Identification Of Acyl CoA Thioesterase Functional Protein In Corynebacterium Glutamicum And Its Effect On Fatty Acid Synthesis

Corynebacterium glutamicum,as an important industrial strain,is commonly used in the production of food and drug grade amino acids.It has type I fatty acid synthase and has potential in fatty acid synthesis.Thioesterase exists widely in animals,plants and microorganisms.It can catalyze the hydrolysis of acyl CoA and acyl carrier protein(ACP)to produce coenzyme A,acyl carrier protein and other sulfhydryl compounds,and release free fatty acids.In this study,three genes of C.glutamicum ATCC13032 with acyl CoA thioesterase activity were studied,namely(Cgl2451,NCgl2365),(Cgl1664,NCgl1600)and(Cgl0091,NCgl0090).Through heterologous and homologous overexpression,the key proteins with acyl CoA thioesterase function and their enzymatic properties were identified,and the effects of genes NCgl2365,NCgl1600 and NCgl0090 on fatty acid metabolism in host cells were studied.The results are as follows:(1)The results showed that TE-5(Cgl2451,NCgl2365)had the highest activity on the long-chain fatty acyl CoA substrate,the optimum reaction temperature was 30?,the optimum pH was 8.0,Fe2+,Mn2+,Zn2+,Mg2+and K+had inhibitory effect on the enzyme activity.When TE-5 protease used C12 fatty acyl CoA as substrate,the Michaelis equation wasY=10.1X+0.00731(R2=0.991),the maximum reaction rate was 136.79 ?mol/L/min,Km value was 1.38 mmol/L.TE-6(Cgl1664,NCgl1600)has the highest activity for medium chain acyl CoA,the optimum reaction temperature is 30?,the optimum pH is 8.0,Fe2+,Cu2+,Zn2+,Mg2+and K+have inhibitory effect on the enzyme activity.When TE-6 protease uses C8 fatty acyl CoA as substrate,the Michaelis equation is Y=9.75X+0.00179(R2=0.997),the maximum reaction speed Vmax is 558.6 ?mol/L/min,Km value is 5.45 mmol/L.The results showed that TE-9(Cgl0091,NCgl0090)had the highest activity for short and medium chain acyl CoA,the optimum reaction temperature was 300C,the optimum pH was 9.0,Mn2+enhanced the relative activity of the enzyme,while Fe2+,Cu2+,Zn2+,Mg2+and K+inhibited the activity.When TE-9 protease used C2 fatty acyl CoA as substrate,the Michaelis equation was Y=246X+0.136(R2=0.992),the maximum reaction rate was 7.35 ?mol/L/min,Km value was 1.81 mmol/L.(2)The recombinant strains BL-5,BL-6 and BL-9 were obtained by overexpression of NCgl2365,NCgl1600 and NCgl0090 genes in E.coli BL21.In the heterologous expression genes,we found that the genes NCgl0090,NCgl2365 and NCgl1600 affected the total synthesis of fatty acids in E.coli in descending order.The total fatty acid production of BL-9,BL-5 and BL-6 increased by 58%,38%and 19%respectively.The genes Ncgl2365,NCgl1600 and NCgl0090 promoted the production of saturated fatty acids C16:0,C18:0 and unsaturated fatty acids C17:1.Compared with the control strain,C16:0 and C17:1 were the two main fatty acids in the recombinant strain,and the percentages of C12:0,C16:1 and C18:1 were significantly lower.(3)Recombinant strains G-5,G-6 and G-9 were obtained by homologous expression of genes NCgl2365,NCgl1600 and NCgl0090 in C.glutamicum ATCC13032.The recombinant strains were induced by IPTG with final concentration of 1 mM and cultured for 20 h.The results of fatty acid analysis showed that NCgl0090 gene had the greatest effect on the total fatty acid synthesis,followed by NCgl1600 and NCgl2365 genes,and the recombinant strains G-9,G-6 and G-5 were increased by 59%,46%and 33%respectively.In terms of fatty acid types,NCgl1600 and NCgl0090 increase fatty acid types,while NCgl2365 has little effect on fatty acid types.Compared with the control strain,the proportion of C17:0 fatty acid in the recombinant strain G-5 increased by 80%,followed by the proportion of C16:0 fatty acid increased significantly,and the proportion of C12:0,C17:1,C18:0,C18:1 decreased.The proportion of C17:0 fatty acid in G-6 increased by 62%,followed by the proportion of C17:1 fatty acid doubled,the proportion of C14:0,C15:0,C18:2 and C20:1 also increased significantly,and the proportion of C12:0,C18:0 and C18:1 decreased.Among the recombinant strains G-9,C16:0 had the highest content of fatty acids in the recombinant bacteria,and its proportion increased by 40%.The proportions of C17:1,C18:0,C18:2,C20:1 and C21:0 increased,and the proportion of C17:1 increased more than twice;the proportions of C12:0,C17:0 and C18:1 decreased.