Cloning and characterization of genes responsible for m-fluoro-D,L-phenylalanine resistance in Brevibacterium lactofermentum

Abstract

Two kinds of 3-deoxy-D-arabino-hepturosonate-7-phosphate (DAHP) synthase genes were cloned from an L-Phe-producing mutant of Brevibacterium lactofermentum, AJ11957, which was resistant to m-fluoro-D,L-phenylalanine (mFP) and p-fluoro-D,L-phenylalanine (pFP) and which had DAHP synthase free from feedback inhibition. Both genes were cloned using a vector plasmid, pAJ1844, and the resulting recombinant plasmids were named pAR1 and pAR2. They had different structures on the restriction maps. Both plasmids, pAR1 and pAR2, conferred mFP and pFP resistance and L-Phe and L-Tyr productivity on a wild-type strain of B. lactofermentum, AJ12036. The degrees of L-Phe-analogue resistance and aromatic amino acid productivity of the pAR1-transformant were larger than those of the pAR2-transformant. The introduction of pAR1 to AJ12036 resulted in the alteration of DAHP synthase activity from a feedback-sensitive mechanism to a feedback-insensitive one accompanied by an elevated level of specific activity. A DAHP synthase deficient mutant of Escherichia coli was complemented by pAR2, but not by pAR1. Characteristics of the two kinds of DAHP synthase genes are discussed.