Redirection of pyruvate catabolism in Lactococcus lactis by selection of mutants with additional growth requirements

Based on requirements for acetate or lipoic acid for aerobic (but not anaer obic) growth, Lactococcus lactis subsp. lactis mutants with impaired pyruva te catabolism were isolated following classical mutagenesis. Strains with d efects in one or two of the enzymes, pyruvate formate-lyase (PFL), lactate dehydrogenase (LDH) and the pyruvate dehydrogenase complex (PDHC) were obta ined. Growth and product formation of these strains were characterized. A P FL-defective strain (requiring acetate for anaerobic growth) displayed a tw o-fold increase in specific lactate production compared with the correspond ing wild-type strain when grown anaerobically. LDH defective strains direct ed 91-96% of the pyruvate towards a-acetolactate, acetoin and diacetyl prod uction when grown aerobically in the presence of acetate and absence of lip oic acid (a similar characteristic was observed in an LDH and PDHC defectiv e strain in the presence of both acetate and lipoic acid) and more than 65% towards formate, acetate and ethanol production under anaerobic conditions . Another strain with defective PFL and LDH was strictly aerobic. However, a variant with strongly enhanced diacetyl reductase activities (NADH/NAD(+) dependent diacetyl reductase, acetoin reductase and butanediol dehydrogena se activities) was selected from this strain under anaerobic conditions by supplementing the medium with acetoin. This strain is strictly aerobic, unl ess supplied with acetoin.