Transcriptomics Analysis And Reverse Engineering For The Yarrowia Lipolytica Strains With Improved Glucose-xylose Co-fermentation And Inhibitor Tolerance

Along with rapid global economic development and population growth,issues caused by utilizing fossil resources are attracting increasing attention.Lignocellulosic biomass,one of the most abundant renewable resources on earth,has received widespread attention as it offers an alternative to fossil resources.In recent years,many researchers have tried to develop technologies for production of biofuels and chemicals from lignocellulosic biomass.Such a conversion process using lignocellulosic biomass as a substrate can not only produce biofuels and chemicals sustainably,but also reduce the emission of greenhouse-gas.Structurally,there are three main components in lignocellulosic biomass,which is cellulose,hemicellulose,and lignin.Cellulose and hemicellulose can be hydrolyzed to fermentable sugars for further production of biofuels and chemicals.Due to its recalcitrant structure,lignocellulosic biomass must undergo pretreatment to make it easy for the hydrolysis of cellulose and hemicellulose.However,accompanied with the pretreatment of lignocellulose,undesirable compounds,such as weak acids,furans and phenolics that are toxic to microorganisms,are also generated.Phenolics can destroy the integrity and permeability of microbial cell membrane,as well as disrupt the metabolic activity of the strain.Yarrowia lipolytica,one of the most well-known model non-conventional yeast,has been classified as ‘‘Generally Recognized As Safe''(GRAS)by the American Food and Drug Administration.It is widely distributed in environments where enrich lipids and proteins,and it can also use hydrophobic substrates such as alkanes and glycerol efficiently as sole carbon sources.Y.lipolytica has been used as a safe and robust host to produce single cell proteins,fatty acid-derived biofuels and organic acids at an industrial scale.The lipid in engineered Y.lipolytica strains can even reach up to 90%(w/w).High fatty acids accumulation and similar lipid composition to diesel make Y.lipolytica as an attractive platform for industrial production of biofuel.However,there were two bottlenecks in the process of using lignocellulose to produce bio-oil by Y.lipolytica.One is that Y.lipolytica cannot efficiently utilize the xylose in the lignocellulose hydrolysate,and the other is that the widetype Y.lipolytica strain cannot tolerate the inhibitors in the lignocellulose hydrolysate.Which two bottlenecks hinder the application of Y.lipolytica in the second-generation biorefinery industry.In our previous study,we obtained a glucose/xylose co-utilization domesticated strain which named yl-XYL+*04 through 2-deoxyglucose/xylose competitive domestication.Although xylose utilization of the domesticated strain was significantly improved,the glucose utilization was slightly decreased.In this study,transcriptomic analysis were performed on the evolved strain yl-XYL+*04 and the starting strain yl-XYL+.The single-base mutation of YALI0?E23287g(N373T)and YALI0?E15488g(G270A)genes affected the utilization of glucose in yl-XYL+*04.Which two targets was predicted by homology modeling and molecular docking and confirmed by reverse metabolic engineering.Our work provides new sights for the construction of engineering strains for glucose/xylose co-utilization.In this study,we also performed ferulic acid and vanillic acid adaptive laboratory evolution experiment in Y.lipolytica,respectively.Then we analysied the transcriptome differences between the evolved strain yl-XYL+*FA*4,yl-XYL+*VA*4 and the initial strain yl-XYL+,respectively.Four targets related(YALI0?E25201g,YALI0?F05984g,YALI0?B18854g and YALI0?F16731g)to ferulic acid tolerance and three targets(YALI0?E19426g,YALI0?B18854g and YALI0?F16731g)related to vanillic acid were confirmed by q RT-PCR and reverse engineering.These targets were all first reported in Y.lipolytica.Our research has broadened the horizons in understanding of the mechanism of Y.lipolytica tolerant to inhibitors,and this study also provided new sights for the construction of more robust industial engineering Y.lipolytica strains.In summary,this study has conducted a systematic study on the two bootlenecks in the processing of lignocellulose bioconversion by Y.lipolytica.It was also the first study of Y.lipolytica tolerant to phenolic acid inhibitors.Our research played an important role in promoting the second-generation biorefinery industrial application of Y.lipolytica.