Study On Metabolic Engineering Of Yarrowia Lipolytica Based On Utilization Of Lignocellulose

The massive consumption of fossil fuels and the consequent environmental problems make it imperative to research new renewable energy sources.Biodiesel is considered to be a high-quality alternative to fossil fuels.Microbial oils(mostly in the form of triglycerides)are ideal raw materials for the production of high energy density liquid fuels(such as biodiesel and aviation fuel)due to their high C and H content.Many microorganisms have the ability to synthesize microbial oils.Yarrowia lipolytica is one of the most promising oleaginous yeasts due to its“generally regarded as safe”(GRAS)microorganism,which has a broad substrate spectrum,excellent microbial oil synthesis ability,clear genetic background,and mature genetic manipulation platform.However,the production of microbial oils by Y.lipolytica,especially from lignocellulosic feedstock,still faces some challenges.For example,Y.lipolytica cannot metabolize cheap and widespread substrates such as xylose,arabinose,and cellobiose;has low tolerance to inhibitors in lignocellulosic hydrolysates;and has low lipid production when utilizing lignocellulosic feedstocks.Considering the above issues,this study metabolically engineered Y.lipolytica to better utilize lignocellulose as a feedstock for lipid production,providing a high-performance chassis cell for the lignocellulose valorization of the biodiesel industry.The main research content and results are as follows:(1)Mechanism analysis of glucose-xylose co-utilization in Y.lipolyticaThe utilization rate of xylose in Y.lipolytica yl-XYL+is equivalent to that of glucose,but is inhibited in the presence of glucose.In order to solve this problem,the co-utilization of glucose and xylose is realized.We constructed a domestication system,using 2-deoxyglucose(d G,D-glucose analogue)to compete and domesticate with xylose to obtain mutant strains.After the domestication,four mutant strains were obtained,yl-XYL+*01*3,yl-XYL+*02*9,yl-XYL+*03*5 and yl-XYL+*04*10.Fermentation phenotypes and kinetic tests revealed that synchronous fermentation can be achieved by reducing glucose uptake to improve xylose utilization.Further transcriptome analysis revealed that the expression levels of genes related to the glucose metabolic pathway were down-regulated,whereas the expression levels of YALI0＿C04730g and YALI0＿D00363g genes encoding xylose-specific transporter proteins were significantly up-regulated,which may be the main reason for the enhanced glucose-xylose co-utilization ability of the strain.The recombinant strains overexpressing YALI0＿C04730g and YALI0＿D00363g had excellent glucose-xylose co-fermentation phenotypes,revealing the importance of xylose transporter protein engineering.(2)Analysis of the tolerance mechanism of Y.lipolytica to aromatic aldehyde inhibitorsHigh substrate lignocellulosic hydrolysates usually contain high concentrations of inhibitors that have negative effects on microbial growth and fermentation.Phenolic inhibitors are much more toxic to microorganisms than organic acids and furan derivatives-based inhibitors.In this part,three representative aromatic aldehyde inhibitors(vanillin,syringaldehyde and 4-hydroxybenzaldehyde)were selected as model compounds to improve the tolerance of yl-XYL+strains through adaptive evolution,and the underlying mechanisms were analyzed.Firstly,the tolerance of yl-XYL+to vanillin,syringaldehyde,and 4-hydroxybenzaldehyde was improved through adaptive evolution,and three tolerant strains yl-XYL+*V,yl-XYL+*S and yl-XYL+*H were obtained.The fermentation phenotypic tests revealed that the mutant strains obtained phenolic inhibitor tolerance mainly by degrading aldehyde inhibitors to the corresponding alcohols and acids,thus reducing their toxicity to cells.In addition,transcriptome analysis,enzymatic property analysis and reverse metabolic engineering showed that the expression of aldehyde ketone reductase gene YALI0＿B07117g and aldehyde dehydrogenase gene YALI0＿B01298g can effectively degrade aromatic aldehyde inhibitors.This study firstly reveals the aldehyde degradation mechanism in Y.lipolytica,screens for some of the tolerance genes,and provides a reliable basis for the development of future biorefinery strains tolerant to phenolic inhibitors.(3)Construction of a diploid Y.lipolytica strain for utilizing multiple lignocellulosic sugar componentsCellobiose and xylose are important components of lignocellulosic hydrolysates,but wild Y.lipolytica usually unable to utilize cellobiose and xylose.In this study,the diploid Y.lipolytica NRRL Y-1095(yl-1095)is used as the host strain.First,the cellobiose utilization strain yl-C was created by inserting the cellobiose gene cassette(the cellodextrin transporter cdt-1 and the intracellular-glucosidase gh1-1 from Neurospora crassa)into the yl-1095 genome’s Zeta site.Next,xylose reductase(XR)and xylitol dehydrogenase(XDH)from Scheffersomyces stipites were introduced into the yl-C,along with overexpression of endogenous xylulose kinase(XK),and the transformed strain could grow using xylose as the sole carbon source.The final engineered strain yl-CX,could utilize cellobiose or xylose as the sole carbon source,and could also effectively utilize glucose,xylose,and cellobiose in the mixed sugar medium.Compared with the original strain,the lipid titer and lipid content of mixed sugar fermentation increased significantly by 76.49%and 97.10%,respectively.(4)Construction of a diploid Y.lipolytica strain with high lipid productionIn order to improve the ability of Y.lipolytica to produce microbial lipids,the lipid synthesis pathway,cellobiose utilization pathway,and xylose utilization pathway were integrated and enhanced in the starting strain diploid Y.lipolytica yl-1095,and a diploid Y.lipolytica strain capable of high lipid production using multiple substrates was obtained.First,the introduction of non-oxidized pentose-phosphate pathway phosphoketolase(f PK)and phosphotransacetylase(PTA)increased the flux of acetyl-Co A and alleviated the limitation of nitrogen starvation and initiated lipid production during cell growth.The lipid concentration of the engineered strain yl-FP was 33.3%higher than that of the original strain yl-1095.Then,over-expressing the DGA1 gene of the oil synthesis pathway further increased the lipid production.The obtained engineered strain yl-FPD lipid concentration increased by 39.4%compared with the original strain.In order to replenish cofactors,we introduced heterologous NADP~+dependent glyceraldehyde 3-phosphate dehydrogenase(Gap C),which can convert cytoplasmic NADH into NADPH,and the constructed engineered yeast yl-FPDG showed a 12.4%increase in lipid concentration compared to strain yl-FPD.In addition,we introduced the xylose pathway and the cellobiose pathway into the engineering yl-FPDG to obtain the engineered strain yl-FPDGCX.The lipid production in different simulated hydrolysates is much higher than that of the original strain yl-1095,and the lipid titer was increased by 86.3%(acid simulated hydrolysate)and 95.0%(alkaline simulated hydrolysate),respectively.Finally,the engineering strain yl-FPDGCX could make better use of crude glycerol and lignocellulose hydrolysate to produce lipid,exhibiting good value for industrial application.