The efficient production of the textile dye indigo by fermentation has been a goal since the early 1980's when the first bacterial strains capable of this synthesis were constructed. We report here the development of a recombinant microorganism that directly synthesizes indigo from glucose. This construction involved the cloning and genetic manipulation of at least 9 genes and modifications of the fermentation medium to help stabilize the biosynthetic activity. Directed genetic changes in two operons caused significant increases in reaction rates and in the stability of the catalytic enzymes. This example of whole cell catalysis by a recombinant Escherichia coli represents a novel and environmentally sound approach to the synthesis of a high value specialty chemical.

The ability of P.putida, strain NCIB 9816, to grow with naphthalene (Nah+) and salicylate (Sal+) is correlated with the presence of an 83 kilobase (kb) conjugative plasmid (pDTG1). The genes encoding the upper pathway (Nah->Sal) for naphthalene degradation are located on a 15 kb EcoRI fragment which was cloned into pKT230. The resulting recombinant, pDTG113, was nick-translated and used as a radioactive probe to investigate nucleotide sequence homology between the naphthalene-utilizing organisms, P.putida G7, P.putida NP, and strain PL6. Each of these bacterial strains were isolated from different locations at different times. The results show that all of these organisms contain closely related genes that are involved in naphthalene metabolism.

The ability of P.putida NCIB 9816 to grow with naphthalene (Nah+) and salicylate (Sal+) is correlated with the presence of an 83 kilobase (kb) conjugative plasmid, pDTG1. Derivatives of pDTG1 were obtained from cells after exposure to halogenated analogs of naphthalene or salicylate. The selection of mutants having a Nah−Sal− or a Nah−Sal+ phenotype could be enhanced by the addition of triphenyltetrazolium chloride to the indicator medium. Structurally modified plasmids were characterized by restriction endonuclease digestion and Southern hybridization experiments. The region of pDTG1 DNA that encodes the enzymes responsible for the conversion of naphthalene to salicylate was identified. The structural changes in mutant plasmids were correlated with the absence of essential enzymatic activities.

In pseudomonads, naphthalene is catabolized in a series of reactions to salicylic acid, which is further degraded via the catechol meta-cleavage, ortho-cleavage, or gentisic acid pathway to Krebs cycle intermediates. The naphthalene catabolic genes have been located on self-transmissible plasmids, in most cases, and implicated to have chromosomal locations in other cases. The best-studied naphthalene catabolic plasmid is NAH7. It carries two operons, one of which enables the host to utilize naphthalene and the other to utilize salicylate as a carbon and energy source. The product of another NAH7 gene, nahR, is required to turn on both operons in the presence of the inducer, salicylate. Several different naphthalene and salicylate catabolic plasmids have been shown to share sequence homology with NAH7. These plasmids can undergo structural alterations involving insertions and deletions during conjugations and changes in nutritional conditions. Available evidence suggests that salicylate catabolic plasmids can form from the naphthalene catabolic plasmids by structural alterations of the plasmid DNA. The gene organization and regulation, as well as the genetic instability of the naphthalene catabolic plasmids, are reminiscent of the TOL plasmids and suggest that the naphthalene catabolic plasmids and other catabolic plasmids may have evolved in a short period of time by acquiring and modifying preevolved gene clusters from host chromosomes or other plasmids.

Pseudomonas diminuta strain MG hydrolyzes parathion to diethylthiophosphoric acid and p-nitrophenol. The esterase responsible for this reaction is encoded by a gene located on a plasmid termed pCMS1. The gene was cloned into plasmid pBR322 and the broad host range cloning vector pKT230. Enzyme activity was detected in Escherichia coli strains that contained the recombinant plasmids. A 1.5 kilobase BamHI fragment with single restriction sites for SalI, PstI and XhoI was shown to direct the synthesis of the enzyme. The 1.5 kilobase BamHI fragment was inserted into the high expression vector pUC7, and the resulting recombinant, pCMS40, was used to construct pKT230 derivatives containing the parathion hydrolase gene. Subsequent transfer of the recombinant plasmids into a cured derivative of P. diminuta MG and a p-nitrophenol-utilizing strain of Pseudomonas resulted in the isolation of transconjugants that exhibited parathion hydrolase activity. The highest enzyme activity was observed with P. diminuta MG.