Microbial production of indigo

#The present invention relates to the biosynthetic production of dye stuff, and particularly indigo, from microorganisms. While the biosynthetic production of indole and tryptophan (both precursors to indigo) have been previously described, the present invention provides an efficient, commercially feasible biosynthetic production system whereby an inhibitory compound in the synthesis of indigo, isatin, is reduced or otherwise removed from fermentation broth. The removal of this inhibitory compound during production of indigo provides enhanced production of the desired end product indigo and/or improved characteristics of the indigo so produced. Furthermore, the present invention prevents or reduces the production of indirubin, a red dyestuff which is a by-product of biosynthetic indigo production.

#The blue dye indigo is one of the oldest dyestuffs known to man. Its use as a textile dye dates back to at least 2000 BC. Until the late 1800s indigo, or indigotin, was principally obtained from plants of the genus Indigofera, which range widely in Africa, Asia, the East Indies and South America. As the industrial revolution swept through Europe and North America in the 1800s, demand for the dye's brilliant blue color lead to its development as one of the main articles of trade between Europe and the Far East. In 1883 Alfred von Baeyer identified the formula of indigo: C16H10N2O2. In 1887 the first commercial chemical manufacturing process for indigo was developed. This process, still in use today, involves the fusion of sodium phenylglycinate in a mixture of caustic soda and sodamide to produce indoxyl. The process' final product, indoxyl, oxidized spontaneously to indigo by exposure to air.

#Current commercial chemical processes for manufacturing indigo result in the generation of significant quantities of toxic waste products. Obviously, a method whereby indigo may be produced without the generation of toxic by-products is desirable. One such method which results in less toxic by-product generation involves indigo biosynthesis by microorganisms.

#FIG. 1 shows the diagrammatic methodology used to clone the isatin hydrolase gene from P. putida strain WW2.

#FIG. 2 shows the methodology used to subcdone the isatin hydrolase gene from P. putida strain WW2.

#FIGS. 3a-f shows the nucleotide sequence of the isatin hydrolase gene (Seq ID No. 1) and the deduced amino acid sequence (Seq ID No. 2), along with the 5′ and 3′ untranslated sequence (Seq. ID No. 1 1-144 bp and 924-1006 bp, respectively) and polylinker sequence of the cloning vector.

#Presently available methods of biosynthetic indigo production may employ the bioconversion of indole to indigo utilizing an aromatic mono- or dioxygenase like TMO or NDO, respectively, or other oxygenase enzymes such as an oxidase from Rhodococcus, as described by S. Hart, K. R. Koch and D. R. Woods [(1992) “Identification of indigo-related pigments produced by Escherichia coli containing a cloned Rhodococcus gene,” J. Gen. Microbiology 138:211-216]. These processes necessitate the addition of indole to the culture medium, as no intracellular indole accumulation occurs in such systems. However, indole added to the culture medium may be toxic to microorganisms. E. coli growth may be inhibited when indole is present in media. Bang et al. [(1983) Biotechnology and Bioengineering 25:999-1011] described the effects of adding exogenous indole to E. coli being grown in shake flasks in minimal media. They found that while concentrations of up to 0.025% slowed bacterial growth, the cells acclimated to the presence of indole over time. However, 0.03% indole severely limited growth with no apparent acclimation, and indole concentrations above 0.04% prohibited growth altogether. In addition, Bang et al., supra, found that L-tryptophan synthesis was inhibited when indole was added at concentrations in excess of 0.2 g/L.

#To avoid the inherent limitations of indigo synthesis due to the need for media supplementation with indole, systems capable of endogenous indole biosynthesis have been developed. One such system may employ transferring an exogenous DNA molecule encoding a DNA sequence for a trp operon, modified so as to promote indole production and accumulation (hereinafter “modified trp operon”), into a recombinant host microorganism already capable of expressing an oxygenase (i.e., NDO) activity. Such a system would allow for the production of indigo from glucose or other carbon sources. Optimally, such a system would efficiently convert the endogenously produced indole to indoxyl in a manner avoiding intracellular indole accumulation. A detailed description of this system is found in U.S. Pat. No. 5,374,543, incorporated herein. Such a system in combination with the isatin-removing methods of the present invention is a preferred embodiment of the improved process for indigo production described herein.

#Generally, as shown in U.S. Pat. No. 5,374,543, certain point mutations in trpB genes, particularly at position 382 in the trpB gene (designated trpB382), resulted in stable mutants of the trpb. Such mutants lead to enhanced indole flux in the host cells and such mutants are preferred for the production of indigo as described herein in combination with isatin-removing activity. Furthermore, expression of these tryptophan synthase mutants in conjunction with an aromatic dioxygenase such as NDO resulted in the intracellular production of indole and its conversion to indoxyl, which spontaneously oxidized to indigo.