Effects of enhanced lysine ?-aminotransferase activity on cephamycin biosynthesis in Streptomyces clavuligerus

Abstract

A recombinant strain of S. clavuligerus (LHM100) that contains an additional copy of the gene (lat) encoding lysine epsilon-aminotransferase (LAT) was analyzed and compared to the wild-type for intracellular concentrations of primary metabolites involved in cephamycin C biosynthesis. This strain had been shown previously to produce higher levels of the antibiotic because of increased levels of LAT, a rate-limiting enzyme involved in the production of alpha-aminoadipic acid. The results showed that the overall growth kinetics of the two strains were comparable, including the intracellular concentrations of cysteine, valine and lysine. In contrast, 60% higher antibiotic production was observed in LHM100, which reflected a significant temporal variation in specific metabolite production rate. The time profile of LAT activity was consistently higher in LHM100; however, alpha-aminoadipic acid levels showed unexpected variation during the growth cycle. These results support the proposal that rate-limiting enzymes in cephamycin C biosynthesis are temporally controlled, and indicate that optimization of metabolite production will require differential overexpression of several biosynthetic genes.

Precursor and Cofactor as a Check Valve for Cephamycin Biosynthesis in Streptomyces clavuligerus

“…The chemically defined medium of Aharonowitz and Demain was used for all experiments (Aharonowitz and Demain, 1978). Batch cultures were carried out at 29 °C and 250 rpm in baffled Erlenmeyer shake flasks as described previously (Malmberg et al, 1995; Zhang et al, 1989). Spores (10 8 ) were innoculated into 50 mL of seed medium in a 250 mL Erlenmeyer flask.…”

“…Analysis of Intracellular Amino Acids and α‐Ketoglutarate. The amino acid concentration in the deproteinated cell extract was determined by HPLC after derivatization with o ‐pthaldialdehyde as described previously (Malmberg et al, 1995). A protein content of 0.55 mg of protein/mg of dry cell weight and a specific cell volume of 2 μL/mg dry cell weight was assumed (Malmberg and Hu, 1991), to convert the amino acid measurement to an intracellular concentration.…”

Contributions of Microorganisms to Industrial Biology

“…Two enzymes of cephamycin biosynthesis, d-(L-a-aminoadipyl)-L-cysteinyl-Dvaline synthetase (ACVS) and lysine-e-aminotransferase (Lat) were found to be limiting in Streptomyces clavuligerus. Cloning of lat increased cephamycin C production by 2-to 5-fold [70]. Replacement of the native promoter of the ACVSencoding gene in Aspergillus nidulans increased penicillin production 30-fold [71].…”

Biochemical Engineering of Natural Product Biosynthesis Pathways

“…An excellent example of pathway-specific metabolic engineering comes from the detailed metabolic flux analysis of precursors required for cephamycin and cephalosporin biosynthesis in Streptomyces clavuligerus by David Sherman and his colleagues. Finding that :-aminoadipic acid was a limiting precursor, they cloned an extra copy of the lysine =-aminotransferase (LAT) gene into the chromosome of S. clavuligerus, achieving both increased enzyme activity and significantly greater levels of the desired product cephamycin C (Malmberg et al, 1993(Malmberg et al, , 1995Khetan et al, 1996).…”