Production of pyruvate in Saccharomyces cerevisiae through adaptive evolution and rational cofactor metabolic engineering

摘要

Pyruvate-decarboxylase (Pdc)-negative Saccharomyces cerevisiae has been proven as a suitable metabolic engineering platform to produce organic acids. S. cerevisiae BY5419 Pdc~(-) strain cannot grow in batch cultures on synthetic medium with glucose as the sole carbon source, yet grows well on synthetic medium with ethanol or acetate. In this study, by combining adaptive evolution and cofactor engineering, we obtained a series of engineered yeasts that can produce pyruvate using glucose as sole carbon source. Differential expression of noxE, encoding a water-forming NADH oxidase from Lacto-coccus lactis, arid udhA, encoding a soluble pyridine nucleotide transhydrogenase from Escherichia coli, was investigated. Of all the constructed recombinant strains, G2Ul-A_(o) was able to produce 75.1 gl~(-1) pyruvate, increased 21% compared with the original strain A_(o). The production yield of this strain reached 0.63 gofpyruvategofglucose~(-1). This study demonstrates that the fine regulation of intracellular NADH/NAD~(+) ratio is critical for cell metabolism and pyruvate production. Combining the adaptive evolution and fine regulation of intracellular NADH/NAD~(+) ratio provides a new strategy for improving the Pdc~(-) strain engineering platform.