Effect of gene disruptions of the TCA cycle on production of succinic acid in Saccharomyces cerevisiae

Arikawa, Yukihiko; Kuroyanagi, Tomoko; Shimosaka, Makoto; Muratsubaki, Haruhiro; Enomoto, Keiichiro; Kodaira, Ritsuko; Okazaki, Mitsuo

doi:10.1016/s1389-1723(99)80004-8

Cited by 96 publications

(65 citation statements)

References 24 publications

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“…Therefore, the accumulation of succinate instead of ethanol in K. lactis suggests a bond between the expression of the KlSDH1 gene on glucose and efficient fermentation. The production of succinate was previously observed to a lower extent under very different growth conditions (15% glucose) also on the S. cerevisiae sdh1⌬ mutant (2). According to the authors of that study, in this yeast succinate accumulation was not dependent on the glyoxylate cycle but on the activation of the oxidative part of the Krebs cycle (2).…”

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confidence: 59%

“…The production of succinate was previously observed to a lower extent under very different growth conditions (15% glucose) also on the S. cerevisiae sdh1⌬ mutant (2). According to the authors of that study, in this yeast succinate accumulation was not dependent on the glyoxylate cycle but on the activation of the oxidative part of the Krebs cycle (2). Since this cycle is the main source of the precursors, in particular ␣-ketoglutarate and oxaloacetate, necessary for the synthesis of amino acids, nucleotides, heme, etc.…”

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confidence: 61%

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The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

et al. 2004

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We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.Succinate dehydrogenase (SDH) is a component of complex II of the respiratory chain that catalyses the oxidation of succinate to fumarate in the Krebs cycle and feeds electrons to the ubiquinone pool. The complex, which is highly conserved through evolution, is located in the inner mitochondrial membrane and consists of two catalytic and two structural subunits, all encoded by nuclear genes (38). In Saccharomyces cerevisiae, the four genes (SDH1 to SDH4) coding for SDH have been isolated and characterized (26,27,45,47). The flavoprotein subunit (11, 42) responsible for the oxidation of succinate to fumarate is encoded by two paralogous genes, SDH1 and SDH1b, although only SDH1 is necessary for growth on respiratory carbon sources (11). SDH2 codes for the iron-protein subunit (31) that contains three different iron-sulfur centers (22) and, together with the protein Sdh1p, constitutes the catalytic core of the SDH complex, which conveys electrons from the covalently attached flavin adenine dinucleotide (FAD) of Sdh1p first to the iron-sulfur centers and then to ubiquinone. SDH3 and SDH4 code for two small hydrophobic peptides, which anchor the complex to the inner mitochondrial membrane (10, 15). In humans, the mutations in the SDH genes have been associated to several mitochondrial-related pathologies suggesting, beside the enzymatic activity of the complex in the Krebs cycle, its involvement in superoxide handling (39,43).In S. cerevisiae, the expression of the SDH genes is repressed by glucose and derepressed on respiratory carbon sources (31, 45), and the loss of SDH functions results in the inability of cells to grow on any respiratory carbon sources (12,47).In this paper we report the isolation of the KlSDH1 gene (EMBL accession number AJ555233) encoding the Kluyveromyces lactis fla...

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The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

et al. 2004

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“…Strategies to redirect carbon fluxes into the respiratory central metabolism by altering expression levels of single enzymes of the central metabolism were not promising (9,44) or even resulted in impaired growth (2,9,17). However, interference with the expression levels of genes involved directly in the glucose repression cascade has been shown to have the potential to redirect the respirofermentative flux distribution.…”

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Shifting the Fermentative/Oxidative Balance in Saccharomyces cerevisiae by Transcriptional Deregulation of Snf1 via Overexpression of the Upstream Activating Kinase Sak1p

Raab

Hlavacek²,

Bolotina³

et al. 2011

Appl Environ Microbiol

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With the aim to reduce fermentation by-products and to promote respiratory metabolism by shifting the fermentative/oxidative balance, we evaluated the constitutive overexpression of the SAK1 and HAP4 genes in Saccharomyces cerevisiae. Sak1p is one of three kinases responsible for the phosphorylation, and thereby the activation, of the Snf1p complex, while Hap4p is the activator subunit of the Hap2/3/4/5 transcriptional complex. We compared the physiology of a SAK1-overexpressing strain with that of a strain overexpressing the HAP4 gene in wild-type and sdh2 deletion (respiratory-deficient) backgrounds. Both SAK1 and HAP4 overexpressions led to the upregulation of glucose-repressed genes and to reduced by-product formation rates (ethanol and glycerol). SAK1 overexpression had a greater impact on growth rates than did HAP4 overexpression. Elevated transcript levels of SAK1, but not HAP4, resulted in increased biomass yields in batch cultures grown on glucose (aerobic and excess glucose) as well as on nonfermentable carbon sources. SAK1 overexpression, but not the combined overexpression of SAK1 and HAP4 or the overexpression of HAP4 alone, restored growth on ethanol in an sdh2 deletion strain. In glucose-grown shake flask cultures, the sdh2 deletion strain with SAK1 and HAP4 overexpression produced succinic acid at a titer of 8.5 g liter ؊1 and a yield of 0.26 mol (mol glucose)؊1 within 216 h. We here report for the first time that a constitutively high level of expression of SAK1 alleviates glucose repression and shifts the fermentative/oxidative balance under both glucoserepressed and -derepressed conditions.The Crabtree-positive yeast Saccharomyces cerevisiae ferments under aerobic conditions in the presence of excess glucose. This may be a major disadvantage in biotechnological production processes, in particular when respiratory metabolism is required. Being able to shift the respirofermentative flux distribution toward a more respiratory metabolic state of the cell could be beneficial for many industrial applications of baker's yeast, since it leads to improved growth characteristics and the reduced formation of fermentation by-products.The formation of ethanol under aerobic conditions can be overcome by growing yeast cells under conditions of sugar limitation. Strategies to redirect carbon fluxes into the respiratory central metabolism by altering expression levels of single enzymes of the central metabolism were not promising (9, 44) or even resulted in impaired growth (2, 9, 17). However, interference with the expression levels of genes involved directly in the glucose repression cascade has been shown to have the potential to redirect the respirofermentative flux distribution. When overexpressing the Hap4p activator subunit of the Hap2/3/4/5 transcriptional complex, which is involved in the carbon-source-dependent regulation of the respiratory status, an increase of the respiratory capacity was observed for glucose-grown cells. Hap4p overexpression resulted in increased growth rates and biomass formation...

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“…It has been reported that aerobic succinic acid production was attempted by constructing S. cerevisiae recombinant strains in which genes related to the TCA cycle, such as SDH1, SDH2, SDH3, SDH4, and FUM1, were disrupted. 33,34) Recently, aerobic production of succinic acid through the oxidative TCA cycle and a glyoxylate shunt by a recombinant strain of *Corresponding author. Email: shimizu@ist.osaka-u.ac.jp + -dependent and NADP + -dependent mitochondrial isocitrate dehydrogenases, respectively, as well as the SDH1 and SDH2 genes were disrupted, was reported.…”

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confidence: 99%

Metabolic engineering of Saccharomyces cerevisiae to improve succinic acid production based on metabolic profiling

Ito

Hirasawa

Shimizu

2014

Bioscience, Biotechnology, and Biochemistry

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We performed metabolic engineering on the budding yeast Saccharomyces cerevisiae for enhanced production of succinic acid. Aerobic succinic acid production in S. cerevisiae was achieved by disrupting the SDH1 and SDH2 genes, which encode the catalytic subunits of succinic acid dehydrogenase. Increased succinic acid production was achieved by eliminating the ethanol biosynthesis pathways. Metabolic profiling analysis revealed that succinic acid accumulated intracellularly following disruption of the SDH1 and SDH2 genes, which suggests that enhancing the export of intracellular succinic acid outside of cells increases succinic acid production in S. cerevisiae. The mae1 gene encoding the Schizosaccharomyces pombe malic acid transporter was introduced into S. cerevisiae, and as a result, succinic acid production was successfully improved. Metabolic profiling analysis is useful in producing chemicals for metabolic engineering of microorganisms.

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Effect of gene disruptions of the TCA cycle on production of succinic acid in Saccharomyces cerevisiae

Cited by 96 publications

References 24 publications

The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

Shifting the Fermentative/Oxidative Balance in Saccharomyces cerevisiae by Transcriptional Deregulation of Snf1 via Overexpression of the Upstream Activating Kinase Sak1p

Metabolic engineering of Saccharomyces cerevisiae to improve succinic acid production based on metabolic profiling

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