE 7 Construction of pGFM1146 Plasmid Vector The plasmid pCFMl146 is an E. ggli cloning vector similar to pCFM4722 (Burnette et al., Bio[Technology Q: 699l988)). The starting material for the construction of the pCFMll46 plasmid was the pCFM836 plasmid. A detailed description of the construction of expression vectors, including pCFM836, is described in U.S. Patent No. 4,710,473, which is hereby incorporated by reference in its entirety.

The pCFM836 plasmid contains a heat inducible promoter, a restriction site bank (cloning cluster), plasmid origin of replication, a transcription terminator, genes regulating plasmid copy number, and a gene conferring kanamycin resistance but no synthetic ribosome binding site immediately preceding the cloning cluster. The pCFMll46 plasmid (A.T.C.C. accession no. 67672, deposit date April 5, 1988) was derived from pCFM836 by substituting the small DNA sequence between the unique glgl and gbgl restriction sites with the following oligonucleotide ’ CGATTTGATT 3' 3. TAAACTAAGATC 5, and by destroying the two endogenous Nggl restriction sites by cleavage with Ndgl and then end-filling with T4 polymerase enzyme, followed by blunt end ligation. is known to complement each PmKRl mutant strain for toluene utilization according to Example 5 and thus is likely to contain at least one of the PmKR1 toluene monooxygenase genes. The plasmid pUCl9 carrying the 9.4 kb fragment was radiolabeled and used as a probe to select hybridizing fractions from the sucrose gradient.

The hybridizing fractions were pooled to provide a population of DNA fragments enriched in PmKRl toluene monooxygenase genes. This enriched population of DNA fragments was used to construct the _S_agI library in _E;. coli HB10l. They were randomly cloned into the _S_a_gI site of plasmid pKMY235 as follows.

Transformed colonies were tested for PmKR1 toluene monooxygenase genes by the conjugation and complementation assay of Example 3.

EXAHPLQ 9 Isolation of 20.4 kb fig Fragment A number (10) of the transconjugates which utilized toluene as a sole carbon source were further characterized by isolating the plasmid DNA, transforming _E_J. coli I-115101, and conjugating into PmYle001 to test for growth on toluene according to Example 5. An E. coli 1-[B101 transformant containing a recombinant pKMY235 plasmid (designated pKMY266, A.T.C.C. accession no. 67671, deposit date fragments of 10.2 kb and 10.3 kb, respectively). One of the 10.2 kb Plasmid pKMY266 containing the ~20.4 kb (previously designated .5 kb) gagl insert described above, allowed the utilization of toluene by PmY4001. Despite the complementation pattern, no TMO enzyme activity (according to the alternative method of Example 11) could be detected in E. ggli HB101 carrying pKMY266. Since the insert in pKMY266 consisted of two different fiagl fragments of identical size, each was subcloned and expressed in the high-copy- ggli expression vector pUCl9 described by Yanisch-Perron number E. -119 (1985). This experiment led to the successful mapping of the TMO genes as described in Example 10 below.

EXAMPLE 10 Construction of Recombinant Plasmids to Map the Toluene Honooxygenase Genes This example presents results on the mapping of the TMO genes and determination of THO activities in various recombinant strains based on earlier constructs and TMO assay procedure (Example 11, Part A). Further mapping of the TMO genes and determination of THO activities in more recently constructed strains based on the alternative TMO assay procedure described in Example ll, Part B is presented in Example 16.

One of the figgl fragments described in Example 9, when cloned into pUCl9 (pKMY277) and expressed in 5. coli JM109, led to the synthesis of a blue pigment which was chloroform soluble and water insoluble. Production of the blue pigment was also observed from _E. putida cells harboring pKMY266 and was dependent on the presence of indole. This blue pigment was identified as indigo. A low level of TMO enzyme activity was detected from the E. coli JMl09 carrying pKMY277 (Table I). Further mapping of the toluene monooxygenase genes correlated the indigo-producing property with the presence of toluene monooxygenase activity. (See Table I in Example ll and also Table III in Example 16). series of restriction enzymes and a partial restriction map was generated as shown in Figure 3. Based on this restriction map, a series of DNA fragments were deleted from one end of the 10.2 kb S_a1_gI fragment in pKMY277 to generate plasmids pKMY280, pKMY28l, p1 plasmid pMY404. The pUCl8 plasmid is identical to pUCl9 except the polycloning site is in an opposite orientation with respect to the Q; promoter. As a result, the -4.7 zh_oI fragment was inserted into the pUCl8 plasmid in an opposite orientation to that in the pUCl9 of pKMY277 was also cloned into the broad host range plasmid vector pMMB66EH described by Furste et al., Gene 321: ll9-131 (1986) to construct the plasmid pMY402.

In addition, as shown in Figure 3, an -29. ~5.9 kb fiagl - zmal fragment of pKMY282 by digesting pKMY282 DNA with figgl and figlll, filling the ends with the large fragment of E. ggl; DNA polymerase I and ligating the ends. The resulting plasmid was designated pMY400.

As shown in Table I (according to the assay of_Example ll), pMY402 containing cells responded to IPTG for induction of the toluene monooxygenase genes. This result located the toluene monooxygenase genes in the ~4.7 kb Xhgl fragment and revealed the direction of transcription of the toluene monooxygenase genes as from left to right shown in Figure 3. The difference in the orientation of the ~4.7 kb Xhgl fragment in pMYA0l and pMY404 as well as the difference in toluene monooxygenase activity in pMYh0l and pMY404 containing cells (Table I) are also consistent with this transcriptional direction of the toluene monooxygenase genes. In order to express the toluene monooxygenase genes at a high level, the ~4.7 kb Xhgl fragment of pKMY282 was also cloned into the Xhgl E. ggli expression vector pCFMll46 (as described in site of the Example 7) to construct pKMY287.

ELAAZLEL1 Toluene Monooxygenase Assays A. Conditions for Assay Cells were grown in PAS medium containing 0.4% glutamate or in L broth to saturation. They were resuspended into an appropriate volume of the same medium to an O.D.5m of 3.0. An aliquot of the cells was used for the determination of protein concentration by the method of Bradford, Anal. Biochem. 1g: 248 (1976) using the Bio- Rad Protein Assay. An aliquot of 0.5 ml of cells was mixed with 4 .30. ;moles of p—cresol in lo pl and 15 nmole of radioactive toluene (toluene—ring-“C, Sigma Chemical Co., 56.3 mCi/mmole) in 5 pl and the mixture was incubated at room temperature with occasional vortexing for 20 minutes. After incubation, 20 pl of the mixture were spotted on a small piece of a thin~layer chromatography plate and the plate was air-dried for twenty minutes. The nonvolatile radioactivity remained on the filter was determined in a liquid scintillation counter and was used to calculate the amount of toluene degradation product on the plate and the specific activity of toluene monooxygenase. The results are presented in Table I.

B. Conditions for Alternative Assay Alternatively, toluene monooxygenase activity could be assayed by a procedure similar to the assay described for naphthalene dioxygenase by Ensley et al. , in Microbial Metabolism and the Carbon Qlcl_e (Hagedorn et al., eds.), Harvard Academic Publishers, New York (1988) at p. 6.37. Late log-phase cells were diluted into L-broth to a density of O.D.550-0.5 for the assay. The reactions were initiated by adding 15 nmole of “C-toluene (Sigma, 40-60 mCi/mmol) to 0.5 ml of cells in L-broth. After incubation at room temperature for 5 minutes, aliquots of 20 pl were spotted on small strips of thin- layer chromatography plate. The plates were air-dried for 20 minutes and counted in a scintillation counter to determine the remaining radioactivity. Specific activity of TMO was expressed as moles of nonvolatile material produced from “C-toluene per minute per milligram of whole cell protein. The protein concentration was determined by the method of Bradford, Anal. Bioghem. _7_2_: 248, using the Bio—Rad protein assay kit obtained from Bio-Rad Laboratories, CA 94801». For the protein determination, cells were Richmond, resuspended in 0.1 N NaOH and incubated in a boiling water bath for The results of this minutes, then assayed with the kit. alternative TMO assay are presented in Table III, Example 16.

TABLE I Expression of the Toluene Honooxygenase (THO) genes in E. coli and g. mendocina EXAMPLE L2 Conversion of Certain Phenyl Compounds to Certain Phenolic Compounds A. Conversion by PmKRl Cells Many phenyl compounds, including toluene, methylphenylacetic acid, ethylphenylacetic acid, 2-phenylethanol, acetanilide, fluorobenzane and ethylbenzene, may serve as substrates and thus be converted to phenolic compounds via para-hydroxylation by the toluene monooxygenase system of PmKRl. The following schemes illustrate several possible conversions: Scheme A CH3 CH3 wherein: I is toluene II is p-cresol Scheme B CHZCOOCHJ CHZCDOCH3 OH II I IV wherein: III is methylphenylacetic acid IV is p—hydroxymethy1phenylacetic acid Scheme C cH2cH,oH cH,cH,oH OH V V! wherein: V is 2-phenylethanol V1 is p-hydroxy—2—phenylethanol For each conversion, a phenyl compound substrate (for example, Formulas I, III, or V) was mixed with PmKRl cells, incubated for a period sufficient to effect the bioconversion and then assayed for the presence of phenolic compounds as follows.

Pseudggonas mendocina KR1 cells were grown at 25°C-30°C in 50 ml PAS medium supplemented with 0.4% glutamate to stationary phase (12-16 hours) in the presence (induced) or absence (uninduced) of toluene vapor supplied from 2.5 ml toluene. An aliquot of 5-50 ml cells were resuspended into the same volume of the same medium or concentrated 2.5 fold in the same medium. A given amount of the substrate equivalent to form a 15-30 mM solution was mixed with the cells and the mixture was incubated at 25°C-30°C with vigorous shaking for 1-24 hours. Typically the mixture was incubated for S- 6 hours. Formation of phenolic compounds was determined according to the assay method of Gupta et al., Clin. Biochem. l6 (4): 220- 221 (l983). The assay results for conversion of several phenyl substrates to phenolic compounds at various times and temperatures of incubation are shown in Table II.

TABLE II Synthesis of Phenolic Compounds by Toluene Honooxygenase of Pseudomonas mendocina KRI Substrate (Time and Temperature O.D.&m reading of Incubation) in Assay acetanilide (6 hrs., 25°C) 1.07 fluorobenzene (24 hrs., 25°C) 0.73 methylphenylacetate (6 hrs., 30°C) 0.23 ethylphenylacetate (6 hrs., 30°C) 0.13 ethylbenzene (6 hrs., 30°C) 0.37 2—phenylethanol (5 hrs., 30°C) 0.16 substrate in uninduced culture 0.03 B. Conversion by Microorganism Host Cells Containing Recombinant Plasmids encoding PmKRl Toluene Monooxygenase Genes The same conversions according to Part A may be accomplished by using microorganism host cells containing the recombinant plasmids of Examples 10, 16, and 19. Any of the recombinant plasmids (except pKMY283 or pMY400) which encode functional PmKRl toluene monooxygenase genes as described in Example 10 may be used to transform an appropriate microorganism host cell. A preferred method is to use pMY402 as the recombinant plasmid, E. coli H3101 as the microorganism host cell and IPTG as the inducer, as described in Example 11. The resulting strain was designated EcY5072 (HBl0l/pMY402). Another preferred method is to use pKMY287 or pKMY336 as the recombinant plasmid, E. ggli FMS as the microorganism host cell and heat (42°C for 1.5 or 3 hrs.) as the inducer} The resulting strains were designated EcY5082 (FMS/pKMY287) and EcY5236 (FMS/pKMY336), respectively. A particularly preferred method is to use pKMY342 as the recombinant plasmid, PpY2500 as the microorganism host cell and sodium salicylate (0.35 mM in L-broth throughout cell growth) as the inducer. The resulting strain was designated PpY25ll (PpY2500/pKMY342).

For each conversion, a phenyl compound (for example, Formulas EcY5082 mixed with EcY5072 (HBl0l/pMYh02), I, III or V) is (FMS/pKMY287), EcY5236 (FMS/pKMY336) or PpY25l1 (PpY2500/pKMY342) cells. The mixture is incubated for a period sufficient to effect the bioconversion and then assayed as described in Part A for the presence of phenolic compounds. For each bioconversion with EcY5072 (HB101/pMY402) cells, the cells are grown and assayed in the presence of IPTG to induce PmKRl toluene monooxygenase activity as follows. Cells are grown in PAS medium containing 0.4% glutamate and 1 mM IPTG or grown in L broth with 1 mM IPTG to saturation. The cells are resuspended in an appropriate volume of the same medium to an O.D.550 of 3.0 and. incubated with substrate and assayed as described in Part A. For each bioconversion with EcY5082 (FMS/pKMY287) or EcYS236 (FMS/pKMY336) cells, the cells are grown under the following temperature conditions to induce PmKRl toluene monooxygenase EcY5082 activity. (FMS/pKMY287) or EcYS236 (FM5/pKMY336) cells are grown in L broth to an 0.D.5w of 0.4. The cultures are incubated with shaking at h2°C for 3 hours and then shifted to 30°C to incubate for another 2 hours. Cells are resuspended in fresh L broth to an O.D.5m of 3.0 and incubated with substrate and assayed as described in Part A. For indigo production (Example 15) using these two strains, the cells are incubated at ° for 24 hours after induction at 42°C. For each bioconversion with the PpY25l1 strain, the cells are grown and induced under the following conditions. The cells are grown in L broth to saturation in the presence of 0.35 mM sodium salicylate to induce toluene monooxygenase production. Cells are resuspended in the same medium to an O.D.%o of 3.0 and incubated with substrate and assayed as described in Part A. .37- EYE L3 Conversion of Toluene to p-Hydroxyphenylacetic Acid A. Conversion by PmKR1 Cells For the conversion of toluene substrate to p- hydroxyphenylacetic acid, toluene is mixed with a PmKRl mutant defective dehydro genase as containing p - hydroxyb enz aldehyde described in Example 4 and incubated for a period sufficient to effect the conversion of toluene to p-hydroxybenzyl alcohol. In the second step, the cell mixture containing the p-hydroxybenzyl alcohol intermediate is reacted with nickel (Ni) and carbon monoxide (CO) in such concentrations and at such temperatures sufficient to convert the p-hydroxybenzyl alcohol to p-hydroxyphenylacetic acid, according to the methods of U.S. Patents 4,482,497; 4,659,518; 4,631,348, which are hereby incorporated by reference. The conversion scheme is illustrated as follows: CH3 CH5 toluene m°“°°XY9°“°3° p—-cresol hydroxylcse —-——-—-> ——————> OH toluene p—cresol CHZCOZH Ni/CO CHZOH OH p-Hydroxybenzyl alcohol p—Hydroxyphenylocetic acid B. Conversion by Microorganism Host Cells containing Recombinant Plasmids encoding PmKR1 Toluene Monooxygenase Genes The same conversion according to Part A may be accomplished by using microorganism host cells harboring recombinant p1asmid(s) carrying the p-cresol hydroxylase gene and the TMO genes. The p- cresol hydroxylase genes may be isolated by cloning of restriction fragments from PmKR1 or plasmid pND50 (Hewetson et al., Genet. Res.

Camb. gg: 249-255, 1978) which allow p-cresol hydroxylase defective mutants of PmKR1 (Example 4) to use p-cresol as a carbon and energy source. Alternatively, it may be isolated by using the sequence of TMO genes as a probe to clone overlapping restriction fragments that contain the gene. The possibility exists that the ~10.2 kb gggl fragment containing the TMO gene cluster (Example 10) contains the p-cresol hydroxylase genes. For use in the bioconversion described in Part A, the p-cresol hydroxylase genes may be cloned and expressed in plasmid pMY402, pKMY287, pKMY336 or pKMY342 (Examples , 16, and l9) each of which contains a functional TMO gene cluster. Alternatively, the p-cresol hydroxylase genes may be cloned and expressed in another plasmid.which can be introduced into strains which contain plasmid pMY402, pKMY287, pKMY336 or pKMY342.

For the conversion as illustrated in Part A, toluene is mixed with induced cells containing the p-cresol hydroxylase genes and the TMO genes. The mixture is incubated for a period sufficient to effect the conversion of toluene to p-hydroxybenzyl alcohol, and then is reacted with Ni and CO according to Part A to effect the conversion to p-hydroxyphenylacetic acid. .39.

EXAMPLE L4 conversion of Hethylphenylacetic Acid to p-Hydroxyphenylacetic Acid A. Conversion by PmKR1 Calls For the conversion of methylphenylacetic acid substrate to p- hydroxyphenylacetic acid, methylphenylacetic acid is mixed with PmKRl grown as described in Example 12 and incubated for a period sufficient to effect the conversion of methylphenylacetic acid to p- hydroxymethylphenylacetic acid. In the second step, the cell mixture containing the p-hydroxyphenylacetic acid intermediate is subjected to acid hydrolysis at acid concentrations and temperatures sufficient to convert the p-hydroxymethylphenylacetic acid to p- hydroxyphenylacetic acid. The conversion scheme is illustrated as follows: CH2C02CH3 CH,CO,CH3 toluene _ . monooxygencse acid hydrolysis —-——- > H phenylacetic acid P“l"Yd"°"YPhe">’l°°9tlC acld methyl ester methyl 9-Star cH2<:o2H OH p-hydroxyphenylccetic acid B. Conversion by Microorganism Host Cells Containing Recombinant Plasmids encoding PmKRl Toluene Monooxygenase Genes The same conversion according to Part A may be accomplished by using microorganism host cells containing the recombinant plasmids of Examples 10, 16, and 19 that carry a functional TMO gene cluster isolated from PmKRl. A preferred method is to use EcY5072 (HBlOl/pMY402) cells. Another preferred method is to use EcY5082 (FM5/pKMY287) or EcY5236 (FMS/pKMY336) cells. A particularly preferred method is to use PpY25ll (PpY2500/pKMY342) cells.

For the conversion as illustrated in Part A, methylphenylacetic acid is mixed with: EcY5072 (HBIO1/pMY402) cells grown and induced with IPTG, EcY5082 (FMS/pKMY287) or EcYS236 (FMS/pKMY336) cells grown and induced with heat, or, PpY25ll (PpY2500/pKMY342) cells grown and induced with sodium salicylate, as described in Example 12. The mixture is incubated for a period sufficient to effect the bioconversion of p-hydroxymethylacetic acid and then the mixture is subjected to acid. hydrolysis at acid concentrations and temperatures sufficient to yield p- hydroxyphenylacetic acid.

EXAMPLE 15 Conversion of Indole to Indigo A. Conversion by PmKRl Cells For the conversion of indole substrate to indigo, 50 pg/ml indole was mixed with PmKRl cells grown as described in Example 12 and incubated for a period sufficient to effect the conversion of indole to indigo, generally 48 hours. The indigo may be isolated from the cell mixture by the procedure described by Ensley in Example 5 of U.S. Patent No. 4,520,103.

B. Conversion by Microorganism Host cells Containing Recombinant Plasmids encoding PmKR1 Toluene Monooxygenase Genes The same conversion according to Part A may be accomplished by using microorganism host cells containing the recombinant plasmids of Examples 10 and l6 that carry a functional TMO gene cluster isolated from PmKRl. A preferred host strain is one that produces indole endogenously in the presence of an inexpensive carbon source, coli or a such as glucose. An example of such a host is E. particular strain of E. coli with an enhanced rate of indole synthesis. A preferred method is to use EcY5082 (FMS/pKMY287) or EcY5236 (FMS/pKMY336) cells.

EcY5082 illustrated in Part A, For the conversion as (FMS/pKMY287) or EcY5236 (FM5/pKMY336) cells were grown in L-broth and induced with heat as described in Example 12. The mixture is incubated for a period sufficient to effect the bioconversion of The indigo may be isolated from the cell mixture indole to indigo. according to the procedure of Part A.

EXAMPLE l6 Mapping and Nucleotide Sequence Analysis of tmoABCDEF Gene Cluster A. Mapping The region of the ~l0.2 kb figgl fragment (see Example 10) encoding TMO proteins was determined by deletion mapping. Deletion mapping was accompanied by DNA sequencing to reveal restriction sites. Various regions of the gggl fragment (Figures 3 and 4) were cloned individually into the Q. ggli expression vector pCFMlll¢6 (Example 7) which can express foreign genes from a heat-inducible phage PL promoter. Each of the recombinant plasmids was introduced into the E. coli strain FMS, which contains the integrated phage lambda repressor gene C1857 (Sussman and Jacob, Compt. Rend. Acad.

Sci, 153: 1517-1519 (1962)), as described by Burnette et al., (su a). The resulting strains were assayed for TMO activity by the alternative assay described in Example 11 under induced and uninduced conditions .

Several intermediate plasmids were involved in the construction of pMYA37 (Figure 4). Deletion of an ~O.9 kb _S_spI fragment downstream from the t:moABCDE genes in plasmid pMYl+0l (Example 10) produced plasmid pMY424. Insertion of a )_(_hgI linker into the S_spI site of pMY42h generated plasmid pMY/+36. Substitution of an @718-Q1 fragment of pMYl+2l containing the tmoDE genes with the corresponding $3718-Q1 fragment of pMY436 generated plasmid pMY437.

Construction of plasmid pMYl+l+8 (Figure 1») involved using the plasmids pMY476 and pKMY336. Insertion of the -0.8 kb 1i_ndIIl fragment within the tmoE gene (Figure 4) into the figal site of pUCl9 produced pMY476. Substitution of the Asp7l8-B_a_1_nHI fragment of pMY421 containing the tmoDE genes with the longer A__sp718-B_a;.HI fragment of pKMY282 (previously designated pKY282 in Example l0; Figures 3 and 4) containing the tmoDEF genes produced plasmid pKMY336. Substitution of the figbfll-II fragment of pKMY336 containing part of the tmoE gene with the ~0.76 kb Espl-@111 fragment of pMYls76 produced plasmid pMY448 (Figure 4). Deletion of an ~l.2 kb M31 fragment from the 5’ end of the tmoA gene in pKMY287 produced plasmid pMY429 (Figure 4).

Construction of plasmid pKMY3ls0 involved using the plasmids pKMY277 (previously designated pKY277 in Example 10) and pMY42l (described above). Deletion of an ~2.3 kb _I3an_11-II fragment from the region downstream from the tmoABCDEF gene cluster in pKMY277 generated a plasmid designated pKMY280 (previously designated pKY280 in Example 10). Replacement of the -2 kb gs_p7l8-fllil fragment of pl-{V1421 containing the tmoDE genes with the ~4.8 kb gsp_7l8-Lagfll fragment of pKMY280 containing the tmoDEF genes produced pKMY340.

Inducible TMO activity (as measured by the alternative assay described in Example 11) was observed from a strain carrying any of the recombinant plasmids pKMY287 (Example 10), pMY437, pl‘{Y42l, pKMY336 or pKMY3l+0, but not from the strain carrying pMY429 or pMY448 (Figure 4, Table III). This result further demonstrated transcriptional direction of the TMO genes and defined more precisely the minimal DNA region required for TMO activity. The TMO genes are transcribed from left to right based on the map shown in Figure 4. Plasmids pKMY287, pMYl+37 and pMYl+2l gave similar levels There is a perfect correlation between the presence of TMO activity and the indigo-producing capability among these strains tested. Indigo was produced only in strains having TMO activity but not in strains lacking TMO activity (Tables I and III). The indigo- plus strains all contain the intact tmoAECDE gene cluster and each of the indigo-minus strains misses an essential TMO component gene.

Indigo production can therefore serve as a good indicator for the presence of the TMO gene cluster when these genes are investigated.

TMO Activities and Indigo-Forming Properties of Recombinant E. coli Plasmids Carrying Different PmKRl DNA Fragments.

Specific Activity of mob Indigo Plasmid“ (nmole min” mg”) Formation° pCFMl146, Induced 0.1 - pMY429, Uninduced 0.1‘ - pMY429, Induced 0.1 — pMY448, Uninduced 0.1 - pMY448, Induced 0.1 - pKMY287, Uninduced 2.0 + PKMY287, Induced 7.0 + pMY437, Uninduced 0.5 + pMY437, Induced 7.3 + pMY42l, Uninduced 0.9 + pMY42l, Induced 10.0 + pKMY336, Uninduced 0.6 + pKMY336, Induced l9.0 + pKMY340, Uninduced 0.7 + pKMY340, Induced 20.0 + “Each plasmid listed except pCFMll46 is the E. coli expression'vector pCFMl1h6 carrying TMO genes. The different inserts in these plasmids are defined in Figure 4.

“THO specific activities in toluene—induced and uninduced PmKR1 cells are 30 and 0.5 nmole of non-volatile material formation from toluene per minute per mg of protein, respectively. °+, indigo formation; -, absence of indigo formation entirety in both orientations. The nucleotide sequence of this Xhol fragment carrying the TMO genes was determined by the dideoxy method of Sanger et al., Proc, Natl, gcggl, Sci. _73: 5463-67 (l977) on double-stranded DNA using the Sequenase" DNA sequencing kit obtained from United States Biochemical Corporation, Cleveland, Ohio 44122.

DNA samples were denatured in 0.2 M NaOH for 10 minutes and neutralized with 0.2 M ammonium acetate (pH 4.5) before use in the sequencing reactions. The ~4.7 kb @I fragment and various deletion derivatives were cloned into pUCl9 or pUCl8 (Yanisch- Perron et al., (s_\1p_1.;a)) for DNA sequencing. Both commercially available and synthetic primers were used for sequencing reactions.

The nucleotide sequence corresponding to the H_h'_1dIII-_S_s;p_I region required to give TMO activity is presented in Figure 5. Five open reading frames were identified in this region.

Each of the five open reading frames was confirmed by determining the N-terminal amino acid sequence of the corresponding gene product produced in E. coli from plasmid pMYb,2l (see Table VI in Example 18 below) and by cloning each of the regions containing an open reading frame and demonstrating corresponding activity (see Example 17 below). The genes defined by these five open reading frames were designated t:moA, tmoB, tmoC, tmoD and t:moE in the order of transcription (Figure 4) . Part of a sixth open reading frame was also detected near the 3’ end of the _X_h_gI fragment. This led to further sequence analysis of the region downstream of the tmoABCDE gene cluster. The sequence of the ent :e sixth open reading frame is also shown in Figure 5. This sixth open reading frame was designated tmoF, continuing in the order of transcription. This open reading frame was confirmed by determining the N-terminal amino acid sequence of the corresponding gene product in E‘ 99;; from plasmid pMYhA0 (see Table VI in Example 18 below). Plasmid pMYA4O encodes the sixth open reading frame and expresses a functional TmoF protein in the E. 99;; FMS host cells. It was constructed by deleting the ~3.4 kb flindlll fragment from the 5' end of the tmoABCDEF gene cluster in pKMY336.

The TMO genes are organized as a very closely—spaced cluster.

In addition to the Shine-Delgarno (S-D) sequence (Nature ;;g; 34- The base composition of the tmoABCDEF cluster is unusual for E. mendocina genes. The G+C content of the DNA fragment presented in Figure 5 is 48.8%. This low value is significantly different from the reported G+C content of 62.8-64.3% for the E. mendocina -[;_8- genome (Palleroni, et al., J. Gen. Microbiol 59: 215-231 (1970)). toluene plasmid pAUTl (Table I of Example 11) in PmKRl. -49.

TABLE IV Codon Usage of the tmoABCDEF Genes.

Amino Acid Codon A B C D E F Total Gly GGG A O 1 0 1 9 15 Gly GGA 5 0 3 3 1 5 17 Gly GGU 14 1 3 2 5 6 31 Gly sec 10 1 A 1 1 3 20 Glu GAG 15 5 5 2 lb 12 53 G1u GAA 24 3 7 10 12 17 73 Asp GAU 19 5 S 5 13 8 55 Asp GAC 15 1 3 2 6 4 31 Val GUG 4 4 1 2 3 10 24 Val GUA 5 3 4 1 3 6 22 Val GUU 9 6 0 2 1 3 21 Val GUC 4 1 3 0 5 5 18 Ala GCG 7 2 1 1 3 9 23 Ala GCA 18 2 1 3 5 7 36 Ala GCU 7 2 1 5 10 6 31 Ala GCC 12 1 2 1 5 5 26 Arg ACG 1 0 O 1 2 1 5 Arg AGA 1 1 0 0 1 1 4 Ser AGU 4 0 2 0 10 3 19 Ser AGC 8 1 3 1 7 1 21 Lys AAG 14 1 2 0 3 7 32 Lys AAA 11 2 3 3 7 11 37 Asn AAU 8 2 2 3 7 9 31 Asn AAC 9 1 2 3 6 7 28 Met AUG 21 4 3 3 12 8 51 Ile AUA 3 1 0 1 1 1 7 Ile AUU 7 0 4 3 7 11 32 Ile AUG 14 1 4 6 2 4 31 Thr ACG 4 0 1 0 3 3 11 Thr ACA 4 O 3 3 3 2 15 Thr ACU 3 2 1 1 4 S 16 Thr ACC 11 1 2 2 5 3 24 Trp UGG 22 O 2 0 13 2 39 End UGA 1 0 O 0 1 0 2 Cys UGU 1 1 3 0 1 2 8 Cys UGC 3 O 2 O 3 5 30 TABLE IV Continued Amino Acid Codon A B C D E F Total End UAG O 1 0 0 0 0 1 End UAA 0 0 1 1 0 1 3 Tyr UAU 14 0 1 1 6 6 28 Ty: UAG 8 1 3 1 8 4 25 Leu UUG 10 1 0 4 10 1+ 29 Leu UUA 2 1 3 1 1 A 12 Phe UUU 12 3 1 2 1+ 11 3 3 Phe UUC 12 2 2 2 4 6 28 Ser UCG 4 1 O 0 4 6 15 Ser UCA 3 1 O 0 4 6 14 Ser UCU 1+ 0 1 0 2 2 9 Ser UCC 4 1 2 1 3 5 16 Arg CGG 2 2 O 1 1 1 7 Arg CGA 0 1 0 1 3 2 7 Arg CGU 15 3 0 1 6 7 32 Arg CGC 7 O 1 3 6 1 18 C111 CAG 14 1 2 5 11 5 38 Gln CAA 7 1 O 1 7 4 20 His CAU 9 2 3 1 3 4 22 His CAC 6 1 2 0 10 1 20 Leu CUG 9 1 1 2 15 11 39 Leu CUA 3 1 1 1 3 6 15 Leu CUU 7 1 1 0 8 5 22 Leu CUC 4 O 1 1 1 6 13 Pro CCC 9 O 1 3 5 7 25 Pro CCA 5 3 1 1 5 A 19 Pro CCU 5 1 1 0 2 5 114» Pro CCC 1+ 0 1 0 5 2 12 Identification cf tmoABs-E as Essential THO Genes A. Plasmid pKMY341 and Sing1e—gene Mutant Derivatives To determine if each of the designated tmoABCDE genes as described in Example 16 encodes a TMO protein component necessary for TMO activity, a single mutation was introduced into each of the genes and its effect on TMO activity was determined. A plasmid, pKMY34l, was initially constructed by cloning the tmoABCDE genes into the E. 991; plasmid T7-5, followed by introducing a DNA sequence change into the individual genes. The pT7-5 plasmid is a QQLEI-based plasmid containing the fl-lactamase gene and a multiple cloning site downstream from a T7 RNA polymerase-specific promoter obtained from S. Tabor (see, e.g., Tabor and Richardson, Proc. Natl. tmoABCDE genes was removing the overhangs and ligation generated the mutation in plasmid pMY472.

Enzyme assays revealed that each of the mutations completely eliminated TMO activity in _E_. c_o_l_i cells as shown in Table V.

TABLE V Complementation Between Individually Cloned tmo Genes and the tmoABGDE Gene Cluster Carrying Corresponding Mutations.

Bacterial Specific Activity of Strain (EcY#)“ Plasmid? tmo Genes TMO (nmole min’1 mg'1) 5246 pKMY34l ABCDE 10.7 5283 pMY459 A'BCDE 0.06 5282 pMY458 AB'CDE 0.04 5286 pMY482 ABC'DE 0. 08 5287 pMY484 ABCD'E 0.05 5285 pMY472 ABCDE' 0.10 5258 pMYA38 A 0 .09 5265 pMYla-47 B 0.03 5288 pMY474 C 0 .04 5289 pMY479 D 0.02 5224 pKMY327 E 0.04 5291 pMY438, A, ACBCDE 2.0 pMY4S9 pMYl+h7, B, AB'CDE 3. 9 pMYh58 pMY47h, C, ABC'DE 7.4 pMY4B2 pMY479, D, ABCD'E 2.0 pMY484 S292 pKMY327, E, ABCDE’ 10.3 pMY472 ‘Each of the strains was constructed by introducing an appropriate plasmid or plasmids into the E. coli strain H5101. All cultures were grown in the presence of 0.35 mM sodium salicylate which induced the TMO genes cloned into pKMY3l9. bConstruction of all plasmids is described in Examples 16 and l7. .53.

B. Complementation Tests Between Mutant Derivatives of Plasmid pKHY34l and Plasmids Carrying a Single THO Gene To determine whether lack of TMO activity was due to lack of expression of the TMO genes in the presence of a polar mutation which itself is not located in a THO gene, complementation tests pMYh82, pMY484 and pMY472 pMY459, pMY458, between plasmids (constructed as described in part A above) and plasmids carrying only one of the tmoABCDE genes were conducted. Plasmids pMY438, pMY447, pMYh7h, pMY479, and pKMY327 were constructed to contain the tmoA, B, C, D, and E genes, respectively, in the broad host range expression vector pKMY319. Plasmid pKMY3l9 is described and claimed in co-pending and co-assigned U.S. Patent Application Serial No. 07/590,280, filed on September 28, 1990, hereby incorporated by reference in its entirety. Plasmid pKMY3l9 is a plasmid vector in which expression of foreign genes can be regulated by the NahR protein and an inducer, such as sodium salicylate. pMY479, and pMY327 are Plasmids pMY438, pMY4A7, pMYh7h, derivatives of pKMY3l9 that carry tmoA, tmoB, tmoC, tmoD, and tmoE, respectively, and were constructed as follows. For the construction of plasmid pMY438, pMY430 was initially constructed by cloning an ~l.6 kb Qrgl fragment containing the tmoA gene (Figure 4) into the fimgl site of pUCl9 in an orientation that placed the Xbgl site of pUCl9 at the 5’ end of the tmoA gene. Plasmid pMY438 was constructed by cloning the ~l.6 kb Xggl-fiagl fragment of pMY430 carrying the tmoA gene into the Xbgl and gggl sites of pKMY3l9.

Plasmids pKMY332 and pMY446 were used in the construction of Construction of plasmid pMY47h involved constructing of pUC19 produced intermediate plasmid pMY4l4. Deletion of an -0.1 generated intermediate plasmid pMY426. Ligation of a gagl linker containing the tmoAB genes and the 5’ end of the tmoC gene (Figure plasmid pMY466. Deletion of a l.8 kb Qlgl fragment upstream from the tmoC gene in pMY466 generated plasmid pMY474.

Intermediate plasmids pMYh04, pMY470, and pMY478 were generated and used in the construction of plasmid pMY479. Cloning ) generated intermediate plasmid. pMY470. An ~0.4- kb fligdlll Plasmid pKMY327 was constructed from the intermediate plasmid pKMY324. Cloning of the Xhgl fragment of pKMY282 carrying the tmoABCDE genes into pKMY3l9 produced intermediate plasmid pKMY324.

Deletion of an ~3.l kb glgl fragment of pKMY324 upstream from the tmoE gene (Figure 4) generated plasmid pKMY327.

Each of these plasmids was introduced by transformation into a strain which contained a corresponding member of the plasmids pMY459, pMY458, pMY482, pMY48h, and pMY472 and the TMO activity in each of the resulting strains was determined after induction as shown in Table V above. A plasmid carrying a mutation in any member of the tmoABCDE genes was complemented by the plasmid which carries that particular gene in synthesizing the TMO enzyme (Table V). This result demonstrated that the mutation in each of the tmo genes, did not abolish the expression of the downstream tmo genes. Since each of the mutations prevented the synthesis of a functional TMO system (Table V), this indicated that each of the tmoABCDE genes plays an essential role in directing the synthesis of the THO enzyme system.

EXAHELE 18 Purification and Analysis of THO Proteins TMO proteins except TmoF were partially purified from PmKRl or E. 99;; FMS cells carrying the plasmid pMY42l by DEAE cellulose chromatography according to Whited, (supra), except that TEGD buffer (50 mM Tris, pH 7.45; 10% glycerol; 10% ethanol; 1 mM dithiothreitol) instead of PEG buffer was used in the column. The TmoF protein was purified from E. coli FMS cells carrying the plasmid pMYh40, by isolating the protein after polyacrylamide gel electrophoresis of the FMS/pMYhAO lysate.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed essentially according to Laemmli, Nature _zZ: 680-685 (1970).

Protein samples were heated at 65°C for 15 minutes in.a loading buffer containing 2% SDS, 5% 2-mercaptoethanol, % glycerol, 0.02% bromophenol blue, and 62.5 mM Tris—Cl (pH 6.8) before they were loaded on the gel.

For determination of N-terminal amino acid sequences, partially purified TMO proteins were further purified by SDS~PAGE and electroblotted onto polyvinyldifluoride membrane according to Matsudaira, J. Biol. Chem. gggz 10035-38 (1987) with slight modifications. The bands protein immobilized on the polyvinyldifluoride membrane were visualized by Coomassie blue staining and destained with 50% methanol and 7% acetic acid. The stained bands were excised with a razor blade and sequenced in an Applied Biosystems Model 477 automated protein sequencer as described by Lu et a1., Int. J. Peptide Protein Res. ;;: 237-49 (1989). -57.

Conditions to generate tryptic fragments were described by Klein et al., Arch. Biochem, fiiophys, gzgz 531-537 (1990). The tryptic peptides were isolated by reverse~phase HPLC in an acetonitrile-trifluoroacetic acid gradient elution system. The isolated peptides were pooled and loaded onto a glass-fiber disc precycled with polybrene for automatic sequence analysis. in situ cyanogen bromide cleavage of protein samples and sequence analysis of the cleaved mixture were also described by Klein et al., (supra).

The nucleotide but not for the tmoC gene product. products, sequence of the tmoC gene has capacity to encode a protein with a molecular weight of approximately 12,000 as shown in Table VI.

However, on SDS-PAGE the observed molecular weight of the TmoC protein was approximately 25,000 (Table VI), almost exactly twice A 25 kilodalton (kDa) the size of the expected molecular weight. protein was also isolated from PmKRl and identified to be the same kDa t:moC product isolated from the recombinant E. coLi by comparing the N-terminal amino acid sequences. Further characterization of the 25 kDa protein demonstrated that it is encoded solely by the mac‘ gene. This protein was isolated from E. colj and further purified from SDS polyacrylamide gels. Fragments of the protein were obtained in separate experiments after trypsin digestion or cyanogen bromide treatment. N-terminal amino acid sequence analysis demonstrated that all of the fragments were cleavage products of the TmoC protein. This result suggested that the 25 kDa protein was a dimer of the t:moC product not fully reduced under the conditions used for the SDS-PAGE.

TABLE VI N-terminal Sequence and Molecular Weights of the t:moABCDEF Gene Products Produced from Recombinant E. gci, FMS Host Cells Carrying Plasmid pMY4-21‘ or pMY440b P oduct Molecu We ' Predicted Estimated Region of From From N - terminal Sequence Nucleotide Nucleotide Purified Determined From Gene Sequence Sequence Protein Purified Protein“ tmoA° 37-1536 57,982 55,000 AMEIPRIOIDWYELTR tmoB° 1558-1809 9,588 9,500 SAFPVHAAFEXDFLVQLVV VDLNDSMDQVA t:moC“ 1818-2153 1.2, 326 25,000 SFH{IXSlDDDNGEME.'I'FEIS tmoD‘ 2217-2525 11,618 11,500 STLADQALHNNNVGPIIR IIRAGD t:moE‘ 2539 - 3519 38,386 35,000 SI-‘ESKXPMRTWSXL tmoFb 3548-1675 35,983 38,000 MFNIQSDDLLHHFE °The letter X indicates undetermined amino acid. .59- The definitive functions of each of the TMO genes is not known. However, there is evidence at least to suggest a role for the tmoC gene. Comparison of the amino acid sequences deduced from the TMO genes with those of known proteins reveal homology between the tmoC product and several other ferredoxin proteins functioning in dioxygenase systems. Among the 114 amino acid residues in the TmoC protein, 36 residues (31.6%) are identical to those of the benzene dioxygenase ferredoxin protein at corresponding positions and 14 residues (12.3%) are represented by evolutionarily related amino acids in the benzene dioxygenase ferredoxin at corresponding positions (Figure 6). Similar homology exists between the TmoC protein and the naphthalene dioxygenase ferredoxin protein (Figure 6). The ferredoxin component of the toluene dioxygenase system from PpFl differs from the benzene ferredoxin protein by only six amino acid residues (Zylstra and Gibson, J. Biol, Chem. gggz 14940-46 (1989)). It therefore shares similar homology with the TmoC protein, The region of maximum homology between the TmoC protein and the other ferredoxins is located between positions 53 and 77 (Figure 6). Among the 23 amino acid residues in this region, the Tmoc protein shares 10 (43%) with the benzene dioxygenase ferredoxin and 9 (39%) with the naphthalene dioxygenase ferredoxin (Figure 6).

In addition, the two dioxygenase ferredoxins share 13 (56.5%) amino acids in this region (Figure 6). It is interesting to note that this region contains two conserved cysteine residues (at positions 53 and 74, respectively) each of which is followed by a conserved histidine in the vicinity. Benzene dioxygenase ferredoxin (Geary et al., (supra)) and toluene dioxygenase ferredoxin (Subramanian at [2Fe-2S] clusters coordinated to four cysteine residues. It has been suggested by Cline et al., J, Biol, Chem. QQQ: 3251-54 (1985), that in addition to cysteines, histidine residues may provide nitrogen ligands to the [2Fe—2S] cluster which may contribute to the higher redox potential of the cluster. It is likely that this region of maximum homology between the two dioxygenase ferredoxins is involved in the binding of the [2Fe~2S] cluster. The fact that the TmoC protein shares overall homology with these two ferredoxins and does so especially in this region suggests that it is a ferredoxin of the toluene monooxygenase system.

EXAMPLE 19 Degradative Bioconversion of TCE by Microorganism Host Cells Containing Recombinant Plasmids Carrying PmKRI Toluene Monooxygenase Genes TCE degradation catalyzed by the toluene monooxygenase of PmKRl has been demonstrated by Winter et al., Bio/Technology it 282- (l989) and claimed in co—assigned U.S. Patent Application Serial No. 177,640 incorporated by reference. Plasmid pKMY342 is plasmid pKMY3l9 carrying two copies of the tmoABCDEF gene cluster and is described in co-pending and co—assigned U.S. Patent Application Serial No. 07/590,280, filed on September 28, 1990. It gives higher TMO activity that the recombinant plasmids previously described for TCE degradation and can replicate in all Gram-negative bacteria tested. to date. It is therefore a particularly preferred and improved plasmid for use in TCE degradation. Other recombinant .51. plasmids such as pKMY336 and pKMY340 (Example 16) that give higher TMO activity than the plasmids described in U.S.S.N. 177,640 are also useful for TCE degradation.

In a particularly preferred embodiment, Pseudomonas pggida Y25ll cells harboring recombinant plasmid pKMY342 are grown to mid- log phase in L-broth with 0.35 mM sodium salicylate as inducer.

The cells are washed in L-broth for the degradative bioconversion of TCE, as follows. Cells are resuspended to an 0D5w of ~0.5 in L- broth, and 4 ml of the cell suspension is added to serum vials. TCE (Aldrich, (Milwaukee, Wisconsin), spectrophotometric grade) is diluted in N,N-dimethylformamide (DMF) (Aldrich, spectrophotometric grade) to 10 mM or 20 mM and 4 pl added to cell suspension to give a final TCE concentration of 10 pM (1.3 ppm) or 20 pM (2.6 ppm).

Vials are stoppered, vortexed, and 10 pl of gas phase are withdrawn using a gas-tight syringe at various times for analysis. Gas phase samples are analyzed on a Hewlett-Packard 5890A gas chromatograph equipped with a 25 meter 5% phenyl methyl silicone column (Hewlett Packard, Palo Alto, California) and a “Ni electron capture detector.

The injector, oven, and detector temperatures are l20°, 100°, and 300°, respectively. The carrier gas is helium and the makeup gas is 95% argon-5% methane. Peak areas are calculated by’ a Hewlett- ?ackard 3392A Integrator. Data are reported as the percentage of TCE remaining at various times after addition to the cell suspension. The amount of TCE present at zero time is taken to be %.

Claims (1)

1. An isolated nucleotide sequence encoding the tmoABCDE gene product, said product having the sequence of