The present invention relates generally to the microbial production of dyestuffs and more particularly to microbial production of indigo by organisms in indole-free media.

Indigo, or indigotin, occurs as a glucoside in many plants of Asia, the East Indies, Africa, and

South America, and has been used throughout history as a blue dye. Principally obtained from plants of the genera Indiqofera and Isatus, indigo was used to dye blue the earliest known textiles, linen mummy wrappings dating from 2000 BC. By the middle of the 19th century, indigo had become a principal item in trade between Europe and the Orient. Prior to elucida- tion of the structure and the synthesis of the indigo molecule, the use of natural indigo involved protracted fermentation processes to liberate the dye for introduc¬ tion into fabric in a soluble, colorless form, indican. By steeping the fabric and indican in a vat, the soluble indican was easily hydrolyzed to glucose and indoxyl. Mild oxidation, such as exposure to air, would convert the indoxyl to indigo, regenerating the pigment in the fibers of the fabric.

During the 19th century considerable effort was directed towards determining the structure of this valuable compound. The chemical structure of indigo, corresponding to the formula ^c ιg ^H in ^N ₂ °2' ^waS announced in 1883 by Adolf von Baeyer after eighteen years of study of the dye. However, a commercially feasible manufacturing process was not developed until approximately 1887. The method, still in use throughout the world, consists of a synthesis of indoxyl by fusion of sodium phenylglyσinate in a mixture of caustic soda and sodamide. All industrially successful processes also involve the final step of air oxidation of indoxyl to indigo. To date, indigo has been principally used for dying cotton or wool shades of navy blue. The

compound also has potential use in processes for solar energy collection. [See British patent 1,554,192].

Pertinent to the background of the invention are prior observations of microbial production of a blue pigment. Using selective methods of cultivation, one experimenter in 1927 isolated a soil organism (Pseudomonas indoloxidans) that could decompose indole with the formation of blue crystals. The blue particles that appeared in cultures containing that bacterium were insoluble in water, alcohol, ether, xylol, and benzol, but did dissolve in strong sulfuric acid to give a blue solution which dyed silk blue. The experi¬ menter concluded that indoxyl was probably not formed within the cells of this organism, but rather that the blue crystal formation was due to the production of an exoenzyme diffusing out from the bacterial growth. This organism could not use indole as a' source of energy and could not oxidize indole to indigotin without an additional source of carbon, but could oxidize indole if given a supply of carbon. A high carbon to nitrogen ratio appeared to be most suitable to the growth of Pseudomonas indoloxidans and the produc¬ tion of indigotin. Further observations made by the experimenter were that indole appeared to depress the growth of the organism and that the organisms multiplied rapidly as soon as the indole had been consumed. The oxidation of indole was observed to take place only during the early stages of growth of the organism. No trace of indoxyl was found in cultures, and the indigotin was not apparently further oxidized to isatin. The experimenter also noted that two other soil organisms, Mycobacteriu globerulum and Micrococcus piltonensis, could also produce small amounts of indigotin on indole agar only. [See: Grey, P.H., "Formation of Indigotin from Indole by Soil Bacteria," Roy.Soc.Proc.. B, 102; 263-280 (1927)].

A single mutant culture of Schizophyllum commune fungus producing a "blue pigment" has also been described. The culture was grown on a chemically defined, synthetic medium containing glucose, (NH ₄ ) ₂ HP0 ₄ , thiamine, KH ₂ P0 ₄ , K ₂ HP0 ₄ , and MgS0 ₄ "7H ₂ 0. The ammonium ion was the nitrogen source. Both a red and a blue pigment were harvested from mycelial macerates. The identification of the blue pigment extracted from the macerates with chloroform was obtained by solubility tests, absorption spectroscopy, and chemical analyses. The results of these tests were all consistent with the conclusion that the blue pigment was indigo. [See: Miles, P., et al., "The Identification of Indigo as a Pigment Produced by a Mutant Culture of Schizophyllum commune ," Archives of Biochemistry and Biophysics, 62.: 1-5 (1956)].

In 1962, a study was performed on the bio¬ genesis of the pigment violacein by the organism Chromo- bacterium violaceum, which readily converted L. trypto- phan to violacein, but did not utilize this amino acid for growth. The experimenters created a novel microbiological assay, specific for L. tryptophan, in which the quantity of violacein produced was a function of the amount of . tryptophan present in the test sample. It was observed that when L. tryptophan was incubated with lyophilized cells, indole was tran¬ siently formed and, after a forty-eight-hour incubation, a deep blue pigment was synthesized. The pigmented material was identified as indigo on the basis of its color, absorption spectra, and RF values in thin layer chromatog aphy. The experimenters concluded that indoxyl was an intermediate of the indigo pathway in this bacterium, and found that Chromobacterium violaceum metabolized L. tryptophan to indole by the action of tryptophanase or tryptophan synthetase. This microorganism synthesized violacein not only from . tryptophan but also from indole. When the

enzymes of the violacein pathway were inactivated by rapid lyophilization, both L. tryptophan and indole were metabolized to indigo. [See: Sebek, 0. and Jaeger, H. , "Divergent Pathways of Indol Metabolism in Chromo- bacterium Violaceum," Nature. 196: 793-795 (1962)].

In a more recent report, experimenters isolated an organism from soil by the enrichment culture tech¬ niques using indole as the sole source of carbon and nitrogen. An aerobic gram positive coccus, which rapidly decomposed indole when grown in a medium contain¬ ing indole, KH ₂ P0 ₄ , K ₂ HP0 ₄ , NaCl, MgS0 ₄ , water, and yeast extract, produced a blue pigment which was not released into the culture medium. It was noted that the indole in the medium was used up very rapidly and more indole was added several times during the culture period. The cells, when harvested, were very blue and decomposed indole with the consumption of eleven to thirteen atoms of oxygen per mole of the substrate. When anthranilic acid, glucose or glycerol was substituted for indole in culturing the organism, the cells showed no ability to decompose indole, indicat¬ ing that the activity was inducible. When grown on indole, the microorganism decomposed indole to hydroxy- indole, anthranilic acid, and catachol. A cell-free extract of this organism contained an enzyme, dihydroxy- indole oxygenase, which catalyzed the conversion of dihydroxyindole to anthranilate plus CO-. The dihydroxy- indole oxygenase was determined to be an inducible enzyme which appeared only when the organism was grown on indole. The pathway proposed for degradation of indole by these experimenters was: indole to indoxyl to dihydroxyindole to anthranilic acid to catachol. [Fujioka, M. and Wada, H. , "The Bacterial Oxidation of Indole," Biochemica et Biophysica Acta, 158: 70-78 (1968)].

To date, none of the above organisms has been put to use in the large-scale microbial synthesis of indigo. This is likely to be due, in large part, to unfavorable economic factors involved in providing indole as a substrate or otherwise maintaining precise nutrient balances in the growth medium.

Enteric bacteria (e.g., E.coli) indigenous to the intestinal tracts of animals are capable of accumulating indole [see, e.g.. Post, et al., P.N.A.S. USA, 7j6: 1697-1701 (1979)] by the activity of the enzyme tryptophanase produced by the tryptophanase structural gene. Tryptophanase, believed to be a catabolic enzyme, catalyzes the degradation of tryptophan, resulting in the stoichiometric production of indole, pyruvate, and ammonia. An associated enzyme, tryptophan synthe- tase, can also catalyze the synthesis of tryptophan, from indole glycerol phosphate, and serine. In E.coli, synthesis of tryptophanase is inducible by tryptophan. The tryptophanase structural gene tnaA of E.coli K12 has been cloned and sequenced. See, Deeley, M., et al., "Nucleotide Sequence of the Structural Gene for Trypto¬ phanase of E.coli K12," J.Bacteriology, 147: 787-796 (1981); and Deeley, M., et al., "Transcription Initia¬ tion at the Tryptophanase Promoter of E.coli K12," J.Bacteriology. 151: 942-951 (1982) . While enteric bacteria are capable of growing on simple media, they do not possess the enzymatic wherewithal to convert indole to indigo.

Of particular interest to the background of the present invention is the inventor's copending U.S. patent application Serial No. 419,953, filed September 20, 1982, entitled "Method and Materials for the Microbiological Oxidation of Aromatic Hydrocar¬ bons," the disclosures of which are specifically incor- porated by reference herein. In this copending applica¬ tion, the applicant describes, inter alia, a trans¬ missible plasmid pE3l7 containing a DNA sequence of

Pseudomonas putida origin which codes for expression in a host microorganism of enzymes participative in the oxidative degradation of naphthalene to salicylate. Most briefly put, the copending application discloses use of plasmids such as pE317 and others to transform microorganisms such as E.coli and imbue them with the capacity to produce and accumulate selected valuable intermediates ordinarily only transitorily formed in the microbial mineralization of aromatic compounds such as naphthalene. Included in the enzymes coded for by plasmid pE3l7 is a naphthalene dioxygenase enzyme. This enzyme catalyzes the transformation of naphthalene to cis-l,2-naρhthalene dihydrodiol. Applicant and his coworkers had previously performed an exhaustive study of the oxidation of naphthalene by a multi-component enzyme system from Pseudomonas sp.NClB 9816 [see Ensley, et al., J.Bacteriology, 149: 948-954 (1982)] and characterized the initial reaction in naphthalene oxidation as involving an enzyme system comprised of three protein components. At present, therefore, the art has not been provided with any reliable description of efficient microbiological production of indigo. This is the case, despite knowledge of the existence of certain microorganisms having the capacity to synthesize and accumulate indole and certain other organisms having the capacity to employ indole as a substrate for indigo synthesis.

BRIEF SUMMARY

The present invention provides the first instance of microbiological production of indigo in a genetically-transformed microorganism grown in an indole-free medium. In one of its aspects, the invention provides a process for microbiological production of indigo in a selected microorganism having the metabolic capacity

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to produce and accumulate indole. The process involves stably genetically transforming the microorganism to incorporate the capacity to synthesize one or more aromatic dioxygenase enzymes. Dioxygenase enzymes operable in the present invention are those which will catalyze microbial oxidative tranformation of an aromatic hydrocarbon to a cis-dihydrodiol. The transformed microorganisms are grown under conditions which facilitate the dioxygenase enzyme catalysis of the oxidative transformation of indole. The presump¬ tive oxidative transformation reaction product is cis-indole-2,3-dihydrodiol.. This product, in turn, is believed to rearrange to indoxyl, which then condenses to indigo in the presence of air. Indigo is then isolated from the microorganism or its growth medium.

In one of its presently most preferred forms, microbiological production of indigo in a microorganism already having the metabolic capacity to produce and accumulate indole is accomplished using E.coli as the host cell microorganism. Included in the genetic transformation step is transformation with a DNA vector including a DNA sequence coding for the expression of naphthalene dioxygenase, an aromatic dioxygenase enzyme of Pseudomonas origin, derived from the P.putida naphthalene mineralization plasmid nah7. A suitable expression vector for this purpose is pE317 described in copending application Serial No. 419,953.

Practice of processes of the invention may include the additional steps of stably genetically transforming the microorganism to incorporate the capacity to synthesize tryptophanase enzyme and growing the microorganisms under conditions facilitative of tryptophanase catalyzed degradation of tryptophan into pyruvate and indole. Genetic transformation of host microorganisms to develop (or enhance). the capacity to synthesize tryptophanase enzyme as well as aromatic dioxygenase enzymes may be accomplished

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by transformation with a single DNA vector including DNA sequences coding for both types of enzymes.

The present invention thus provides processes for microbiological production of indigo in selected microorganisms which do not have the metabolic capacity to produce and accumulate indole, as well as in those that do. "Multiply-transformed" microorganisms can be grown under conditions facilitative of both trypto¬ phanase enzyme catalysis of the transformation of tryptophan to indole, and dioxygenase enzyme catalyzed oxidative transformation of indole to an oxidized form further "processed" within the cell to indigo. Indigo can thereafter be isolated from the microorganisms and/or the surrounding culture medium. Also provided by the present invention, therefore, are novel DNA transformation vectors compris¬ ing DNA sequences coding for microbial synthesis of both an aromatic dioxygenase enzyme and a tryptophanase enzyme. An "indigo operon" may be incorporated into a single vector, in which operon both the tryptophanase and dioxygenase enzyme coding regions are under the control of a single promoter/regulator. The promoter/ regulator of the operon can enable simultaneous opera¬ tion of both enzymes in the microbial host, thus creat- ing a microbial "sink" in which continuous catalysis of tryptophan to indole, and indole to cis-indole- 2,3-dihydrodiol and ultimately to indigo occurs. Desirably, the promoter/regulator would be sensitive to an inducer or a change in culture temperature. According to still another aspect of the invention, organisms having the capacity to synthesize one or more dioxygenase enzymes (whether by means of expression of genomic or plasmid-bo ne DNA sequences) are genetically altered to have the capacity to produce indigo upon growth in an indole-free medium. Such genetic alteration involves stable transformation to incorporate the capacity to synthesize a tryptophanase ^enzvme *

Further aspects and advantages of the present invention will become apparent upon consideration of the following detailed description of presently preferred embodiments thereof.

DETAILED DESCRIPTION The methods and materials which provide an illustration of the invention and which comprise the presently preferred embodiment relate specifically to plasmid-borne DNA sequences of Pseudomonas putida origin which can be employed to transform a desired indole-producing host microbial species, such as E.coli. Cells transformed according to this embodiment of the process and the DNA transformation vector express the DNA sequences in the form of synthesis of an initial dioxygenase enzyme (or enzyme system) which is capable of converting accumulated indole to cis-indole-2,3- dihydrodiol. The dihydrodiol, in turn, rearranges to form indoxyl, which also accumulates in the selected host microorganism. The latter product, in the presence of air, is transformed to indigo.

DNA sequences coding for dioxygenase enzymes useful in practice of the invention may be secured by recombinant methods practiced on microbial species displaying the capacity to synthesize and accumulate one or more enzymes catalyzing microbial oxidative transformation of an aromatic hydrocarbon to a cis-di- hydrodiol form. Especially likely to provide DNA sequences for use in the invention are those organisms empirically determined to display the capacity to transform indole supplied to the growth medium into indigo. One such organism is Pseudomonas putida PpG7 containing a transmissible naphthalene degrading plasmid, nah7. This organism served as the parent strain for development of plasmid pE317 employed in the selective procedures for aromatic hydrocarbon oxidation set out in copending application Serial No. 419,953.

Also expected to provide suitable DNA sequences is P.pu ida NCIB 9816, containing a transmissible naph¬ thalene-degrading plasmid similar to (and possibly identical to) nah7. Both these organisms have now been observed to produce indigo when indole is supplied as a component of their growth medium.

Also expected to provide useful sources of DNA sequences coding for dioxygenase enzymes for practice of the invention are those organisms possessing a capacity for oxidative mineralization of aromatic hydrocarbons other than naphthalene (e.g., toluene, benzene and the like) , whether or not the gene coding for the enzyme is plasmid-borne or genomic. As examples of such organisms may be cited Pseudomonas putida "TOL" described by Yeh, et al., Biochem. & Biophys. Res. Comm. , 78: 401-410 (1977) and P.putida 39/D described by Gibson, et al., Biochem. , 9.: 1626-1630 (1970). Each of these organisms displays the capacity to synthe¬ size ^' a dioxygenase enzyme catalyzing the formation of cis-toluene-2,3-dihydrodiol as a product of the oxidation of toluene. Each of these organisms has now been observed to display the capacity to produce indigo when indole is supplied as a component of their growth medium. When a DNA sequence coding for a dioxygenase enzyme is transformed by an appropriate vector into a microorganism, such as E.coli, which has its own tryptophanase enzyme, the microorganism can produce indigo from tryptophan. The following illustrative examples treat: (1) the identification of the blue pigment produced by the vector-harboring microorganism of the present invention during growth in defined medium containing ampicillin; (2) the determination that naphthalene dioxygenase enzyme produced by the DNA coding region of the DNA vector is reacting with indole produced endogenously by E.coli; and (3) measure¬ ment of the rate of indigo synthesis by the recombinant E.coli.

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Example 1 Plasmid pE317 was prepared as described in Example 4 of U.S. Patent Application, Serial No. 419,953. When E.coli HB101 was transformed with pE3l7 and grown in Luria broth containing 200 μg/ml ampicillin, a blue pigment was observed to form in the culture medium and cells after overnight incubation.

The blue pigment was purified and identified by the following procedure. E.coli HB101 containing pE3l7 was grown for 18 hours in two one-liter flasks containing 250 ml of mineral salts medium composed of (g/L) 10 g K ₂ HP0 ₄ , 3.5 g Na(NH ₄ )HP0 ₄ ^* 4H ₂ 0, 2.0 g citric acid'H-O, 0.2 g MgS0 ₄ ^* 7H 0 supplemented with 0.25% glucose, 25 mg/L proline and leucine, 2.0 mg/L ampicillin. The flasks were shaken at 250 RPM and kept at 30°C.

After growth, the cells were separated from the spent medium by centrifugation, resulting in a dark blue cell pellet and a clear, straw-colored super- natant. The cell pellet was extracted 8 times with 25-ml volumes of boiling chloroform. The organic extracts were pooled and the volume reduced to 10 ml under a stream of argon gas. The organic extract was dried over anhydrous sodium sulfate and applied to the top of a silica gel 60 column (2.5 x 5 cm) previously equilibrated in chloroform. The blue pigment was washed through the column with chloroform and 4.0 ml fractions were collected. Fractions containing blue pigment were analyzed for purity by chromato- graphy on thin layer chromatography (TLC) sheets (EM Reagents, Silica gel 60 F ₂ _ ₄ ) developed in a solvent system of chloroform : acetic acid : methanol 40:2:1 (vol/vol) . Those blue fractions which contained a single UV-absorbing spot after analysis by TLC were pooled and the solvent removed under vacuum. This procedure resulted in 26 mg of dark blue crystals. The crystals were dissolved in a small volume of chloro-

form and subjected to analysis. The blue pigment had identical chromatographic properties, visible, ultraviolet, mass and infrared spectra to that of synthetic indigo (Kodak) . This data indicates that indigo is produced by the recombinant E.coli during growth under the described conditions.

Example 2 The indication that the enzymes synthesized from the cloned naphthalene dioxygenase genes are reacting with indole produced endogenously by E.coli is consistent with the following observations.

1. After several serial passages in nonselec- tive (i.e., ampicillin-free) medium, the recombinant organism loses the ability to produce indigo. When these cultures are analyzed for the ability to oxidize naphthalene, a parallel loss in naphthalene oxidizing activity is observed. Since untransformed E.coli is unable to produce the blue pigment, these experiments demonstrate the essential nature of the naphthalene dioxygenase genes in blue pigment formation.

2. Blue pigment formation is enhanced if the recombinant E.coli is grown in culture medium supplemented with 10 mM tryptophan or 1 itiM indole. 3. No blue pigment formation is observed if the recombinant E.coli is grown in a medium supple¬ mented with 1% glucose. High levels of glucose cause catabolite repression of tryptophanase synthesis in E.coli [Botsford, J.L. and R.D. DeMoss, J.Bacteriol. 105.: 303-312 (1971)].

4. Blue pigment formation is observed if Pseudomonas putida PpG7, which carries the naphthalene dioxygenase genes on the Nah7 plasmid, is incubated with indole in the culture medium. This organism does not possess a tryptophanase enzyme system and does not produce indole during the normal course of metabolism.

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Example 3 The rate of indigo synthesis by the recombi¬ nant E.coli was measured by the following procedure. Transformed and untransformed E.coli was grown in two flasks containing the mineral salts medium described in Example 1. Ampicillin was omitted from the mineral salts medium used to grow untransformed E.coli. Growth of the organisms was monitored by measuring the absor- bance at 500 nm. Indigo synthesis was monitored by removing 1.0-ml samples from each culture at various time intervals. The cultures were extracted with 2.0 ml of ethyl acetate, σentrifuged to break the emulsion, and a portion of the upper (ethyl acetate) layer was transferred to a cuvette. The optical density of each organic extract at 600 nm was measured. Indigo was empirically determined to have a visible absorption maximum at 600 nm in ethyl acetate. Easily measured synthesis of indigo during growth was observed in the culture containing the transformed cells, while no indigo synthesis could be measured in the culture containing untransformed E.coli.

The foregoing examples demonstrate that endogenous tryptophanase enzyme in E.coli cells examined converts tryptophan to indole (and, likely, pyruvate and ammonia) . As a consequence of the transformation of the E.coli with a DNA vector including a DNA sequence coding for microbial synthesis of naphthalene dioxy¬ genase, an aromatic dioxygenase, is produced within the cells. The specific intermediates formed during conversion of indole to indigo in practice of the invention have not as yet been dispositively identified. It is likely, however, that the initial product of dioxygenase enzyme catalyzed transformation of indole is cis-indole-2,3-dihydrodiol. Rearrangement of the diol yields indoxyl and condensation of indoxyl in the presence of air yields indigo.

The amount of indigo formed in such procedures can be considerably increased if the organisms are additionally transformed to stably incorporate a DNA sequence coding for the tryptophanase enzyme. See, Deeley, et al., supra.

Although E.coli has its own tryptophanase enzyme coding region, that region and its regulatory mechanism are on a chromosome providing only one copy per cell. On a high copy number DNA plasmid vector, many copies of the tryptophanase enzyme coding region may be dispersed within the cell, providing higher efficiency and a higher rate of conversion of tryptophan to indole. This increased metabolism of tryptophan should activate E.coli's endogenous tryptophan synthe- tase enzyme regulatory mechanism to convert indole glycerol phosphate and serine to tryptophan. When the DNA vector coding regions for both tryptophanase and dioxygenase enzymes are on the same DNA vector and under the control of the same promoter, they may both be activated simultaneously. Once the E.coli bacteria cells harboring such vectors are grown to an optimal level, both enzyme coding regions on the plasmid may be simultaneously activated to convert the tryptophan in the cells to indole and pyruvate, and the indole to indoxyl and ultimately to indigo until all of the tryptophan produced in the cells is consumed. The indigo so produced frequently crys¬ tallizes in the medium as well as within the cells themselves, and may be extracted by simple chemical and mechanical methods.

Where the host microorganism does not have endogenous metabolic capacity to produce and accumulate indole, the transformation of the microorganisms by the DNA vector containing both DNA sequences coding for tryptophanase enzyme and an aromatic dioxygenase enzyme will function to enable the microorganism to

initially convert typtophan to indole, and thereafter convert indole to indigo.

In a preferred form, an "indigo operon" DNA transformation vector of the invention would contain both tryptophanase and dioxygenase enzymes under simul¬ taneous control of a promoter/regulator. One such indigo operon, like pE3l7, may consist of a small portion of the P.putida naphthalene mineralization plasmid nah7, which includes operon-containing DNA fragments retaining the capacity to direct naphthalene dioxygenase enzyme expression. Associated with the dioxygenase gene on the DNA transformation vector would be a tryptophanase enzyme coding region such as described by Deeley, et al., supra. An example of a temperature sensitive promoter/ regulator potentially useful in construction of such an indigo operon is phage λPL under cl 857 control. This highly efficient promoter (PL) can be regulated by the λ repressor protein cl, a product which is regulated autogenously in E.coli λ lysogens. Mutant repressor protein cl 857 inactivates the PL promoter at temperatures below 32°C. At temperatures between 32°C and 41°C, cl 857 is inactivated, thereby turning on transcription under control of the PL promoter. See: Shimatake, H., et al.. Nature. 292: 128-131

(1981); and Sussman, R. , et al., Acad.Sci.Paris, 254: 1517-1519 (1962). While the benefits of use of a temperature sensitive promoter/regulator in coordination of cell growth and gene expression are abundantly clear, they must be considered in the light of potential drawbacks in terms of diminished activity of trypto¬ phanase and/or dioxygenase.

The foregoing illustrative examples and detailed description have principally been directed to securing indigo production by organisms lacking the genetic wherewithal to "process" indole in a manner leading to the formation of indigo, i.e., lacking

the capacity to .synthesize a suitable dioxygenase enzyme. It will be apparent to those skilled in the art that the present invention also comprehends securing production of indigo by cultured growth of organisms already having the capacity to synthesize a suitable dioxygenase enzyme. This is accomplished by genetic transformation of such organisms to stably incorporate DNA sequence(s) specifying synthesis of a tryptophanase enzyme, thus allowing for processing and transformation of cellular tryptophan into an indole substrate for action of the dioxygenase enzyme. As was the case with augmenting the endogenous tryptophanase synthesiz¬ ing capacity of an organism by ^' inserting multiple "extra" copies of a tryptophanase gene, substantial benefits are expected to attend augmenting the endog¬ enous dioxygenase synthesizing capacity of selected host cells by insertion of multiple copies of a plasmid comprising an "indigo operon."

At present, the best prospective host cells for practice of this aspect of the invention are the Pseudomonas putida previously discussed as appropriate sources of dioxygenase gene, i.e., PpG7, NCIB 9816, "TOL" and 39/D.

Numerous other modifications and variations of the invention as above-described are expected to occur to those skilled in the art and consequently only such limitations as appear in the appended claims should be placed thereon.

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