Isolation of sake yeast strains possessing various levels of succinate- and/or malate-producing abilities by gene disruption or mutation
ABSTRACT Succinate and malate are the main taste components produced by yeast during sake (Japanese alcohol beverage) fermentation. Sake yeast strains possessing various organic acid productivities were isolated by gene disruption. Sake fermented using the aconitase gene (ACO1) disruptant contained a two-fold higher concentration of malate and a two-fold lower concentration of succinate than that made using the wild-type strain K901. The fumarate reductase gene (OSM1) disruptant produced sake containing a 1.5-fold higher concentration of succinate as compared to the wild-type, whereas the α-ketoglutarate dehydrogenase gene (KGD1) and fumarase gene (FUMI) disruptants gave lower succinate concentrations. The Δkgd1 disruptant exhibited lower succinate productivity in the earlier part of the sake fermentation, while the Δfum1 disruptant showed lower succinate productivity later in the fermentation, indicating that succinate is mainly produced by an oxidative pathway of the TCA cycle in the early phase of sake fermentation and by a reductive pathway in the later phases. Sake yeasts with low succinate productivity and/or high malate productivity was bred by isolating mutants unable to assimilate glycerol as a carbon source. Low malate-producing yeasts were also obtained from phenyl succinate-resistant mutants. The mutation of one of these mutant strains with low succinate productivity was found to occur in the KGD1 gene. These strains possessing various succinate- and/or malate-producing abilities are promising for the production of sake with distinctive tastes.
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ABSTRACT: Biotechnological production of chemicals from renewable feedstocks offers a sustainable alternative to petrochemistry. Understanding of the biology of microorganisms and plants is increasing at an unprecedented rate and tools with which these organisms can be engineered for industrial application are becoming ever more powerful. However, biotechnological production processes that are cost-competitive with petrochemistry still have to be developed for many types of chemicals. In this thesis, the ability of Saccharomyces cerevisiae (baker’s yeast) to produce C4-dicarboxylic acids (fumarate, malate and succinate) is investigated. These acids, currently produced from oil in relatively small quantities and mainly applied for human consumption, have interesting properties for roles as commodity platform chemicals. First, S. cerevisiae was metabolically engineered for malate production via a pyruvate carboxylase-dependent pathway. While titers of nearly 60 g per liter were achieved, the fermentation process required oxygen, a significant drawback. Therefore, the second part of the research focused on improving process energetics by using phospho-enol-pyruvate carboxykinase or malic enzyme as alternative carboxylating enzymes. Interestingly, it was found that either enzyme could replace the anaplerotic function of pyruvate carboxylase, which offers the perspective of anaerobic and more efficient production of C4-acids with S. cerevisiae.
01/2000; 38(1):30-36. DOI:10.1271/kagakutoseibutsu1962.38.30
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ABSTRACT: The yeast Yarrowia lipolytica is able to produce high amounts of several organic acids such as pyruvic, citric, isocitric, alpha-ketoglutaric, and succinic acid. Here we report on the influence of the reduced activity of succinate dehydrogenase in Y. lipolytica on its ability to produce succinate. The recombinant strains Y. lipolytica H222-AZ1 and H222-AZ2 were created by exchange of the native promoter of the succinate dehydrogenase subunit 2 encoding gene by inducible promoters. During the cultivation of the strain Y. lipolytica H222-AZ1 in shaking flask experiments, it was found that the promoter exchange resulted in an increase in succinic acid (SA) production. Moreover, it was found that the production of SA depends on an additional limitation of oxygen. Fed-batch cultivations in 1-l bioreactors confirmed this fundamental finding. Y. lipolytica H222-AZ1 produced 2 g l(-1) of SA with oxygen supply and 9.2 g l(-1) under the limitation of oxygen after 165 h. By using a less active promoter in Y. lipolytica H222-AZ2, the production of SA was increased to 25 g l(-1) with a productivity of 0.152 g (l*h)(-1) and a selectivity of 67 % after 165 h. Yields of 2.39 g SA per gram biomass and 0.26 g SA per gram glycerol were found.Applied Microbiology and Biotechnology 12/2014; 99(4). DOI:10.1007/s00253-014-6252-z · 3.81 Impact Factor