METABOLIC ENGINEERING OF LACTOCOCCUS-LACTIS - INFLUENCE OF THE OVERPRODUCTION OF ALPHA-ACETOLACTATE SYNTHASE IN STRAINS DEFICIENT IN LACTATE-DEHYDROGENASE AS A FUNCTION OF CULTURE CONDITIONS

The als gene for alpha-acetolactate synthase of Lactococcus lactis MG1 363 was cloned on a multicopy plasmid under the control of the inducib le L. lactis lacA promoter. More than a hundredfold overproduction of alpha-acetolactate synthase was obtained in L. lactis under inducing c onditions as compared with that of the host strain, which contained a single chromosomal copy of the als gene. The effect of alpha-acetolact ate synthase overproduction on the formation of end products in variou s L. lactis strains was studied under different fermentation condition s. Under aerobic conditions and with an initial pH of 6.0, overexpress ion of the als gene resulted in significant acetoin production that am ounted to more than one-third of the pyruvate converted. However, the effect of the alpha-acetolactate synthase overproduction was even more pronounced in the lactate dehydrogenase-deficient strain L. lactis NZ 2700, Anaerobic cultivation of this strain resulted in a doubling of t he butanediol formation of up to 40% of the converted pyruvate. When c ultivated aerobically at an initial pH of 6.8, overexpression of the a ls gene in L. lactis NZ2700 resulted in the conversion of more than 60 % of the pyruvate into acetoin, while no butanediol was formed. Moreov er, at an initial pH of 6.0, similar amounts of acetoin were obtained, but in addition, approximately 20% of the pyruvate was converted into butanediol, These metabolic engineering studies indicate that more th an 80% of the lactose can be converted via the activity of the overpro duced alpha-acetolactate synthase in L. lactis.