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Cloning of a transaminase gene of Citrobacter freundii and the gene dosage effect on L-phenylalanine production through transaminase reaction.

In a preceding paper (Anazawa et al., 1991), we reported a process for the enzymatic production of Lphenylalanine from phenylpyruvic acid and ammonium fumarate. In this process, the cells of Citrobacter freundii were used as the source of two enzymes for a coupling reaction with aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) and transaminase(s) (Fig. 1). Oxaloacetic acid produced as a byproduct in the transaminase reaction was rapidly decomposed enzymatically and/or nonenzymatically to pyruvic acid, after which the reaction inclined toward L-phenylalanine production. Under adequate conditions for production, the transaminase reaction was the limiting one rather than that of aspartase. We then tried to clone the transaminase gene with the aim to improve the process for L-phenylalanine production. Some genes of C. freundii have been expressed in Escherichia coli (Blumenberg and Yanofsky, 1982; Karube et al., 1984; Yamamoto et al., 1983). Moreover, it is known that the tyrB gene-directed and ilvE gene-directed transaminases of E. coli have broad specificities on several amino acids including L-phenylalanine (Gelfand and Steinberg, 1977; Jensen and Calhoun, 1981) (Table 1) and are important for enzymatic L-phenylalanine production with this bacterium (Calton et al., 1986). Next, we considered that if C.