Construction Of Yarrowia Lipolytica Strain For Producing Triacetic Acid Lactone From Lignocellulose

As an important platform compound,triacetic acid lactone（TAL）is a“bridge”connecting biological transformation and chemical catalysis,and has wide applications in the fields of food,medicine,materials,and fuel additives.The construction of a microbial cell factory to synthesize TAL has the advantages of low raw material prices,low production costs,environmental friendliness,and fewer by-products,and is considered a viable alternative to chemical synthesis and plant extraction methods.Yarrowia lipolytica has a high-throughput metabolic flow of acetyl-Co A,which is the precursor of polyketones,making it a dominant host for production of triacetic acid lactone.However,its low utilization efficiency of xylose limits the comprehensive utilization of cheap lignocellulosic materials in synthesis of TAL.By constructing an integrated strain of Yarrowia lipolytica that can stably and efficiently produce TAL using xylose,this research laid a foundation for the production of TAL using cheap lignocellulosic raw materials.The specific research contents include:1.The construction and optimization of an engineered Yarrowia lipolytica strain that can efficiently metabolize xylose to produce TAL.The three genes of xylose metabolism pathway（ss.xyl1,ss.xyl2,yl.xyl3）and2-pyrone synthase（gh2ps）were integrated into the genome of Y.lipolytica.The integrated strain can use YPX medium to produce TAL,and the TAL yield could reach 1.59 g/L.The expression of gh2ps was enhanced by increasing the copies of gh2ps in the form of free plasmid in the integrated strain,indicating that the best effect was to increase the extra two copies of gh2ps.The citric acid complement pathway was constructed by expressing citric acid lyase ACL1 and ACL2,indicating that overexpression of ACL1 had most obvious effect on the yield improvement.Two copies of gh2ps and ACL1were separately continued for a new round of integration,and it was found that the strain Yali05-10#integrated with extra two copies of gh2ps had the highest yield,with the TAL of 1.75 g/L using YPX medium,and the productivity increased by 26.8%.It indicated that the integrated strain Yali05-10#can better utilize xylose to synthesize TAL.2.The optimization and regulation of the fermentation process for the production of TAL using glucose and xylose by the integrated strain Yali05-10#.To realize the utilization of lignocellulosic hydrolysate,this research explored the co-fermentation process of glucose and xylose based on the integrated strain Yali05-10#.By optimizing the ratio of glucose to xylose,it was determined that when the ratio of glucose to xylose was 3:1,the TAL yield was the highest,reaching 2.32 g/L.In combination with the addition of cerulenin,the synthesis of by-product fatty acid was inhibited,and the TAL yield reached 3.39 g/L.Using the fatty acid responsive promoter P_pox,a CRISPR-d Cas9 system was constructed to replace cerulenin in inhibiting the expression of fatty acid synthase,resulting in a TAL yield of 2.69 g/L.3.The optimization and regulation of the fermentation process for the production of TAL using lignocellulosic raw materials by the integrated strain Yali05-10#.Based on the above research,this paper explored the ability of the integrated strain Yali05-10#to synthesize TAL from lignocellulosic hydrolysate by adding cerulenin,and the effect of the integrated strain to synthesize TAL from lignocellulosic hydrolysate by a constructed CRISPR-d Cas9 system under the control of the promoter P_pox.Using both non detoxified or detoxified lignocellulosic hydrolysate as raw materials,the TAL yield was stably above 3 g/L after adding cerulenin,indicating that the integrated strain Yali05-10#could better utilize lignocellulosic hydrolysate to synthesize TAL.Substituting cerulenin with the constructed CRISPR-d Cas9system,the TAL yield was 2.73 g/L and 3.04 g/L by using the non detoxified and detoxified lignocellulosic hydrolysate,respectively.After introducing xylanase（xln D）from Aspergillus niger and xylosidase（xyn2）from Trichoderma reesei into the integrated strain Yali05-10#,the engineered strain was able to directly synthesize TAL using xylan as the substrate,with a yield of 0.74 g/L,which laid a foundation for the research on the direct synthesis of TAL from lignocellulosic materials.