Modify The Metabolic Pathway Of Saccharomyces Cerevisiae To Produce Fatty Acid Ethyl Ester

Fatty acid ethyl ester（FAEE）is the most potential new energy since the new century.Compared with the current common chemical synthesis methods,the production of FAEE in microbial cell factories has many advantages,such as low environmental pollution and low production cost.Saccharomyces cerevisiae has the potential to produce ethanol and aliphatic acyl coenzyme A,and is an important substrate cell for the construction of high-yielding FAEE cell factories.In this paper,S.cerevisiae cells were rationally modified by means of metabolic engineering to construct recombinant S.cerevisiae cells with efficient synthesis of FAEE.The main research contents include:1.Codon optimization and chemical synthesis of wax ester synthase encoding gene WS2;and expressed in S.cerevisiae BY4741.FAEE was synthesized by glucose to construct recombinant yeast cells.Furthermore,through homologous recombination,the cumulative knockout of sterol ester pathway（ΔARE1,ΔARE2）,triacylglycerol pathway（ΔDGA1,ΔLRO1）andβ-oxidation pathway（ΔPXA2）was performed to block the competitive pathway to increase the accumulation of adipoyl coenzyme A,a precursor intermediate of FAEE.The results showed that the knockout of the three pathways greatly increased the yield of FAEE.Compared with the expression of WS2 in the original strain BY4741,that is,strain BYW2 was increased by 9 times,and the yield of shaking flask reached 11.72 mg·L^-1.2.The FAEE fermentation process of recombinant yeast strain BYD5W2 was optimized by shaking flask level.The effects of ethanol and rapeseed oil addition,fermentation time and ethanol addition strategy on FAEE yield were investigated.The results showed that:The addition of ethanol and rapeseed oil significantly increased the yield of FAEE.The optimal fermentation time was 60 h,and the ethanol addition mode was to add 2%of the fermentation volume every 6 h starting from 20 h.At this time,the yield of FAEE reached 144.4 mg·L^-1,which was 12.3 times that of FAEE before optimization.The yield of FAEE was 480 times that of the original strain BY4741.Furthermore,the effects of nutritional deficiency type and glycerol pathway on the ability of FAEE synthesis by recombinant bacteria were explored.The results showed that the FAEE flask yield of uracil protrophic recombinant strain BYD5W2*was significantly increased,reaching 192 mg·L^-1;However,knockout of glycerol pathway reduced FAEE production,which was only 76.4 mg·L^-1.Increasing the expression level of acetyl-Co A carboxylase gene ACC1 had no significant effect on the yield of FAEE,which was 144.9 mg·L^-1.The effect of ELO2 knockout on the length of FAEE carbon chain was investigated.The results showed that ELO2 gene knockout had no effect on the total yield of FAEE,and the FAEE content of C₁₆ was increased to a certain extent,while the FAEE content of C₁₈ was decreased.3.The FAEE synthesis ability of uracil protrophic recombinant strain BYD5W2*in 5 L fermentation tank was investigated.Under the condition of batch feeding fermentation,the final yield of FAEE was 1.35 g·L^-1.A 15 L fermenter was used to further expand the culture.The results showed that the maximum cell biomass of the recombinant strain BYD5W2*reached 40.3(OD₆₀₀)and the maximum yield of FAEE reached 1.53 g·L^-1,which was the highest in the reported FAEE production by S.cerevisiae.