Combinatorial metabolic engineering and tolerance evolution of Yarrowia lipolytica for improved γ-decalactone synthesis

Abstract

This study highlights an effective strategy based on the synergistic complementarity of metabolic engineering and adaptive evolution.First, the chassis strain YL013 was obtained by deleting five acyl-CoA oxidase genes to reduce the degradation of γ-decalactone(GDL). Then, adaptive laboratory evolution with increasing concentrations of γ-decalactone was employed to obtain the strain YL013–250 with increased γ-decalactone tolerance of up to 250 mg/L. Moreover, this strain produced 664.5 mg/L γ-decalactone, representing a 142 % improvement over the parental strain YL013. Furthermore, four candidate fatty acyl-CoA synthetase genes, FAA1, FAA2 FAA3 and FAT1, were modulated using the strong, constitutive TEFin promoter to investigate their roles in GDL production. The results showed that overexpression of FAA1, FAA2 and FAA3 can effectively improve the ability of Yarrowia lipolytica to synthesize GDL using ricinoleic acid as substrate, among which overexpression of FAA1 had the most significant effect on GDL yield. Finally, gene of alcohol acyltransferase (AAT1) from Prunus persica was introduced into Y. lipolytica, leading to a GDL titer of 2.2 g/L, which was a 1.32-fold increase compared with the parental strain without AAT1. Taken together, this study provides a combinatorial strategy to improve GDL production through rational regulation of pathway genes and non-rational adaptive laboratory evolution.