Expression Of Key Enzyme Genes For Oil Synthesis From A Walnut Endophyte In Escherichia Coli And The Regulation On Free Fatty Acid Synthesis

Microbial oils have become an important oil source in biodiesel development in recent years,cloning of key enzyme genes for oil synthesis from oleaginous microorganisms and heterologous expression in Escherichia coli,which will be of great significance to obtain high-yield engineering bacteria and efficient production of microbial oil.Based on the analysis of key enzyme genes for oil synthesis metabolic pathway of a walnut endophyte Bacillus subtilis HB1310 with industrial oil-producing potential,the acetyl-Co A carboxylase aca,acb genes,desaturase de1,de2 genes,malic enzyme ME gene and 6-phosphogluconate dehydrogenase gnd gene were cloned and heterologous expressed in E.coli BL21(DE3)in this paper,the effects of these enzyme genes expression on enzyme activities and fatty acid production and composition of engineered strains were explored,and the regulation on free fatty acid production in E.coli were revealed,the research results were as follows:(1)aca,acb genes of acetyl-Co A carboxylase carboxyltransferaseαandβsubunits from walnut endophyte were overexpressed in E.coli respectively,and ACA,ACB engineered bacteria were obtained,fusion expression of aca and acb genes were further developed to obtain the AC engineered bacteria.The results showed that the acetyl-Co A carboxylase activity of ACB engineered bacterium reached 4.11±0.03 U/m L under the optimized expression condition of acb gene,which was 39.7%higher than that of the wild strain;The monitoring results of enzyme activity changes in the fermentation process of engineered bacteria ACA and ACB showed that the acetyl-Co A carboxylase activity of ACB engineered bacterium was 16.56%higher than that of the wild strain at12 h,and the acetyl-Co A carboxylase activity of engineered bacterium ACA was 9.24%higher than that of the wild strain at 24 h,which indicated that carboxyα-CT andβ-CT were acetyl-co A carboxylase subunits with good biological activity.The results of fatty acid content and components of ACA,ACB and AC engineered bacteria fermented for24 h showed that the total fatty acid content of ACA,ACB and AC increased by 26.17%,45.56%and 88.97%,respectively as compared with the wild strain.The fatty acid components analysis results exhibited that the unsaturated fatty acid contents of ACA,ACB and AC engineered bacteria reached 23.72%,27.28%and 43.96%,respectively,indicating the single expression and coexpression of aca and acb genes of acetyl-Co A carboxylase could not only improve the content of fatty acids,but also promote the alteration of fatty acid components to unsaturated fatty acids,which declared that the key enzyme gene expression of acetyl-co A carboxylase could effectively regulate the free fatty acids synthesis in E.coli.(2)de1 and de2 genes of walnut endophyte desaturase were overexpressed in E.coli,and the DE1 and DE2 engineered bacteria were obtained.Monitoring results of desaturase activities of engineered bacteria DE1 and DE2 showed that the enzyme activity of engineered bacterium DE1 was the highest at 48 h of fermentation,which was 2.03 times of that of the wild strain,and the enzyme activity of engineered bacterium DE2 was the highest at 12 h,which was 3.27 times of that of the wild strain.Comparative analysis results of fatty acid contents between the engineered bacteria and the wild strain showed that the fatty acid contents of engineered bacteria DE1 and DE2reached 41.59 mg/L and 50.735 mg/L,respectively,both of which were significantly higher than that of the wild strain.Fatty acid composition analysis results showed that overexpression of desatase de1 and de2 genes could also promote the conversion of octadecanoic acid into oleic acid,thus increase the proportion of monounsaturated fatty acid.(3)The malic enzyme ME gene and 6-phosphogluconate dehydrogenase gnd gene were overexpressed in E.coli,and the ME and GND engineered bacteria were constructed.The results showed that the malic enzyme activity of the engineered ME strain was 9.65 U/m L after 12 h fermentation,which was 30.44%higher than that of the wild strain,and the NADPH content of this strain was 1.87 times higher than that of the wild strain after 24 h fermentation;the NADPH content of GND engineered bacterium reached 4.298 nmol/10~6cell after 12 h fermentation,which was 2.70 times that of the wild strain.Further analysis of fatty acid content and components showed that the fatty acid content of ME engineered bacterium reached 42.63 mg/L,18.19%higher than that of the wild strain;overexpression of gnd gene could improve the content of unsaturated fatty acids.The unsaturated fatty acids content in GND engineered bacterium was not only significantly higher than that in the wild strain,but also increased by 31.90%compared with that in engineered ME strain,accounting for11.57±0.55%of total fatty acid content.It can be seen that the expression of ME and gnd genes can promote the synthesis of NADPH,thus promoting the increase of total fatty acid content and unsaturated fatty acid proportion,which is crucial for the synthesis of free fatty acids in E.coli.