Mechanism of suppression of pyruvate and acetolactate formation by use of yeast of modified mitochondrial transportation system

Technical Session 16: Yeast III Session
Hiroshi Kitagaki, National Saga University

ABSTRACT: Residual pyruvate and acetolactate during alcoholic fermentation leads to synthesis of off-flavor diacetyl. Therefore, suppression of these substances during alcoholic fermentation is desirable. In order to circumvent this problem, we came up with the idea of modifying the mitochondrial transportation system, fortifying the transportation of pyruvate from the cytosol to the mitochondria, and decreasing the amount of pyruvate and acetolactate. To accomplish this, we isolated mutants of sake yeast resistant to ethyl alpha-trans-cyanocinnamate, an inhibitor of mitochondrial pyruvate transport. The brewery yeast of sake, the Japanese traditional rice wine, was used as the parent strain. This strain indeed exhibited a decreased amount of pyruvate and acetolactate during sake brewing on a factory scale. This was the first success of development of a brewery yeast that produces a decreased amount of pyruvate and acetolactate without deteriorated fermentation ability. However, although we supposed that pyruvate transportation from the cytosol to the mitochondria during alcoholic fermentation lowered the pyruvate content, its mechanism had not been elucidated. Therefore, we constructed sake yeasts that overexpress various mitochondrial transporters and investigated the resistance of the strains to ethyl alpha-trans-cyanocinnamate. As a result, the strain overexpressing the mitochondrial ATP/ADP translocator gene AAC1, exhibited resistance to ethyl alpha-trans-cyanocinnamate. This strain also exhibited a low pyruvate-producing ability during sake brewing. These results suggest that transportation of ATP from the cytosol to the mitochondria enhances pyruvate turnover within mitochondria during alcoholic fermentation. It can also be inferred that since an electron transport system using molecular oxygen does not occur during alcoholic fermentation, ATP within mitochondria is depleted, and mitochondria need to import ATP from the cytosol, where ATP is synthesize through glycolysis. This research is the first to propose a role of ATP transport from the cytosol to the mitochondria to modify fermentation characteristics and suggest a novel strategy for developing brewery yeasts that produce decreased amounts of pyruvate and acetolactate.

Hiroshi Kitagaki received a Ph.D. degree from the University of Tokyo. He began employment as a brewing analyst at the National Taxation Bureau in 1995. He moved to the National Research Institute of Brewing as a researcher in 2001. He worked as a visiting researcher at the Medical University of South Carolina from 2005 to 2006. He is now an associate professor at the National Saga University. He is the president of the Symbiotic Microbial Fermentation Engineering Forum and has been selected as an associate member of the Science Council of Japan and a program officer of the Ministry of Education, Culture, Sports, Science and Technology, Japan. He has received the Young Scientists’ Award in the Commendation of Science and Technology from the Minister of Education, Culture, Sports, Science and Technology, Japan; The Foundation of Agricultural Sciences of Japan; and The Society for Biotechnology, Japan.