Fumaric acid overproduction in yeast mutants deficient in fumarase

Food Research Institute, Bratislava, Czechoslovakia.
FEMS Microbiology Letters (Impact Factor: 2.12). 04/1992; 70(2):101-6. DOI: 10.1016/0378-1097(92)90667-D
Source: PubMed


Abstract A nuclear mutant of Saccharomyces cerevisiae deficient in mitochondrial fumarase has been identified through the in vitro biochemical assay of enzyme activity after visual selection due to an increased acidification ability of its colonies. Cells of the fumarase-deficient mutant fermenting glucose accumulated extracellular fumaric acid. This accumulation was observed only in growing cultures and required functional mitochondrial electron transport from succinate dehydrogenase to oxygen.

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    • "In the second strategy, FA can be produced via an oxidative TCA cycle and the engineered strain is stable in the fermentation process. It was reported that cells of a fumarase-deficient mutant accumulated extracellular FA when fermenting glucose [11]. Similarly, a concentration of 3.62 g L–1 at a yield of 0.11 moles of succinic acid per mole of glucose was achieved for oxidative production of succinic acid in yeast by deletion of the SDH1, SDH2, IDH1 and IDP1 genes [8]. "
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    ABSTRACT: Fumaric acid (FA) is a promising biomass-derived building-block chemical. Bio-based FA production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here we report on FA production by direct fermentation using metabolically engineered Saccharomyces cerevisiae with the aid of in silico analysis of a genome-scale metabolic model. First, FUM1 was selected as the target gene on the basis of extensive literature mining. Flux balance analysis (FBA) revealed that FUM1 deletion can lead to FA production and slightly lower growth of S. cerevisiae . The engineered S. cerevisiae strain obtained by deleting FUM1 can produce FA up to a concentration of 610±31 mg L<sup>–1</sup> without any apparent change in growth in fed-batch culture. FT-IR and <sup>1</sup>H and <sup>13</sup>C NMR spectra confirmed that FA was synthesized by the engineered S. cerevisiae strain. FBA identified pyruvate carboxylase as one of the factors limiting higher FA production. When the RoPYC gene was introduced, S. cerevisiae produced 1134±48 mg L<sup>–1</sup> FA. Furthermore, the final engineered S. cerevisiae strain was able to produce 1675±52 mg L<sup>–1</sup> FA in batch culture when the SFC1 gene encoding a succinate–fumarate transporter was introduced. These results demonstrate that the model shows great predictive capability for metabolic engineering. Moreover, FA production in S. cerevisiae can be efficiently developed with the aid of in silico metabolic engineering.
    Full-text · Article · Dec 2012 · PLoS ONE
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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.
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    ABSTRACT: Organic acids are essential flavor components in alcoholic beverages such as wine and sake (Japanese rice wine). The effects of fumarase deficiency on the productivity of malic, fumaric and succinic acids in Saccharomyces cerevisiae were examined using the FUM1 gene disruptant. The single nuclear gene FUM1 encoding fumarase in S. cerevisiae, DBY746, was disrupted by a site-directed one-step gene disruption technique. The fumarase activity of the isolated FUM1 disruptant was abolished. The FUM1 disruptant having a pet phenotype showed no growth on minimal medium containing glycerol as a carbon source even after prolonged incubation. The productions of organic acids by the FUM1 disruptant in YPD liquid culture containing 10% glucose and in sake mash were analyzed by HPLC. The analytical results of the FUM1 disruptant showed there were no differences in extracellular malate productivity, fumarate productivity had increased, and that succinate productivity was reduced compared with those of the control strain. In the production of sake using the FUM1 disruptant, fermentation ability as monitored by CO2 evolution was not different from that of the control strain. These results indicate that site-directed FUM1 disruption alters organic acid production without any loss of fermentation ability in yeast.
    No preview · Article · Jan 1995 · Journal of Fermentation and Bioengineering
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