| Nervous acid（C24:1）is an ultra-high-value very-long-chain monounsaturated fatty acid,which can not only prevent and treat diseases related to the nervous system and brain tissue,but also play a huge role in food additives,health products,cosmetics and other aspects.Nervous acid is scarce in nature,with high cost and low yield.Therefore,the synthesis of nervonic acid by microorganisms such as yeast has become a research hotspot.In this project,Saccharomyces cerevisiae was taken as the research object to explore the effect of very-long-chain fatty acid elongase on the synthesis of nervonic acid in Saccharomyces cerevisiae and the mechanism of low temperature induction on nervonic acid biosynthesis,and to improve the production of nervonic acid through single factor and response surface optimization.1.To explore the effects of ultra-long-chain fatty acid elongases La KCS,Mo KCS,Elovl3 and FAE1 on the synthesis of nervonic acid.Successfully constructed expression vectors p YES2.0-PTEF1-La KCS-TCYC-PPGK-FAE1-TCYC,p YES2.0-PTEF1-Mo KCS-TCYC-PPGK-FAE1-TCYC,p YES2.0-PTEF1-Elovl3-TCYC,p YES2.0-PTEF1-Mo KCS-TCYC,PSPGM2-PTEF1-Elovl3-TCYC-PPGK-FAE1-TCYC,and introduced them into Saccharomyces cerevisiae YS58 andΔelo2 host strains.Through preliminary fermentation culture,the results showed that the engineered bacteria could successfully produce nervonic acid and increase the content of unsaturated fatty acids,among which the engineering bacteria expressing p YES2.0-PTEF1-Mo KCS-TCYC-PPGK-FAE1-TCYChad the highest nervonic acid content,reaching 1.26%.2.The mechanism of low temperature-induced promotion of nervonic acid synthesis in Saccharomyces cerevisiae was explored.The control group was cultured at a constant temperature of 28°C.After induction at 18°C for 2h and 4 h,the cell membrane fluidity and fatty acid content were measured,and the transcriptome sequencing and q PCR verification were performed.The results showed that low temperature induced increased cell membrane fluidity by increasing the content of unsaturated fatty acids,and over time,cells synthesized more very-long-chain monounsaturated fatty acids to adapt to the low temperature environment.Transcriptome sequencing and q PCR verification indicated that a large number of genes were differentially expressed.Through the GO database,KEGG and functional enrichment analysis of the genes,it was found that the differentially expressed genes were concentrated in carbon metabolism,energy conversion,glycolysis and amino acid synthesis,among which the up-regulation of lipid metabolism genes such as OLE1,MGA2,and ERG9 led to unsaturation Increased fatty acid content and improved cell membrane fluidity,in turn,increased nervonic acid production.In addition,the up-regulation of SNF1 expression improved cellular metabolic efficiency and energy utilization,and improved cellular adaptability to low temperature.Analysis of fatty acids showed that low temperature induction increased the nervonic acid content of engineered bacteria by 200%compared with 28℃constant temperature culture.3.The fermentation conditions of the engineered bacteria for nervonic acid production were optimized.The effects of six single factors,including carbon-nitrogen ratio,exogenous addition of Asn,Leu,His,Trp and oleic acid,on the production of nervonic acid by engineered bacteria were investigated.Then,three factors with greater influence were selected:Leu,His and oleic acid,and the response surface optimization method was used to explore the interaction of multiple factors,and the software predicted the best conditions for nervonic acid fermentation culture:exogenous Adding Leu 3 g/L,His 0.5g/L and oleic acid 0.5 mmol/L,the production of nervonic acid can reach up to57 mg/L. |
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