Evolutionary Engineering Improved <scp>d</scp>-Glucose/Xylose Cofermentation of <i>Yarrowia lipolytica</i>

Abstract

The recombinant strain Y. lipolytica XYL+ can utilize xylose to produce microbial lipids efficiently. However, its xylose uptake is severely delayed in the presence of d-glucose. In this study, adaptive laboratory evolution (ALE) of strains in the medium containing xylose and d-glucose analogue 2-deoxyglucose (dG) is performed. After four stages of evolution over a total of 64 days, for the first time, we obtained an Y. lipolytica strain (yl-XYL+*04*10) that can cometabolize xylose and glucose, with diminished glucose repression. Xylose uptake kinetics showed that it could efficiently utilize xylose in the presence of 10 g/L of dG or d-glucose. Transcriptional profiling analysis revealed that the relative expression level of xylose-specific transporter genes (encoded by YALI0_C04730g and YALI0_D00363g) was significantly upregulated. In addition, missense mutations N373T and G270A in YALI0_E23287g (encoding a d-glucose transporter) and YALI0_E15488g (encoding a hexokinase) respectively, were found. It implied that these are important gene targets responsible for improved xylose utilization in evolved Y. lipolytica. Our work provides a new approach for breeding Y. lipolytica and paved the way for future pentose metabolic engineering.