Two single-base-pair substitutions causing desensitization to tryptophan feedback inhibition of anthranilate synthase and enhanced expression of tryptophan genes of Brevibacterium lactofermentum

Abstract

A 5-fluorotryptophan-resistant mutant, termed 1041, was isolated from Brevibacterium lactofermentum AJ12036. The anthranilate synthase of 1041 was insensitive to feedback inhibition by tryptophan, and the specific activities of the anthranilate synthase and anthranilate phosphoribosyltransferase of 1041 were 29-and 23-fold higher than those in parental strain AJ12036, respectively. A single-base change (adenine to cytosine) that resulted in a Ser-to-Arg substitution was found in the trpE structural gene of 1041. This substitution was identified as the cause of the desensitization to feedback inhibition by tryptophan of anthranilate synthase in 1041. Another substitution (guanine to adenine) was found at a position in which a mutation would destabilize the p-independent terminator structure within the putative attenuator. The enhanced synthesis of tryptophan enzymes in 1041 could be caused by this substitution in the attenuator.Brevibacterium lactofermentum is an industrially important gram-positive bacterium used for the production of various amino acids. We have focused our efforts on the tryptophan biosynthesis of this microorganism, because tryptophan is an important ingredient in medicines and animal feed as an essential amino acid.Tryptophan biosynthesis in Brevibacterium spp. has been found to be regulated by two mechanisms, feedback inhibition by tryptophan of anthranilate synthase (AS) (6), anthranilate phosphoribosyltransferase (10), and tryptophan synthase (9) and repression by tryptophan of the synthesis of AS and the other tryptophan enzymes (8), as is the case for Escherichia coli and Bacillus subtilis. Furthermore, analysis of the nucleotide sequence of the B. lactofermentum trp operon revealed the presence of operator-and attenuatorlike sequences (3, 5). Thus, we have assumed that repression of the synthesis of tryptophan enzymes in B. lactofermentum is carried out by both the repressor-operator system and the attenuation system, as in E. coli (13). To breed improved tryptophan-producing strains, it is necessary to remove these regulation mechanisms. For this purpose, we have isolated a 5-fluorotryptophan-resistant (4,000 ,g/ml) spontaneous mutant from wild-type B. lactofermentum AJ12036.The spontaneous mutant, termed 1041, was revealed to have an altered AS, which was fully active even in the presence of 10 mM tryptophan, while the activity of the wild-type AS from AJ12036 under the same conditions was less than 1% of that in the absence of tryptophan. Furthermore, the specific activities of AS and anthranilate phosphoribosyltransferase in 1041 were 29-and 23-fold higher than those in parental strain AJ12036, respectively. Determination of the mutation site in 1041 should be useful in clarifying the molecular mechanisms of the regulation of tryptophan biosynthesis in B. lactofermentum.Recombinant plasmids ptrpE97 and ptrpE36 carry the trp operon of 1041 and the 5'-proximal region of the trp operon of AJ12036, respectively (2) (Fig.

Analysis of feedback-resistant anthranilate synthases from Saccharomyces cerevisiae

“…Mutations in the N-terminal part of anthranilate synthase causing feedback resistance have been characterized in Brevibacterium lactofermentum, E. coli, and Salmonella typhimurium (5,16,17). In those studies, several mutations revealed a particular element, a Leu-Leu-Glu-Ser (LLES) stretch, which is one of the few highly conserved sites in the upstream part of the large subunit of anthranilate synthase.…”

Towards bacterial strains overproducing l-tryptophan and other aromatics by metabolic engineering

“…The distribution and organization of these biosynthetic genes on the genome are different from those in E. coli, whereas the gene arrangement of the trp operon (NCgl2927-2932) of C. glutamicum closely resembles that of E. coli. The trp operons of both bacteria have been extensively studied for its organization and regulation (Umbarger 1978;Somerville 1983;del Real et al 1985;Katsumata and Ikeda 1993;Matsui et al 1986;Matsui et al 1987aMatsui et al , 1987bMatsui et al , 1987cSano and Matsui 1987).…”

Amplification of trpEG: adaptation of Buchnera aphidicola to an endosymbiotic association with aphids.

“…If the endosymbiont overproduces tryptophan, modifications would have to occur to permit tryptophan synthesis in the presence of elevated levels of this amino acid. Among the possible adaptations are constitutive synthesis of the tryptophan biosynthetic enzymes and modifications of AS so as to overcome the effects of feedback inhibition by tryptophan (27). In studies of directed evolution, mutations to constitutivity are common (28); in addition, in some species the tryptophan biosynthetic pathway appears to be constitutive (20,29 *To whom reprint requests should be addressed.…”