The combination of NAD+-dependent deacetylase gene deletion and the interruption of gluconeogenesis causes increased glucose metabolism in budding yeast

Metabolic engineering focuses on rewriting the metabolism of cells to enhance native products or endow cells with the ability to produce new products. This engineering has the potential for wide-range application, including the production of fuels, chemicals, foods and pharmaceuticals. Glycolysis manages the levels of various secondary metabolites by controlling the supply of glycolytic metabolites. Metabolic reprogramming of glycolysis is expected to cause an increase in the secondary metabolites of interest. In this study, we constructed a budding yeast strain harboring the combination of triple sirtuin gene deletion (hst3∆ hst4∆ sir2∆) and interruption of gluconeogenesis by the deletion of the FBP1 gene encoding fructose-1,6-bisphosphatase (fbp1∆). hst3∆ hst4∆ sir2∆ fbp1∆ cells harbored active glycolysis with high glucose consumption and active ethanol productivity. Using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) analysis, hst3∆ hst4∆ sir2∆ fbp1∆ cells accumulated not only glycolytic metabolites but also secondary metabolites, including nucleotides that were synthesized throughout the pentose phosphate (PP) pathway, although various amino acids remained at low levels. Using the stable isotope labeling assay for metabolites, we confirmed that hst3∆ hst4∆ sir2∆ fbp1∆ cells directed the metabolic fluxes of glycolytic metabolites into the PP pathway. Thus, the deletion of three sirtuin genes (HST3, HST4 and SIR2) and the FBP1 gene can allow metabolic reprogramming to increase glycolytic metabolites and several secondary metabolites except for several amino acids.