Overproduction of indole acetic acid in Azospirillum lipoferum using the Escherichia coli trp operon.

A recombinant plasmid carrying the trp operon from Escherichia coli, which synthesizes tryptophan from chorismate, was constructed by using a broad host range plasmid vector pRK290; a mutant trp plasmid for tryptophan overproduction was then selected. The physiological, biochemical, and genetic properties of the Azospirillum lipoferum KY6, a potential nitrogen fixer of rice, harbouring the recombinant trp plasmid pMJC1 and its mutant pMJC101, were compared with those of the wild-type bacteria. Anthranilate synthetase is known to be the trpE gene product which plays a key role in the regulatory step in the feedback control of tryptophan biosynthesis. The enzyme activity of the Azospirillum lipoferum KY6 carrying pMJC1 or pMJC101 was respectively 7- and 30-fold higher than that of the wild type in the presence of 10(-4)M tryptophan. As expected, the amount of tryptophan biosynthesis in A. lipoferum KY6 (pMJC101) was increased approximately 100-fold as compared with the wild type, which led to overproduction of indole acetic acid even without addition of exogenous tryptophan. Moreover, the recombinant trp plasmid was fairly stable in A. lipoferum KY6 host, showing only 25% loss of the plasmid itself or the trp insert after 40 generations.