Study On Overexpression Of Acetyl-CoA Carboxylase To Increase Fatty Acid Synthesis In Recombinant Strains Of Corynebacterium Glutamicum

Fatty acids are one of the essential organic substances in microorganisms.According to different classification methods,fatty acids can be divided into different types.Fatty acids can regulate the fluidity of cell membranes,maintain metabolic balance in the body,and can also be used as energy sources to supply energy to the body.Fatty acids have a wide range of applications,involving medical,cosmetics,food,chemicals and other fields.Corynebacterium glutamicum,as a model strain,has the characteristics of easy cultivation,fast reproduction,and high safety.Acetyl-Co A carboxylase of Corynebacterium glutamicum is a key enzyme in fatty acid synthesis,which catalyzes the synthesis of malonyl-Co A from acetyl-Co A,thereby Acetyl-Co A carboxylase regulating the synthesis of fatty acids.In this experiment,Corynebacterium glutamicum ATCC 13032 was used as the research object,and acetyl-Co A carboxylase α subunit and β subunit recombinant strains were constructed,and through induced expression and response surface optimization,the yield of fatty acids was further increased.The results obtained as follows:1.Recombinant Escherichia coli BL-BC and BL-D1 were constructed by means of genetic engineering,and acetyl-Co A carboxylase α subunit(acc BC)was heterologously expressed in E.coli as β subunit(acc D1).The protein expression and fatty acid synthesis were analyzed.The results showed that the recombinant E.coli BL-BC and BL-D1 proteins showed protein bands in accordance with the theoretical values,which were approximately 63 k Da and58 k Da,respectively.Heterologous expression has a greater impact on the synthesis and composition of fatty acids.The fatty acid yields of recombinant E.coli BL-BC and BL-D1 are 45.57 mg/g dry cell weight and 18.81 mg/g dry cell weight,respectively,compared with the control strain The increase was215% and 30.35% respectively.Heterologous expression significantly increases the content of unsaturated fatty acids C16:1 and C17:1,while reducing the content of saturated fatty acids.2.Overexpressing its own acc BC and acc D1 genes in Corynebacterium glutamicum,SDS-PAGE results show protein bands consistent with theoretical values.Compared with the control strain G-19,the fatty acid synthesis of recombinant Corynebacterium glutamicum G-BC and G-D1 increased by162% and 33.07%,respectively,and the yields were 33.77 mg/g dry cell weight and 17.14 mg/g bacteria,respectively.Body weight.The endogenous expression of acc BC and acc D1 genes in Corynebacterium glutamicum increases the content of unsaturated fatty acids,and the total amount of unsaturated fatty acids can reach 50% of the total fatty acids.In both heterologous expression and homologous expression,it was found that the acc BC gene has a significantly greater influence on fatty acid synthesis in the recombinant strain than the acc D1 gene,but the influence on the fatty acid composition is the same.3.Perform single factor and response surface optimization experiments on recombinant Corynebacterium glutamicum G-BC and G-D1 respectively,and determine the optimal conditions for fatty acid synthesis as follows: the induction concentration is 1 m M,the culture temperature is 32 ℃,and the fermentation time is 20 h,the highest yields of recombinant Corynebacterium glutamicum G-BC and G-D1 fatty acids were 34.56 mg/g dry cell weight and17.32 mg/g dry cell weight,respectively.