Connecting central carbon and aromatic amino acid metabolisms to improve de novo 2-phenylethanol production in Saccharomyces cerevisiae

The organic compound 2-phenylethanol (2PE) has a pleasant floral scent and is intensively used in the cosmetic and food industries. Microbial production of 2PE by phenylalanine bioconversion or de novo biosynthesis from sugar offer sustainable, reliable and natural production processes compared to chemical synthesis. Despite the ability of Saccharomyces cerevisiae to naturally synthesize 2PE, de novo synthesis in high concentration and yield remains a metabolic engineering challenge. Here, we demonstrate that improving phosphoenolpyruvate supply by expressing pyruvate kinase variants and eliminating the formation of p-hydroxy-phenylethanol without creating tyrosine auxotrophy significantly contributed to improve 2PE production in S. cerevisiae. In combination with the engineering of the aromatic amino acid biosynthesis and Ehrlich pathway, these mutations enabled better connection between glycolysis and pentose phosphate pathway optimizing carbon flux towards 2PE. However, attempts to further connect these two parts of central carbon metabolism by redirecting fructose-6P towards erythrose-4P by expressing a phosphoketolase-phosphotransacetylase pathway did not result in improved performance. The best performing strains were capable of producing 13mM of 2PE at a yield of 0.113 mol mol-1, which represents the highest yield for de novo produced 2PE in S. cerevisiae and other yeast species.