Isolation of sake yeast strains possessing various levels of succinate- and/or malate-producing abilities by gene disruption or mutation

Section of Clinical Biochemistry, School of Health Science, Kyorin University, Miyashita, Hachioji, Tokyo 192-0005, Japan
Journal of Bioscience and Bioengineering (Impact Factor: 1.88). 02/1999; 87(3):333-339. DOI: 10.1016/S1389-1723(99)80041-3


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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    • "However, deletion of both SDH1 and SDH2 genes is required for the complete loss of SDH activity (Kubo et al., 2000). Arikawa et al. (1999a, 1999b) reported that in YPD medium with 15% glucose under aerobic conditions, deletion of KGD1 results in a lower level of succinic acid production while deletion of SDH1 results in an increased level of succinic acid production. Under anaerobic conditions, Δsdh1 produced almost the same level of succinic acid as the wild-type yeast (Arikawa et al., 1999b). "
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    ABSTRACT: Succinic acid produced by yeast during bread dough fermentation can significantly affect the rheological properties of the dough. By introducing mutations in the model S288C yeast strain, we show that the oxidative pathway of the TCA cycle and the glyoxylate shunt contribute significantly to succinic acid production during dough fermentation. More specifically, deletion of ACO1 and double deletion of ACO1 and ICL1 resulted in a 36 and 77% decrease in succinic acid levels in fermented dough, respectively. Similarly, double deletion of IDH1 and IDP1 decreased succinic acid production by 85%, while also affecting the fermentation rate. By contrast, double deletion of SDH1 and SDH2 resulted in a two-fold higher succinic acid accumulation compared to the wild-type. Deletion of fumarate reductase activity (FRD1 and OSM1) in the reductive pathway of the TCA cycle did not affect the fermentation rate and succinic acid production. The changes in the levels of succinic acid produced by mutants Δidh1Δidp1 (low level) and Δsdh1Δsdh2 (high level) in fermented dough only resulted in small pH differences, reflecting the buffering capacity of dough at a pH of around 5.1. Moreover, Rheofermentometer analysis using these mutants revealed no difference in maximum dough height and gas retention capacity with the dough prepared with S288C. The impact of the changed succinic acid profile on the organoleptic or antimicrobial properties of bread remains to be demonstrated. Copyright © 2015 Elsevier B.V. All rights reserved.
    Full-text · Article · Mar 2015 · International journal of food microbiology
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    • "However , the newly constructed strains also showed an increased production of succinate, which was most probably due to the reduced SDH2 expression or the reduced SDH activity. It was already described for S. cerevisiae that a deletion or mutation of particular SDH genes resulted in an accumulation of succinate (Arikawa et al. (1999); Kubo et al. (2000); Raab et al. (2010); Szeto et al. (2007)). An accumulation of succinate was also observed in the yeast Kluyveromyces lactis after deletion of SDH1 (Saliola et al. 2004). "
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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.
    Full-text · Article · Dec 2014 · Applied Microbiology and Biotechnology
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    • "These enhancements were not observed under strictly anaerobic or sake brewing conditions. Absence or limitation of oxygen resulted in decreased succinate production in sdh1 and/or fum1 deletion strains [31]. In another study on sake yeast strains, the deletion of genes encoding for succinate dehydrogenase subunits (SDH1, SDH2, SDH3, and SDH4) also resulted in increased succinate production only under aerobic conditions [46]. "
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    ABSTRACT: Succinate is a promising chemical which has wide applications and can be produced by biological route. The history of the biosuccinate production shows that the joint effort of different metabolic engineering approaches brings successful results. In order to enhance the succinate production, multiple metabolical strategies have been sought. In this review, different overproducers for succinate production, including natural succinate overproducers and metabolic engineered overproducers, are examined and the metabolic engineering strategies and performances are discussed. Modification of the mechanism of substrate transportation, knocking-out genes responsible for by-products accumulation, overexpression of the genes directly involved in the pathway, and improvement of internal NADH and ATP formation are some of the strategies applied. Combination of the appropriate genes from homologous and heterologous hosts, extension of substrate, integrated production of succinate, and other high-value-added products are expected to bring a desired objective of producing succinate from renewable resources economically and efficiently.
    Full-text · Article · Apr 2013
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