Combination of Three Methods to Reduce Glucose Metabolic Rate for Improving N-Acetylglucosamine Production in Saccharomyces cerevisiae

Previously, the production of N-acetylglucosamine (GlcNAc) in Saccharomyces cerevisiae was improved by deletion of the genes encoding phosphofructokinase 2 (PFK-2) isoforms, which reduced the glycolytic flux by eliminating the pathway to produce fructose-2,6-bisphosphate, an allosteric activator of phosphofructokinase 1 (PFK-1). We further examined the effects of an additional reduction in glucose metabolic rate on N-acetylglucosamine production. Glucose uptake rate was lowered by expressing a gene encoding truncated glucose-sensing regulator (MTH1-ΔT). In addition, catalytically dead Cas9 (dCas9) was introduced in order to down-regulate the expression levels of PFK-1 and pyruvate kinase-1 (Pyk1). Finally, the three strategies were introduced into S. cerevisiae strains in a combinatorial way; the strain containing all three modules resulted in the highest N-acetylglucosamine production yield. The results showed that the three modules cooperatively reduced the glucose metabolism and improved N-acetylglucosamine production up to 3.0 g/L in shake flask cultivation.