Analysis Of Fatty Acid Synthesis Mechanism And Expression Of Key Genes Of Corynebacterium Glutamicum

Fatty acids are an important platform compound for further synthesis of high-energy-ratio biofuels and high-value-added oleochemicals.Corynebacterium glutamicum is a recognized food-grade safe strain with a type I fatty acid synthesis system,without theβ-oxidation pathway of fatty acid degradation,and has the potential for fatty acid synthesis.In this study,Corynebacterium glutamicum ATCC13032 are used as a cell model.In order to promote the synthesis of fatty acids,cell samples from logarithmic growth phase to stable phase were collected for transcriptome analysis.Providing a new direction for further research.Fatty acid synthase and acetyl-Co A carboxylase in Corynebacterium glutamate are key enzymes in fatty acid synthesis.The physicochemical properties and domains of fatty acid synthase FasA were analyzed by bioinformatics.After confirming its operability at the gene level,the FasA recombinant vector was constructed.At the same time,the acetyl-Co A carboxylase AccBCD1 recombinant vector was constructed.The effects of FasA and AccBCD1 on host bacterial fatty acid metabolism were investigated to improve fatty acid synthesis by homologous and heterologous expression.The main results are as follows:（1）The transcriptome analysis showed that Corynebacterium glutamate increased exponentially,the enzyme system was active and the metabolism was exuberant at 12 hours.Between 16 and 20 hours is an important node of Corynebacterium glutamicum,its growth,catalysis and other processes begin to stagnate and enter a rapid secondary metabolite production period.The expression of genes related to fatty acid synthesis reached the maximum among12-16 hours.The genes related to fatty acid biosynthesis pathway began to be down-regulated after 16 hours,when the genes related to fatty acid degradation pathway began to be up-regulated.At 20 hours,fatty acid degradation genes continue to be up-regulated,and related to lipid metabolism pathway are also up-regulated.At 24 hours,fatty acid biosynthesis genes were significantly down-regulated,and related to lipid metabolism pathways such as glycerol phospholipid and glycerol ester metabolism began to down-regulate,lipid metabolism entered the final stage,and biofilm began to form.（2）Through bioinformatics analysis,the molecular formula of FasA is C₁₃₉₄₄H₂₂₀₆₇N₃₈₅₅O₄₃₅₃S₅₅.The molecular weight of FasA is 315128.12 Da,which composed of 2969 amino acids.There is no transmembrane domain,and it does not belong to secretory protein.FasA is a large soluble complex enzyme located in the cytoplasm,with six domains,among which the 3-oxoacyl-ACP synthetase domain and hot dog domain can catalyze the synthesis of unsaturated fatty acids.（3）The strains of heterologous expression and homologous expression of FasA were constructed by means of genetic engineering.The total fatty acid yield was about 98.1mg/g DCW,which was 14.76%higher than that of the control strain.Over-expression significantly increased the contents of C16:1（Palmitoleic acid）and C18:1（oleic acid）in Escherichia coli,which were19.3 mg/g DCW and 23.3 mg/g DCW,respectively,compared with the control strain,it increased by 6.46 and 14.08 times,respectively.The total yield of fatty acids of homologous expression strain changed little,which was about 61.0 mg/g DCW.The homologous expression decreased C18:1by 42.71%and increased C18:0 by 4.59 times.（4）The strains of heterologous expression and homologous expression of AccBCD1 were constructed by means of genetic engineering.The total fatty acid yield reached 132.4 mg/g DCW,which was 57.24%higher than that of the control strain.Among them,the contents of C16:0,C16:1,and C18:1 fatty acids were significantly increased,compared with the control strain,it increased by1.22 times,8.78 times and 26.96 times,respectively.Overexpression of the AccBCD1 had a marked effect on the fatty acid composition of E.coli.Compared with the control strain,the homologous expression strain increased the C16:0 by 27.94%,while the content of C18:1 decreased by18.56%.The total fatty acid content of strain was 54.0 mg/g DCW.