(599d) Improved L-Lactic Acid Production By Regulating Alcohol Dehydrogenase in the Living Culture of Rhizopus Oryzae

Authors: 
Thongchul, N., Chulalongkorn University
Thitiprasert, S., Institute of Biotechnology and Genetic Engineering, Chulalongkorn university

Rhizopus oryzae is becoming more important due to its ability to produce an optically pure L-lactic acid. However, fermentation by this fungus usually suffers from low yield due to the presence of ethanol as a major byproduct. Previous studies revealed ethanol production as the result of limited oxygen during fermentation. Various techniques including cell immobilization have been applied; however, none of them fully controlled ethanol production during fermentation by R. oryzae. 2 key enzymes are responsible for ethanol production. Pyruvate decarboxylase (PDC) converts the key intermediate pyruvate to acetaldehyde (ADH) while alcohol dehydrogenase reversibly converts acetaldehyde to ethanol. Limited ethanol production in the living cell of R. oryzae by regulating ADH was observed in the shake flask culture. ADH was regulated by adding the compounds that showed the competitive inhibition to ADH in the in vitro test. Among the tested compounds, 1,2-diazole and 2,2,2-trifluoroethanol exhibited the high affinity to ADH resulting in the improved lactate yield of about 38% increasing from that from the control fermentation. While fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4’-dithiodipyridine, and 4-hydroxymercury benzoic acid were present. From the enzymatic assay, it was found that both 1,2-diazole and 2,2,2-trifluoroethanol not only strongly affected ethanol formation but they also indirectly regulated lactate production as observed by the decreasing affinity for glucose flux toward lactate and ethanol production. The increase in both ethanol and lactate formation rates revealed 1,2-diazole and 2,2,2-trifluoroethanol not only regulated the reversible redox reaction by ADH, but they also caused the dynamic change in the conversion of all metabolites in the living R. oryzae in order to maintain the balanced flux for cellular growth and maintenance.