Molecular mechanisms of furanone production through the EMP and PP pathways in Zygosaccharomyces rouxii with D-fructose addition.

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) are important aroma chemicals in fermented foods. In this study, transcriptomics, qRT-PCR and enzymology methods were used to study the molecular mechanisms of furanone production through the Embden-Meyerhof-Parnas (EMP) and Pentose Phosphate (PP) pathways in Zygosaccharomyces rouxii based on the results of our previous study. The results indicated that D-fructose addition could significantly enhance Z. rouxii biomass production. In addition, HDMF and HEMF production was increased as a result of D-fructose addition based on HPLC analysis. The significant pathways for furanone synthesis were EMP (zro00010) and PP (zro00030) based on KEGG analysis. At the mRNA level, the differentially expressed genes involved in HDMF and HEMF biosynthesis were HK, PFK1, G6PI, FBA, TPI, 6GPL, TKT, and 6PGDH. Transient overexpression of FBA and 6PGDH in Z. rouxii was significantly increased during furanone production. FBA can regulate the accumulation of dihydroxyacetone phosphate (DHAP), which is one of the precursors of HDMF, while 6PGDH can regulate the accumulation of ribulose-5-phosphate, a precursor of HEMF. In addition, the activities of PFK1, FBA, and 6PGDH were significantly correlated with furanone production. LC-MS/MS results indicated that the primary metabolites for furanone synthesis in the EMP and PP pathways gradually increased with the consumption of D-fructose. These data demonstrate that D-fructose addition can be used to generate furanones through the EMP and PP pathways in Z. rouxii.