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Metabolic engineering of the acid-tolerant yeast Pichia kudriavzevii for efficient L-malic acid production at low pH

ABSTRACT: Soluble pyridine nucleotide transhydrogenase (EcSthA) derived from Escherichia coli was introduced into the L-malic acid producing strain MA009-5 of Pichia kudriavzevii to obtain strain MA009-10. It was found that MA009-10 was superior to MA009-5 in terms of L-malic acid titer, glucose consumption rate, and glucose/L-malic acid conversion rate. To elucidate the effect of EcSthA expression on L-malic acid synthesis, we analyzed the RNA-seq data of both strains.

ORGANISM(S): Pichia kudriavzevii

PROVIDER: GSE202655 | GEO | 2023/05/03

REPOSITORIES: GEO

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Project description:Malic enzymes decarboxylate the tricarboxylic acid (TCA) cycle intermediate malate to the glycolytic end-product pyruvate and are well positioned to regulate metabolic flux in central carbon metabolism. The bacterium Sinorhizobium meliloti has a NAD(P)-malic enzyme (DME) and a NADP-malic enzyme (TME) and DME is required for symbiotic N2-fixation. To help understand the role of these enzymes, we examined growth, metabolic and transcriptional consequences resulting from the deletion of these enzymes. Few effects were observed upon growth with glucose, whereas growth with the gluconeogenic substrate, succinate, resulted in transcriptional and metabolic effects particularly in the dme mutant strains. When grown with succinate, DME mutant cells accumulated hexose sugar phosphates and trehalose, while TME mutants accumulated putrescine. Succinate-grown DME mutant cells also showed increased transcription of genes for gluconeogenesis and for pathways such as amino acid and fatty acid synthesis that divert metabolites away from the TCA cycle. These data suggested that, DME is required to regulate the levels of TCA cycle intermediates and that the activity of TME is insufficient to prevent the accumulation of TCA cycle intermediates in cells utilizing succinate as carbon source. Consistent with this, in short-1-3 hour incubations with succinate, dme mutant cells excreted large amounts of malate whereas little malate was excreted from tme or wild-type cells. These results support the suggestion that DME is required for N2-fixation in alfalfa because it is required for synthesis of pyruvate and acetyl-CoA and the rapid metabolism of C4-dicarboxylates supplied by the plant. RNA expression was measured for S. meliloti in exponential phase grown in MOPS (morpholinpropanesulfonic acid) buffered minimal media under 2 growth conditions: 1) 15 mM succinate, 2) 15 mM glucose; using wild type cells as well as dme and tme mutant strains (6 experiments, 2 replicates: total 12 samples)

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Metabolic engineering of the acid-tolerant yeast Pichia kudriavzevii for efficient L-malic acid production at low pH

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