The Mechanism of High Flux Supply of NAD + in Aerobic Fermentation of Candida glycerinogenes

Nicotinamide adenine dinucleotide (NAD⁺) is a cofactor involved in numerous redox reactions, and its supply is a bottleneck problem in metabolic engineering. Candida glycerinogenes can provide high flux NAD(H), but the mechanism by which transcription factors regulate its high flux synthesis of NAD⁺ is still unclear. This study investigated the role of the transcription factor MIG in the NAD⁺ de novo synthesis pathway in C. glycerinogenes. Tryptophan has been identified as a limiting factor in NAD⁺ synthesis, and its concentration directly affects the productivity of glycerol and ethanol as well as NAD⁺ synthesis. The key genes bna3 and bna6 were found to be rate limiting in the NAD⁺ de novo synthesis pathway, and when inhibited, they significantly affect the production of NAD. This study demonstrates that the transcription factor MIG significantly enhances the expression levels of genes involved in the NAD⁺ de novo synthesis pathway while simultaneously improving the efficiency of NAD synthesis and ethanol production. Its successful application in Saccharomyces cerevisiae ethanol fermentation resulted in a 25.8% increase in the ethanol conversion rate. These findings emphasize the importance of MIG in the production of NAD by C. glycerinogenes, providing valuable insights for metabolic engineering strategies.