WO1995034658A1

WO1995034658A1 - Microbial production of indole-3-acetic acids and substituted analogs thereof via fermentation

Info

Publication number: WO1995034658A1
Application number: PCT/US1995/007174
Authority: WO
Inventors: Matthew W. Chase; Scott D. Power; Gregory M. Whited
Original assignee: Genencor International, Inc.
Priority date: 1994-06-10
Filing date: 1995-06-06
Publication date: 1995-12-21
Also published as: AU2818195A

Abstract

Bacteria of the genus Escherichia, genetically altered to relieve inherent regulatory mechanisms of the tryptophan biosynthetic pathway, carrying DNA containing sufficient genetic information for conversion of anthranilate to tryptophan and for tryptophan to indole-3-acetic acid are useful in the fermentative production of indole-3-acetic acid and substituted analogs thereof.

Description

MICROBIAL PRODUCTION OF INDOLE-3-ACETIC ACIDS AND SUBSTITUTED ANALOGS THEREOF

VIA FERMENTATION

Field of the Invention

The present invention relates to microorganisms carrying DNA constructs useful in the fermentative production of 4-, 5-, 6- and 7-substituted tryptophan and indole analogs and indole 3- acetic acids and substituted analogs thereof. Furthermore, this invention relates to processes for producing and recovering such substituted tryptophans, indoles and indole acetic acids.

Prior Art

Production of L-tryptophan by fermentation has long been looked at as a profitable tool. The biosynthetic tryptophan pathway as shown in Figure 1 has been extensively studied. Many investigators have labored to find or produce suitable microorganisms for the purpose of large scale production of tryptophan which would be economically feasible from an industrial point of view. The prior art of large scale tryptophan production comprises the fermentative cultivation of many microorganisms such as Micrococcus (US Patent No. 3,385,762), Micrococcus, Brevibacteriu , Candida. Arthrobacter, Hansenula, Asperαillus. Penicillium and Streptomvces (US Patent No. 3,591,456), Arthrobacter, Bacillus and Corynebacterium (US Patent No. 3,594,279), Bacillus (US Patent No. 3,700,558), Brevibacterium, Corvnebacterium. Microbacterium and Micrococcus (US Patent No. 3,700,559), Bacillus (US Patent No. 3,801,457), Corvnebacterium (US Patent No. 3,849,251), Escherichia (US Patent No. 4,371,614) and Corynebacterium (US Patent No. 4,742,007). These processes all include microorganisms with tryptophan analog resistance, and/or auxotrophic requirements, or reco binant episomal DNAs which render the organisms tryptophan overproducers. Tryptophan production is an important process to develop as tryptophan has a very extensive worldwide market as an essential amino acid, as well as being important for agricultural purposes. In as much as tryptophan is an important product, tryptophan analogs themselves have found an increasingly important place in the pharmaceutical, agricultural and antimicrobial markets. Likewise, the immediate precursor to tryptophan in the biosynthetic pathway to tryptophan, indole (see Figure 1), also has important uses both physiologically and chemically. Indole and it's substituted analogs have a wide array of important uses ranging from pharmaceuticals, pharmaceutical intermediates, uses in flavors and fragrances, pest deterrent applications, antimicrobials, polymerization compounds, photographic chemical applications, intermediates in dye preparations, as well as a variety of biological activities (W.A. Remers, Heterocyclic Compounds, Vol. 25, 'Indoles, ' Part II, ed. by W.J. Houlihan, John Wiley and Sons, New York, 1972, p. 86) .

Indole-3-acetic acid, a compound which can be made by the present invention, is a naturally occurring plant hormone or auxin. Typical production of this compound utilizes a petroleum derived feed stock, whereas the present invention allows its production from glucose. The auxin indole-3-acetic acid is synthesized by plants as well as certain phytopathogenic bacteria for use in the induction of many types of growth. The auxin activity of indole-3-acetic acids as well as substituted indole 3-acetic acids such as 4-chloro; 5-chloro; 6-chloro; 7-chloro; 4, 6-dichloro; 4, 7-dichloro-; 5,7-dichloro; 6, 7-dichloro; 5-bromo; 7-bromo; 5-fluoro; 5- chloro-7-methyl; 5-methoxy; 5-hydroxy as well as naphthalene acetic acids, has been studied for utility in tissue culture work. K.C. Engvild, Physiol. Plant, 345-346 (1978) .

Tryptophan and indole, as well as their respective substituted analogs or derivatives such as indole acetic acids, are difficult to obtain commercially at economically feasible prices, if at all in large scale, and are not economically feasible to chemically synthesize at large scale. Chemical syntheses of tryptophan and tryptophan analogs have been reviewed (J.P. Greenstein and M. Winitz, 'Chemistry of the A ino Acids, ' Vol. 3, Wiley, New York, 1961, pp. 2316-2347, and T. Kaneko, et al., Synth. Prod. Util. Amino Acids, 206-219 (1974)), but the drawbacks of these syntheses are that a number of contaminants are produced along with the desired product. More convenient and efficient routes to tryptophan synthesis have also been reported, but the economics of large scale chemical production is doubtful (Hengartner, et al., J. Orq. Chem., 3748-3752 (1979); S.V. Thiruvikraman, et al. , Tetrahedron Letters, 2339-2342 (1988)) . Large volumes of chemical syntheses of indole and indole analogs have been published, as well as modifications of these syntheses (US Patent No. 3,976,639; A. Kozikowski, Heterocycles, 267-291 (1981); R. Clark, et al., Heterocycles, 195-221 (1984); M. Moyer, et al., J. Orq. Chem., 5106-5110 (1986); G. Bartoli, et al., Tetrahedron Letters, 2129-2132 (1989); Y. Yang, et al., Heterocycles, 1169-1175 (1992); Y. Murakami, et al. , Chem. Pharm. Bull., 1910-1919 (1993); G. Carrera, Jr. and G. Sheppard, Synlett, 93-94 (1994)) . Of the chemical syntheses described for indole and its analogs, only the last method, the synthesis of 6- and 7-arylindoles via palladium-catalyzed cross-coupling of 6- and 7-bromoindole with arylboronic acids, seems to be amenable to large scale production with economic feasibility.

Based on the shortcomings of these known chemical methods, it would be advantageous to develop a cost effective, environmentally 'sensitive' process for producing tryptophan, substituted tryptophan and analogs thereof including substituted and unsubstituted indole-3-acetic acids, as well as for producing indoles and analogs thereof from a renewable carbon source such as glucose. Accordingly, it is an object of the present invention to provide novel processes for the biosynthetic production of 4-, 5-, 6- and 7-substituted indoles and tryptophans from substituted anthranilates. Another object of the present invention is to provide novel processes for the biosynthetic production of indole-3-acetic acids and substituted derivatives thereof.

Particularly we have found that substituted anthranilates can be used as substrates for enzymatic conversion of the substituted anthranilates to the corresponding substituted indole or tryptophan. Thus, anthranilate substituted at 3-, 4-, 5- or 6- can be used to make the corresponding 4-, 5-, 6- or 7-substituted indole or tryptophan (see Scheme 1) . Alternatively the anthranilate substrate can be used to make indole-3-acetic acid also as shown in Scheme 1.

Scheme 1 .

Tryptophan Indole

wherein R is H, halogen, OH, C1-C5 alkyl, C1-C5 alkoxy, N0₂, NH₂, COOH, CN, sulfur, S0₃, or S0₄ Therefore, one aspect of the present invention relates to a process for the biosynthetic production of substituted indoles, the process comprising:

a) culturing an appropriate microorganism in suitable conditions in the presence of a 3-, 4-, 5- or 6-substituted anthranilate of the formula:

wherein R is a halogen, OH, C1-C5 alkyl, C1-C5 alkoxy, N0₂, NH₂, COOH, CN, sulfur, S0₃ or S0₄; to produce a corresponding 4-, 5-, 6- or 7-substituted indole of the formula:

wherein R is as defined above; and

b) recovering the corresponding substituted indole.

Another aspect of the present invention relates to a process for the biosynthetic production of substituted tryptophan, the process comprising:

wherein R is a halogen, OH, C1-C5 alkyl, C1-C5 alkoxy, N0₂, NH₂, COOH, CN, sulfur, S0₃ or S0₄; to produce a corresponding 4-, 5-, 6- or 7-substituted tryptophan of the formula:

wherein R is as defined above; and

b) recovering the corresponding substituted tryptophan, and

c) optionally culturing an appropriate microorganism with the substituted tryptophan under suitable conditions to convert the substituted tryptophan to substituted indole-3-acetic acid.

Another aspect of the present invention relates to a process for the biosynthetic production of indole-3-acetic acids and substituted analogs thereof the process comprising:

a) culturing an appropriate microorganism in suitable conditions in the presence of a substituted or unsubstituted anthranilate of the formula:

wherein: R is H, halogen, OH, C1-C5 alkyl, C1-C5 alkoxy,

N0₂, NH₂, COOH, CN, sulfur, S0₃, or SO,; to produce a corresponding substituted or unsubstituted indole-3-acetic acid of the formula:

wherein R is as defined above; and

b) recovering the indole-3-acetic acid.

Another aspect of the present invention comprises a process for the production of a substituted or unsubstituted indole-3- acetic acid in a suitable host using a renewable carbon source such as glucose, the process comprising transforming a host cell with suitable DNA as detailed herein and culturing the transformed host in the presence of glucose.

In a preferred embodiment of the present invention the appropriate microorganism for the production of tryptophan or indole is a bacteria of the genus Escherichia, genetically altered to remove certain regulatory mechanisms of the normal tryptophan biosynthetic pathway (for example, trp^~ , tna^~ , trpR^') and to comprise sufficient genetic information to convert anthranilate to either indole or tryptophan, including but not limited to the trp operon. Preferably the microorganism comprises an intact chromosomal trp operon although it may be present in plas ids within the strain.

When the compound of interest is indole or a substituted indole, it is preferable that the microorganism comprise a mutation in the sole trpB gene of the cell, chromosomal or plasmid borne, such as trpB/26, which prevents the condensation reaction between serine and indole such that the substrate anthranilate or substituted anthranilate is converted to indole or substituted indole and no tryptophan is formed.

When the compound of interest is tryptophan or substituted tryptophan, it is preferable that the microorganism comprise the trp£^fbrDCBA genes, either integrated into the chromosome of the cell or plasmid borne.

When the compound of interest is indole-3-acetic acid or a substituted analog thereof, the microorganism must further comprise DNA coding genes for the conversion of tryptophan to indole-3-acetic acid.

In a preferred process aspect of the present invention a two phase fermentation process is employed such that the desired product (substituted indole, substituted tryptophan, indole-3- acetic acid or analog thereof) is effectively partitioned in one phase of the fermentation (i.e., an organic phase) while relatively low levels of the desired product are present in the second phase (i.e., aqueous phase) of the fermentation.

As used herein, "suitable conditions" for culturing the microorganisms with the anthranilate include conditions routinely used for growing Escherichia strains, for example, pH 5-8, temperature 20-40° C, in the presence of minimal or rich medium, provided that no anthranilate is present in such medium. Specific nutritional needs of any auxotrophs will be readily apparent to those skilled in the art.

Brief Description of the Drawings

Figure 1 shows the tryptophan biosynthetic pathway of E. coli ,

Figure 2 shows the plasmid map for pGD007.

Figure 3 shows the plasmid map for pBMW.

Figure 4 shows the plasmid map for pGA007- trp.

Figure 5 shows the plasmid map for pCPJ3.

Figure 6 shows the plasmid map for pBE7.

Detailed Description of the Invention

The development of a biocatalytic synthesis of tryptophan analogs and indole analogs would be a process of considerable importance. As early as 1970 it was found, rather indirectly, that when E. coli tryptophan auxotroph (trpC2) cells were grown overnight in tryptophan limited media and concentrated in an incubation mixture of 7-methylindole, serine, pyridoxal phosphate and glucose in minimal media, that the 7- ethylindole was converted to 7-methyltryptophan. Additionally, no tryptophan was found following the reaction (W.A. Held and O.H. Smith, J. Bacteriology, 209-217 (1970)). In the same set of experiments, the investigators grew an E. coli trpR mutant overnight in the presence of 3- methylanthranilate and found that the cells accumulated 7- methylindole. In addition, when an E. coli tryptophanase- deficient ( tna ) mutant was grown overnight in the presence of 3-methylanthranilate then concentrated, the radioactive 3- methylanthranilate was converted to ³H-7-methyltryptophan when the cells were incubated with ³H-3-methylanthranilate, serine and glucose.

In 1974 it was observed that when tryptophan synthase purified from E. coli trpE (anthranilate synthase) mutants was incubated with pyridoxal phosphate, serine and indole, that tryptophan was produced. In similar assays, substituting indole analogs for indole in the reaction (7-azaindole, 5- fluoroindole, 6-fluoroindole, 5-hydroxyindole, 2-azaindole, 5- ethoxyindole, 6-methoxyindole, 2-methylindole, 5- methylindole, 7-methylindole and pyrimidazole) , all but pyrimidazole yielded the corresponding substituted tryptophan visualized by paper chromatography. It was determined that the reactions did not go to completion, but did reach 80-85% completion (M. Wilcox, Analytical Biochemistry, 436-440 (1974)) .

In 1985 it was shown that a tryptophanase-deficient E. coli strain carrying a tryptophan operon on a high copy number plasmid (pBR322) could produce 5-hydroxytryptophan from 5- hydroxyindole. A precursor fermentation method was developed by which indole or 5-hydroxyindole was added to actively growing cells and the product tryptophan or 5- hydroxytryptophan was produced by the cells (G.S. Skogman and J.E. Sjostrom, Biotechnology Letters, 783-788 (1985)) . However, it was concluded that the economic feasibility of this process was limited by the high cost of the precursors indole and 5-hydroxyindole.

Another set of experiments was one in which purified tryptophan synthase was assayed for the kinetics of the B reaction, or the condensation of indole or an analog with serine to form the corresponding tryptophan. It was found that indole derivatives containing electron-donating groups in the 5-position (i.e., H, CH₃, OH, OCH₃) decreased the k_eat. of the condensation, but increased the K_M. Derivatives that have electron-withdrawing groups in the 5-, 6- or 7- position (i.e., Br, N0₂) were found to decrease the k_cat to near zero while not affecting the K_M (K. Kirshner, et al.. Biochemistry, 472-478 (1991).

As noted previously, both plants and certain bacteria produce indole-3-acetic acid, the exact biosynthetic pathway producing indole-3-acetic acid in plants has not been elucidated. Recent data suggests that plants may use a tryptophan independent biosynthetic pathway (E. Prinsen et al . , Molecular Plant- Microbe Interactions, 609-615 (1993), and J. Normanly et al . , Proc. Natl. Acad. Sci. USA, 10355-10359 (1993)).

Phytopathogenic bacteria such as Pseudomonas savastanoi produce an enzyme known as tryptophan monooxygenase (E.C.1.13.12.3) which converts tryptophan to indole-3- acetamide (T. Kosuge et al . , Journal of Biological Chemistry, 3738-3744 (1966) ) . In a second reaction, an enzyme known as indoleacetamide hydrolase cleaves indole-3-acetamide into ammonia and indole-3-acetic acid. The genes encoding these enzymes were found to be organized on extrachromosomal DNA or plasmid DNA called pIAAl (L. Comai and T. Kosuge, Journal of Bacteriology, 950-957 (1980)). In 1982, the genes for tryptophan monooxygenase, iaaM, and indoleacetamide hydrolase, iaaH, were cloned from Pseudomonas savastanoi and expressed in Escherichia coli (L. Comai and T. Kosuge, Journal of Bacteriology, 40-46 (1982)).

Bacteria other than Pseudomonas savasanoi also produce indole- 3-acetic acid during pathogenesis of plant tissue. The genes involved in indole-3-acetic acid from the bacterium Agrobacteri um tumefaciens were shown to have nucleotide sequence homology with the iaaM and iaaH genes from Pseudomonas savasanoi , and were shown to encode for tryptophan monooxygenase and indoleacetamide hydrolase, confirming a tryptophan mediated biosynthesis (T. Yemada et al . , Proc. Natl. Acad. Sci. USA, 6522-6526 (1985)).

In 1991, the phytopathogenic bacterium Enterobacter cl oacae was shown to produce indole-3-acetic acid via a different biosynthetic route than that of P. savasanoi or A. tumefaciens . It was shown that this bacterium converts tryptophan to indole-3-acetic acid via three steps. These steps include the conversion of tryptophan to indole-3-pyruvic acid, the conversion of indole-3-pyruvic acid to indole-3- acetaldehyde, and the conversion of indole-3-acetaldehyde to indole-3-acetic acid. However, although these conversions were attributed to enzymatic reactions, the enzymes and their respective genes were not identified (J. Koga et al . , Agric. Biol. Chem., 701-706 (1991)). In 1991, an Enterobacter cloacae gene for the enzymatic conversion of tryptophan to indole-3-acetic acid via indole-3-pyruvic acid and indole-3-acetaldehyde intermediates, termed indole-3- acetic acid synthetase was cloned and expressed in E. coli (EP 0 445 658 Al) . The nucleotide sequence as well as the amino acid sequence of the indole-3-acetic acid synthetase enzyme cloned are given in the above mentioned patent application.

The enzymes tryptophan monooxygenase and indoleacetamide have been shown to accommodate a degree of conformational change in substrates. For example, it was shown that tryptophan monooxygenase from P. savastanoi is specific for the L isomer of tryptophan, and that it can produce 5-hydroxyindole-3- acetic acid from 5-hydroxytryptophan at 17% of the efficiency of the conversion of tryptophan to indole-3-acetic acid (T. Kosuge et al . , Journal of Biological Chemistry, 3738-3744 (1966)). Additionally, it was shown that 5-methyltryptophan could be accommodated by tryptophan monooxygenase from P. savastanoi at 89% of the efficiency of tryptophan (L. Comai and T. Kosuge, Journal of Bacteriology, 950-957 (1980)). In 1985, in an attempt to purify and elucidate the properties of the enzymes for indole-3-acetic acid biosynthesis from P. savastanoi , it was determined that tryptophan monooxygenase could utilize a number of substituted tryptophans as substrates, albeit at lower specific activities than with tryptophan. Tryptophans with 4-, 5-, 6-, or 7- position methyl monosubstitutions, 7-azatryptophan, 5-fluorotryptophan, and 5- hydroxytryptophan all were utilized as substrates by the enzyme (S. W. Hutcheson and T. Kosuge, Journal of Biological Chemistry, 6281-6287 (1985)).

The tryptophan biosynthetic pathway has served as one of the most intensely studied pathways in microorganisms. The pathway is highly conserved throughout prokaryotic, as well as eukaryotic, microorganisms. Essentially comprising seven enzymatic reactions, the enzymes of the pathway ultimately catalyze tryptophan from chorismate, which serves as the common intermediate in all aromatic amino acid biosynthetic pathways.

The tryptophan biosynthetic pathway of Escherichia coli is shown in Figure 1. Gene designations for the tryptophan biosynthetic enzymes vary from organism to organism, however, for convenience, without intending to be limited by such, all genetic descriptions herein will correspond to those assigned to E. coli . These E. coli genes and brief descriptions are summarized in Table I below. These genes of the tryptophan pathway are linearly oriented on the chromosome, coordinately regulated and expressed, and comprise what is commonly referred to as the tryptophan operon (F.C. Neidhardt, ed., ' Escheri chia coli and Salmonella typhimuri um Cellular and Molecular Biology, ' pp. 1453-1472 (1987)).

Table I Gene Description trp A Tryptophan Synthase: A Protein (E.C. 4.2.1.20) trpB Tryptophan Synthase: B Protein (E.C. 4.2.1.20) trpC Phosphoribosyl Anthranilate Isomerase-Indoleglycerol

Phosphate Synthase (E.C. 4.1.1.48) trpD Anthranilate Phosphoribosyl Transferase (E.C.

2.4.2.18) trpE Anthranilate Synthase (E.C. 4.1.3.27) trpR trpR Aporeppressor tna Tryptophanase aroG DAHP Synthase

A number of investigators have strived to produce E. coli strains capable of large scale tryptophan production. In 1979 one group studied a series of E. coli mutants produced in an attempt to elucidate the most productive alterations needed in order for tryptophan overproduction. (D.E. Tribe and J. Pittard, Applied and Environmental Microbiology, 181-190 (1979); U.S. Patent Application Serial No. 06/994,194 (Frost et al) shows the effect of blocking various enzymes in the pathway as it relates to accumulation of key intermediates and production of a desired product.).

The previous study involved genetically altering E. coli strains to improve tryptophan production. In 1982 another group used E. coli tryptophan operon-deficient strains (trpAEl) in combination with either tryptophan repressor (trpR) deletions and/or tryptophanase ( tna) deletions. Multicopy (~5) composite plasmids (pSClOl) carrying the tryptophan operon genes, which were desensitized to tryptophan feedback inhibition, were transformed into these strains and the resultant tryptophan productivity assayed. They found that copy number and release of feedback inhibition, as well as trpR^" and tna^' qualities, all effected tryptophan production (S. Aiba, et al., Applied and Environmental Microbiology, 289- 297 (1982)) . The only drawback found in this study was that of plasmid instability.

Prior to the study above, another group had found that multicopy plasmids carrying the tryptophan operon genes exhibited instability with regard to the particular host strain harboring the plasmid. It was found that plasmids carrying the tryptophan operon were either entirely lost or experienced deletions of the tryptophan genes when grown in host cells with trp repressor mutations and/or tryptophanase deletions. These same plasmids lacking the tryptophan genes were stable in all strains tested. It was concluded that the extra cellular burden imposed on these strains via overproduction of the tryptophan biosynthetic enzymes caused lowered growth rates and allowed for trp^" seggregants to become enriched (T. Imanaka, et al., Journal of General Microbiology, 253-261 (1980)) .

A more recent study involving increasing tryptophan production in E. coli involved the amplification of the trp operon genes via chromosomal integration of appropriately mutagenized trp operon genes. In this study the investigators attempt to combat the stringent regulatory factors involved in the tryptophan biosynthetic pathway. They recognized the need for removal of feedback inhibition and repression controls, as well as the problem of trp operon- containing plasmid instability. These researchers successfully integrated trp operons in multicopy into the chromosome of the host strain. The operons integrated were first mutated so as to desensitize the enzymes to feedback inhibition. Following integration they found that the tryptophan operon was integrated into the chromosome at a copy number of three per chromosome and that the resulting strains had increased levels of tryptophan production. Also important in this study was that the resultant strains retained the trp operons and that they were stable for many generations, thus removing both the problem of plasmid stability, as well as the expensive need for antibiotic selection during fermentation (E. Chan, et al. , Applied Microbiological Biotechnology, 301-305 (1993)).

For the purpose of this invention, any microorganism of the genus Escherichia having the following characteristics can be used: Escheri chia which have been genetically altered to remove certain regulatory mechanisms of the normal tryptophan biosynthetic pathway; Escherichia which are deficient (by genetic alteration or otherwise) in the gene product tryptophanase, including those strains wherein the tna gene has been deleted or inactivated; and Escherichia which carry DNA containing the genes for conversion of anthranilate to tryptophan [ trpA, trpB, trpC, trpD and trpE) ; or DNA containing genes for conversion of anthranilate to indole [ trpA, trpB/26 or appropriately mutated trpB, trpC, trpD or trpE); and Escherichia which carry DNA containing the genes for conversion of tryptophan to indole-3-acetic acid (genes coding for the enzyme tryptophan monooxygenase such as iaaM and for the enzyme indole acetamide hydrolase such as iaaH) .

The fermentative production of and 4-, 5-, 6- and 7- substituted tryptophans aspect of the present invention comprises culturing microorganisms of the genus Escheri chia comprising tryptophan pathway regulatory genetic alterations and DNA containing genes for the conversion of anthranilate to tryptophan, in the presence of 3-, 4-, 5- or 6-substituted anthranilates, and recovering the corresponding tryptophan from the culture medium.

Likewise, the fermentative production of 4-, 5-, 6- and 7- substituted indoles aspect of the present invention comprises culturing microorganisms of the genus Escheri chia comprising tryptophan pathway regulatory genetic alterations, such as feedback resistance of pathway enzymes ( trp^ibr ) , trpR and tna^" in the presence of 3-, 4-, 5- or 6-substituted anthranilates, and recovering the corresponding indole from the culture medium. The fermentative production of 4-, 5-, 6- and 7- substituted or unsubstituted indole-3-acetic acids analogs aspect of the present invention comprises culturing microorganisms of the genus Escheri chia comprising tryptophan pathway regulatory genetic alterations and DNA containing genes for the conversion of anthranilate to tryptophan and tryptophan to indole-3-acetic acid in the presence of anthranilate or 3-, 4-, 5-, 6- substituted anthranilates and recovering the corresponding indole-3-acetic acid.

The E. coli strains useful in the present invention are capable of producing tryptophan, indole and indole-3-acetic acids and their corresponding 4-, 5-, 6- and 7-substituted analogs by fermentative procedures. We have surprisingly found that the pathway enzymes (including tryptophan monooxygenase and indoleacetamide hydrolase) can accommodate a very wide degree of conformational change in substrates and can yield quantitative amounts of substituted products. This finding is surprising not only due to the degree of change accommodated by the enzymes, but also by the purity of the products. Most successful chemical syntheses of these same compounds do not reach quantitative yields with regard to total attainable product and are not free of contamination by side-products and by-products, as are the products derived by the present invention.

Most successful bioconversions in the prior art are not quantitative and were not shown to be successful at large scale. In the present invention the production of substituted analogs of tryptophan and indole as well as indole-3-acetic acid or substituted analogs thereof are quantitative and the products are pure, needing only to be separated from cell mass and culture medium.

As used herein, "quantitative" means that each product is produced in a one-to-one ratio with substrate and that the total product is equal to that which is possible from the given substrate. "Pure" is intended to mean that the product formed is not contaminated with unused substrates, intermediates or side products.

Table II shows strains and plasmids used in the following experiments:

Table II

Strain/ Plasmid Description Reference

C534 Derived from E. coli K-12 W3110; trp- , tna- , trpR-

FM5 Derived from E. coli K-12 W1485; prototrophic, : -. lambda cI857 pGD007 E. coli trpE^fbrDCB/26A under tandem Fig. 1 lac UV5; pACYC origin and CAT gene pBMW pGD007 with aroG^fbr under tandem Jac Fig. 2 UV5; pACYC origin and CAT gene pGA007-trp E. coli trpE^fbrDCBA under tandem lac Fig. 3 UV5; pACYC origin and CAT gene

JB102 Derived from C534 (above) trp-, tna-, trp R- , ser A- pCPJ3 P. syringae subsp. savastonoi iaa M b/Fig.5 and iaa H under CAT promoter; pBR322 origin and ampicillin resistant pBE7 E. coli trp operon under lac promoter; Fig. contains aro G and ser A; tetracycline res.; pACYC origin

a) PCT WO87/01130 b) Burnette, et al., BioTechnology, 699-706 (1988)

For the production of substituted indole analogs hereunder, pGD007 and pBMW were used. These two plasmids (shown in Figures 1 and 2 respectively) contain promoter and attenuator deleted trp operons, in addition to containing a trpB/26 gene. The promoter and attenuator deletions in this system remove negative regulation of the tryptophan operon by removing those DNA sequences involved in the down regulation, or shutting down of the pathway. The trpB/26 gene is the result of a point mutation which replaces leucine at amino acid residue 382 of the trpB protein with glycine. This mutation does not interfere with the allosteric interactions of the trpB and trpA subunits of the tryptophan synthase enzyme complex. The active tryptophan synthase complex is a tetramer of two trpA encoded polypeptide subunits and two trpB encoded polypeptide subunits. The trpA subunit catalyzes the conversion of indoleglycerolphosphate to indole and glyceraldehyde 3- phosphate, and the trpB subunit catalyzes the condensation of indole with serine to form tryptophan. The trpB/26 mutation allows for the tryptophan synthase complex to form, but does not allow for the condensation between indole and serine, and so only allows indole formation. Therefore, with these gene constructs anthranilate is converted to indole and no tryptophan is formed.

Indole production was found to be much greater when using the FM5 host strain (from Table II) as opposed to C534 (Table II) . This is believed to be due to the fact that FM5 has an intact chromosomal trp operon, whereas C534 does not. In fact, when used for substituted indole production, FM5/pBMW produced 70% indole and only 30% substituted indole, whereas C534 produced 100% substituted indole and no indole when fed substituted anthranilate.

Escherichia strains grow in the presence of indole up to a level of about 100 mgs/L. For this reason it was necessary to develop a two phase fermentation process such that the indole produced would be effectively partitioned to the organic environment of the fermentation broth while maintaining a viable, low concentration in the aqueous phase of the fermentation broth. Soybean oil exhibits a sufficiently high partition coefficient with indole in water at 37°C, and the E. coli strains described grow at normal rates when in the presence of the oil; therefore, the following experiments were carried out using this oil. However, it is contemplated that other oils having a comparable partition coefficient with indole (or tryptophan) in water may be used.

The following examples are illustrative only and are not intended to limit the scope of the present invention.

Experimental

Example 1 Small Scale Indole from Anthranilate Biotransformation

25 gs of anthranilate were incubated with 3 grams (dry cell weight) E. coli C534/pGD007 ( rpB/26) cells (Table II) in 25 mLs solution consisting of 100 mM KP0₄ buffer with 0.4% D-

Glucose (w/v) in a 250 mL baffled shake flask. The suspension was placed in a 37°C orbital-shaker incubator at 300 rpm. 1 mL samples of whole broth were withdrawn at 0 minutes and at

180 minutes post incubation. Each 1 mL sample was transferred to 1.7 L microfuge tubes and subsequently centrifuged 2 minutes at 14,000 rpm. The cell-free supernatant was removed and diluted with an equal volume of HPLC Mobile Phase A (see below) at pH 4.6. Samples were vortexed briefly (about 10 seconds) and 1 mL aliquots distributed to standard Altech glass HPLC sample vials for subsequent chromatography.

Samples were analyzed for tryptophan, anthranilate and indole, as compared to standards purchased from commercially available sources using the TAI HPLC method described in detail below.

TAI HPLC Conditions:

Column: LC8-DB (15cm x 4.6mm) with 5 micron packing, commercially available from Suppelco. Precolumn: Spheri-5 RP-8 (30 x 4.6mm) with 5 micron packing, commercially available from

Brownlee. Mobile Phase A: 8.952 grams sodium acetate trihydrate

5.88 mL tetrabutyl ammonium hydroxide dissolved in 4 liters MQH₂0 pH adjusted to 4.6 with acetic acid

210.5 mL Mobile Phase B (see below) Mobile Phase B: 5.88 mL tetrabutyl ammonium hydroxide

0.4 mL acetic acid dissolved in 4 liters HPLC grade methanol Flow Rate: 3 mL/minute Injection: 25 microliters Detection: UV 270 nM and UV 254 nM

The HPLC results from Example 1 show the depletion of anthranilate from the aqueous buffer and the production of indole in a quantitative conversion ratio. Furthermore, no detectable levels of L-tryptophan were produced. In a further effort to conclusively identify the indole product, the cell- free supernatant described above was extracted with equal volumes of hexane, concentrated, and analyzed via GC/MS. Conditions of the GC/MS are given below.

GC/MS Conditions:

All products were analyzed by HPLC and then confirmed by gas chromatography/mass spectral analysis (GC/MS) .

Column: DB-5 phase (J & W Scientific)

Film Thickness: 1 μM

Column Dimensions: 60M x 0.324mM

Injector Temperature: 250°C

Detector Temperature: 280°C

Initial Temperature: 50°C

Exponential T Ramp: 10°C/min; Final Temperature 325°C

Run Time: 33.00 min

The results from the GC/MS analysis agreed with the HPLC analysis, identifying the product as indole.

Example 2 Screen for Anthranilate Substitutions which the Tryptophan

Operon can Accommodate for Indole Biotransformations

Part A

Substituted anthranilates ("A" in Table III) were screened for the purpose of identifying substitutions which can be accommodated by the enzymes of the tryptophan operon of E. coli C534/pGD007 (trpB/26) . Experimental procedure followed exactly that found in Example 1. TABLE I I I

A B

(R is as defined herein)

Substituted Anthranilates Screened

Resulting Indole (B) (% Anthranilate

Anthranilate (A) Commercial Supplier Conversion) anthranilate Aldrich indole (100%

4-chloroanthranilate Aldrich 6-chloroindole (13%

5-chloroanthranilate Aldrich 5-chloroindole (13%

6-chloroanthranilate Lancaster 4-chloroindole (30%

5-fluoroanthranilate Aldrich 5-fluoroindole (48%

5-bromoanthranilate Aldrich 5-bromoindole (30%

5-hydroxyanthranilate Aldrich 5-hydroxyindole (29%

3-methylanthranilate Aldrich 7-methylindole (18%

5-methylanthranilate Aldrich 5-methylindole (23%

6-methylanthranilate Aldrich 4-methylindole (60%

3-chloroanthranilate Aldrich 7-chloroindole '*

5-iodoanthranilate Aldrich 5-iodoindole

4-fluoroanthranilate Aldrich 6-fluoroindole

3-hydroxyanthranilate Aldrich 7-hydroxyindole

3-methoxyanthranilate Aldrich 7-methoxyindole 3, 5-dimethylanthranilate Aldrich 5, 7-dimethylindole

*No standard for these indole products available, so quantitation of conversion rates is unavailable.

Table III shows the anthranilate substitutions screened using the tryptophan operon of E. coli C534/pGD007 (trpB/26) . All of these anthranilate substitutions were accommodated by the organism and produced the corresponding substituted indoles, also shown in Table III. Resulting compounds were confirmed by GC/MS. The conditions for the GC/MS are shown in Example 1.

Part B

Substituted anthranilates were also screened as described in Part A above, however, production was increased by incorporation of 1.25 grams of XAD-2 (Sigma) nonionic resin into each screen. At completion of each transformation, resin was filtered, washed and eluted with 6 mis ethanol before analysis on HPLC as above.

Table IV

Resulting Indole (B)

(% Anthranilate

Anthranilate (A) Conversion anthranilate indole (100%

4-chloroanthranilate 6-chloroindole (99%

5-chloroanthranilate 5-chloroindole (60%

6-chloroanthranilate 4-chloroindole (106%

5-fluoroanthranilate 5-fluoroindole (109%

5-bromoanthranilate 5-bromoindole (50%

5-hydroxyanthranilate 5-hydroxyindole (43%

3-methylanthranilate 7-methylindole (100%

5-methylanthranilate 5-methylindole (103%

6-methylanthranilate 4-methylindole (148%

3-chloroanthranilate 7-chloroindole (*

5-iodoanthranilate 5-iodoindole (*

4-fluoroanthranilate 6-fluoroindole (*

3-hydroxyanthranilate 7-hydroxyindole (*

3-methoxyanthranilate 7-methoxyindole (*

3, 5-dimethylanthranilate 5, 7-dimethylindole (*

*No standard for these indole products available, so quantitation of conversion rates is unavailable. *No standard for these indole products available, so quantitation of conversion rates is unavailable. All of the anthranilate substitutions shown in Table IV were accommodated by the organism and produced the corresponding substituted indole.

Example 3 14 Liter Fermentation Scale Up - Anthranilate to Indole

6 liters of minimal media (see Fermentation Media below) were combined with 2 liters of sterile soybean oil in a 14 liter fermentor. 500 mLs of a 6 hr E. coli FM5/pBMW culture (Table II) (see inoculum preparation below) were added to the fermentor aseptically. Following 3 hrs elapsed fermentor time, 10 mgs/mL filter sterilized IPTG (Sigma) were aseptically injected into the fermentor to induce the pBMW containing tryptophan operon genes. At the conclusion of the glucose ramp (see Fermentor Conditions below) , anthranilate was added to the fermentation as a slow drip from a pump at a rate of 1 gram/liter/hr (see Anthranilate Stock Preparation below) . 40 grams of anthranilate were added by the completion of the anthranilate feed. 50 mL samples were taken hourly during the anthranilate feed beginning at 0 hrs so as to monitor aqueous, as well as organic, constituents for anthranilate and indole. Samples were centrifuged as 1 mL aliquots in 1.7 mL microcentrifuge tubes for 5 minutes at 14,000 rpm. Organic and aqueous phases were separated and subsequently centrifuged a second time. 0.1 mLs of the organic phase were dissolved into 0.9 mLs hexane and placed in standard glass Altech HPLC sample vials. 0.5 mLs of the cell- free aqueous phase was diluted with an equal volume of TAI HPLC Mobile Phase A as in Example 1. The organic samples were analyzed on a normal phase HPLC system for indole concentration vs. standards (see Normal Phase HPLC Conditions below) . Aqueous samples were analyzed on the TAI HPLC for anthranilate and indole as in Example 1. Results from the anthranilate to indole fermentation show the gradual increase of indole in the organic phase and a corresponding decrease in anthranilate concentration in the aqueous phase. No anthranilate accumulates in the oil and only trace amounts of indole accumulate in the aqueous phase. Trace amounts of L- tryptophan were also observed in the aqueous phase. The conversion rate corresponds to slightly less than an equimolar ratio and the product was identified by GC/MS (see Example 1) as being indole.

Fermentation Media: 6 Liters

Pre-sterilization:

7.5 g/L K₂P0₄ monobasic

1.2 mL/L concentrated sulf ric acid

2 g/L citric acid anhydrous

5 g/L glucose

5 g/L MgS04.7H20

2 mL/L trace metals (see below) 1.2 mL/L Fe NH₃ citrate (from 270 g/L stock) 2.0 liters soybean oil Post-Sterilization:

6 mL thiamine.HCl (from 1M stock) antibiotic

NH₄OH to pH 7.0

Trace Metal Stock : 10 g/L Na₂S0₄ 2 g/L MnS0₄ . H₂0 2 g/L ZnCl₂ 2 g/L CoCl₃. H₂0 0 . 3 g/L CuS0₄ . 5H₂0 10 g/L FeS0₄ . 7H₂0

Fermentor Inoculum:

500 mL 6-8 hr rich media (see below) culture from 2 liter Fernbach at 37°C.

Rich Media:

16 g/L K₂HP0₄

14 g/L K₂P0₄

5 g/L (NH₄)₂S0₄

15 g/L yeast extract 30 g/L glucose 2 g/L MgS0₄.7H₂0 2 drops P-2000

Fermentor Conditions: exponential glucose feed ramp 0.4 g/min to 1.5 g/min (12 hrs)

DO controlled between 10 and 30% pH held at 7.0 with NH₄OH back pressure held at 0.2 Bar/PSI glucose limitation

Anthranilate Stock Preparation: 40 grams anthranilate

(Aldrich) were dissolved in 500 mLs MQH₂0 by slow addition of 50% NaOH to a pH of 12.5 and the volume brought to 1 liter with MQH₂0. Stock bottle was wrapped in foil to protect from potential reactions with light.

Normal Phase HPLC Conditions:

Column: PAC (4.6mm x 25cm) with 5 micron packing, commercially available from VWR. Mobile Phase: Hexane: ethyl acetate 50:50 (HPLC grade solvents) . Flow: 1.5 mL/minute

Injection: 25 microliters Detection: UV 270 nM

Example 4 14 Liter Fermentation Scale Up - 4-Chloroanthranilate to 6-Chloroindole

Chloroindole Production

40 grams 4-chloroanthranilate were added to a 14 liter fermentor prepared as in Example 3, except that the E. coli strain used was C534/pGD007 ( rpB/26) (Table II) . Results from this example showed the gradual production of β-chloroindole in the organic phase with a corresponding absence of 4- chloroanthranilate in the aqueous phase. The conversion again was slightly less than equimolar and the time required for complete conversion was slightly longer. No 4- chloroanthranilate accumulated in the organic phase and L- tryptophan was present in only trace amounts in the aqueous phase. The 6-chloroindole product was verified via GC/MS (see Example 1) .

Extraction of 6-Chloroindole from Soybean Oil A 6 grams/liter sample of 6-chloroindole in soybean oil was distilled according to standard Kugel Rohr procedure. Greater than 90% of the 6-chloroindole was recovered as a pure, crystalline product verified by GC/MS (see Example 1) after the first distillation. Using standard steam distillation technique, only 50% of the 6-chloroindole was recovered in one distillation.

Extraction of 6-Chloroindole from Emulsion

During fermentations containing soybean oil, approximately one third of the oil forms an emulsion. Emulsion was separated from a 35 mL sample from a fermentation by centrifugation at 10,000 rpm three times, with removal of oil and aqueous phases following each centrifugation. The resulting emulsion pellet was resuspended in an equal volume of hot 100% ethanol and placed in a hot water bath at approximately 80°C for approximately 10 minutes. 1 mL samples of the hot suspension were aliquoted to 1.7 mL microcentrifuge tubes and microfuged at 14,000 rpm for 5 minutes. The resulting two phases were oil and ethanol/aqueous phases. These two phases were analyzed via Normal Phase HPLC (see above) . Both phases contained equal amounts of 6-chloroindole and when combined, equalled the concentration of 6-chloroindole in the organic phase from the fermentor. This result further confirms the conclusion that an equimolar conversion of 4- chloroanthranilate to 6-chloroindole had occurred in the fermentation since, when the emulsion containing 6- chloroindole concentration was summed together with the oil concentration, quantitative values were obtained. Example 5 Reduction of Emulsion Post Biotransformation

Prior to ending and subsequently collecting fermentation contents, concentrated sulfuric acid was slowly added to the fermentor until the pH of the contents equilibrated at pH 2.0.

Following pH equilibration the entire contents of the fermentor were centrifuged as 1 liter quantities at 4,200 rpm.

This sulfuric acid treatment successfully reduced the volume of emulsion, giving a more quantitative volume of soybean oil for analysis and subsequent distillation without lysing cells or perturbing aqueous analysis.

Example 6 Small Scale Biotransformation of Tryptophan from Anthranilate

25 mgs anthranilate and 2.5 grams L-Serine were incubated with

3 grams (dry cell weight) E. coli C534/pGA007 rp in 25 mLs solution consisting of lOOmM KP0₄ buffer with 0.4% w/v D- glucose in 250 mL baffled shake flasks. The remainder of this example follows the procedure described in Example 1. The results showed the depletion of anthranilate from the aqueous buffer and the gradual production of tryptophan in a quantitative ratio. As in Example 1, the product was analyzed by GC/MS and confirmed as tryptophan. To do this, cell-free supernatant was acidified to pH 2.0 with IN HCl and run over a column of activated charcoal. The column was washed twice with pH 3.0 phosphate-citrate buffer and the tryptophan then eluted with hot Etch. The eluent was blown down and the residue subsequently derivatized by adding 0.5 mLs pyridine and 0.5 mLs BSTFA to the residue in a standard glass GC/MS sample vial. The vial was capped and placed in a heating block at 60°C for 20 minutes. This sample was then analyzed via the GC/MS procedure given in Example 1.

Example 7

Screen for Anthranilate Substitutions which the Tryptophan

Operon can Accommodate - Tryptophan Biotransformations

The substituted anthranilates of Table III were screened following the procedure of Example 6 using E. coli

C534/pGA007 rp. All substitutions from Table III were accommodated, producing the corresponding substituted tryptophans. GC/MS analysis was performed via the procedure of Example 1.

Example 8 Small Scale Indole-3-Acetic Acid from Anthranilate

Biotransformation 25 mg of anthranilate were incubated with 3 g (dry cell weight) E. coli C534/pGA007-trp/pCPJ3 cells (Table II) in 25 ml solution consisting of 100 mM KP0₄ buffer with 0.4% D- Glucose (w/v) and 0.2% L-Serine in a 250 ml baffled shake flask. The suspension was placed in a 37°C orbital-shaker incubator at 300 rpm. 1 ml samples of whole broth were withdrawn at 0 minutes and at 180 minutes post incubation. Each 1 ml sample was transferred to 1.7 ml microfuge tubes and subsequently centrifuged 2 minutes at 14,000 rpm. The cell- free supernatant was removed and diluted with an equal volume of HPLC Mobile Phase A as shown in Example 1, at pH 4.6. Samples were vortexed briefly (about iθ seconds) and 1 ml aliquots distributed to standard Altech glass HPLC sample vials for subsequent chromatography. Samples were analyzed for tryptophan, anthranilate and indole-3-acetic acid, as compared to standards purchased from commercially available sources using the TAI HPLC method described in detail in Example 1. The results from the GC/MS analysis agreed with the HPLC analysis, identifying the product as indole-3-acetic acid.

Example 9 Screen for Anthranilate Substitutions Which can be Accommodated in the Indole-3-Acetic Acid Biotransformations Part A

Substituted anthranilates ("A" in Table V) were screened for the purpose of identifying substitutions which can be accommodated by the enzymes of the tryptophan operon of E. coli and the P. savastanoi indole-3-acetic acid biosynthetic enzymes (tryptophan monooxygenase ( iaaM) and indole acetamide hydrolase iaaH) as present in the strain C534/pGA007- trp/pCPJ3. Experimental procedure followed exactly that found in Example 8.

In many cases product standards were unavailable from commercial sources. In these cases, transformation broths were analyzed using TLC for identification of anthranilates and corresponding indole-3-acetic acids by comparison of R_f values. One ml cell free broth supernatant was first acidified to pH -2 with concentrated HCl. This was extracted with 200 u isopropyl acetate. This extract was then applied via capillary tubing to Silica Gel MLF TLC plates (AnalTech Uniplates) . These plates were exposed to a mobile phase consisting of 100% methyl-tertiary butyl ether until the solvent front reached an appropriate height (empirically determined) . Bands were then visualized by exposure to standard Ehrlich's reagent. Under these conditions, anthranilate bands turn yellow, and indole-3-acetic acid bands turn purple. In circumstances where there was no standard available, expected R_f values were calculated from prior art values of various substituent effects on TLC retention.

Table V

λ B

(R is as defined herein) Substituted Anthranilates Screened

Anthranilate (A) Commercial Supplier Resulting Indole Acetic

Acid(B) anthranilate Aldrich indole acetic acid^a

4-chloroanthranilate Aldrich 6-chloroindole acetic acid^a*

5-chloroanthranilate Aldrich 5-chloroindole acetic acid^a*

6-chloroanthranilate Lancaster 4-chloroindole acetic acid^c*

4-fluoroanthranilate Aldrich 6-fluoroindole acetic acid^a*

5-bromoanthranilate Aldrich 5-bromoindole acetic acid³

5-hydroxyanthranilate Aldrich 5-hydroxyindole acetic acid^b

3-methylanthranilate Aldrich 7-methylindole acetic acid^d*

5-methylanthranilate Aldrich 5-methylindole acetic acid^a*

5-iodoanthranilate Aldrich 5-iodoindole acetic acid³*

3-hydroxyanthranilate Aldrich 7-hydroxyindole acetic acid³*

a- product identified by GC/MS, TLC, and HPLC b- product identified by HPLC c- product identified by GC/MS, and HPLC d- product identified by TLC, and HPLC

*No standard for these indole-3-acetic acid products available, so quantitation of conversion rates is unavailable; in these cases, data from all analyses were pooled, as well as substrate utilization rates for production estimates. Example 10 Glucose to Indole-3-Acetic Acid - 14 Liter Fer entator Scale

Six liters of minimal media (see Fermentation Media set forth in Example 3) were added to a 14 liter fermentor. Five hundred ml of a 6 hr E. coli C534/pBE7/pCPJ3 culture (Table II) (see inoculum preparation Example 3) were added to the fermentor aseptically. Following 3 hrs elapsed fermention time (EFT), 10 mg/ml filter sterilized IPTG (Sigma) were aseptically injected into the fermentor to induce the pBE7 containing tryptophan operon genes. 50 ml samples were taken hourly during the glucose feed beginning at 0 hr to monitor aqueous constituents for tryptophan and indole-3- acetic acid. Samples were centrifuged as 1 ml aliquots in 1.7 ml microcentrifuge tubes for 5 minutes at 14,000 rpm. 0.5 ml of the cell-free aqueous phase was diluted with an equal volume of TAI HPLC Mobile Phase A as in Example 8. Aqueous samples were analyzed on the TAI HPLC for tryptophan and indole-3-acetic acid as in Example 8. Results from the glucose to indole fermentation show the gradual increase of indole-3-acetic acid in the aqueous broth. No tryptophan was seen to accumulate, and indole-3-acetic acid accumulated to 3 g/1 in 20 hrs EFT.

Claims

WHAT IS CLAIMED IS:

1. A process for the biosynthetic production of indole-3- acetic acids and substituted analogs thereof, the process comprising:

a) culturing a microorganism capable of converting a substituted or unsubstituted anthranilate to a corresponding substituted or unsubstituted tryptophan and capable of converting said substituted or unsubstituted tryptophan to a corresponding substituted or unsubstituted indole-3- acetic acid, under suitable conditions in the presence of an anthranilate of the formula:

wherein R is H, halogen, OH, C1-C5 alkyl, C1-C5 alkoxy,

N0₂, NH₂, COOH, CN, sulfur, S0₃, or S0₄; to produce a corresponding substituted indole-3-acetic acid of the formula:

wherein R is as defined above; and b) recovering the indole-3-acetic acid.

2. A process of Claim 1 wherein R is H, halogen, OH, C1-C5 alkyl or C1-C5 alkoxy.

3. A process of Claim 2 wherein R is H, halogen, OH, or CH₃.

4. A process of Claim 1 wherein the microorganism comprises genes coding for the tryptophan operon.

5. A process of Claim 4 wherein the genes for the tryptophan operon are carried on a multicopy plasmid used to transform the microorganism.

6. A process of Claim 4 wherein the genes for the tryptophan operon have been integrated into the chromosome of the microorganism.

7. A process of Claim 1 wherein the microorganism further comprises feedback resistance of tryptophan pathway enzymes.

8. A process of Claim 7 wherein the microorganism comprises trpE^ibr .

9. A process of Claim 4 wherein when R is H the microorganism further comprises genes coding for DAHP synthetase, tryptophan monooxygenase and indole acetamide hydrolase.

10. A process of Claim 4 wherein when R is other than H the microorganism further comprises genes coding for tryptophan monooxgenase and indole acetamide hydrolase.

11. A process of Claim 1 comprising culturing the microorganism E. coli C534/pCPJ3/pBE7 in the presence of anthranilate and recovering the resulting indole-3-acetic acid.

12. A process of Claim 1 for the production of substituted indole-3-acetic acid comprising culturing the microorganism E. coli C534/pGA007 rp/pCPJ3 in the presence of a 3-, 4-, 5- or 6-substituted anthranilate and recovering the resulting substituted indole-3-acetic acid.

13. A process for the production of indole-3-acetic acid or a 4-, 5-, 6-, 7-substituted analog thereof in a suitable host cell using a renewable carbon source, the process comprising: a) transforming the host cell with DNA comprising genes coding for one or more enzymes selected from the group consisting of tryptophan synthase, phosphoribosyl anthranilate isomerase-indole- glycerol phosphate synthase, anthranilate phosphoribosyl transferase, anthranilate synthase DAHP synthase, tryptophan monooxygenase and indole acetamide hydrolase; provided that the gene coding for DAHP synthatase is not necessary for the production of a substituted indole-3-acetic acid; and b) culturing the transformed host cell under suitable conditions in the presence of the renewable carbon source.

14. A process of Claim 13 wherein the genes are trpA, trpB, trpC, trpD, trpE, aroG, IaaM and IaaH.