Evolutionary engineering improved D-glucose/xylose co-fermentation of Yarrowia lipolytica

Abstract Background: Yarrowia lipolytica is considered as a promising biorefinery chassis for production of microbial lipids, the important precursors of advanced biofuels. Unfortunately, wild Yarrowia lipolytica is unable to consume xylose, the major pentose in lignocellulosic hydrolysates. A recombinant strain Yarrowia lipolytica yl-XYL+ can utilize xylose to produce microbial lipids efficiently, but its xylose uptake is severely delayed in the presentence of D-glucose. Therefore, it is critical to develop co-fermenting D-glucose and xylose strains and study the underlying mechanisms.Results: In this study, an adaptive laboratory evolution (ALE) is performed to engineering the strains in the medium containing xylose and D-glucose analog 2-deoxyglucose (dG). After four stages of evolution over a total of 64 days, we obtained for the first time a strain of Y. lipolytica (yl-XYL+*04*10) with derepressed xylose metabolism. Xylose uptake kinetics showed that it could efficiently utilize xylose in the presence of 10 g/L dG or D-glucose. Transcriptional profiling analysis revealed that relative expression level of YALI0_C04730g and YALI0_D00363g (both encoding xylose-specific transporter) was significantly up-regulated. Besides, we found that missense mutations N373T and G270A in YALI0_E23287g (encoding a D-glucose transporter) and YALI0_E15488g (encoding a hexokinase) respectively.Conclusions: These results indicate that these are important gene targets responsible for improved xylose utilization in the evolved Yarrowia lipolytica. Our work provides a new approach for breeding Yarrowia lipolytica and paved the way for future pentose metabolic engineering.