The Effect Of Thioesterase On Fatty Acid Synthesis In Rhodococcus Opacus PD630

In the fatty acid synthesis pathway of Rhodococcus opacus PD630, thioesterase hydrolysis fatty-ACP(acyl carrier protein) to generate fatty acid and ACP, which can relieve the feedback inhibition of fatty-ACP on fatty acid biosynthesis regulation. Fatty acids are the key precursers for the synthesis of triacylglycerol, phospholipid and sterol. In addition,thioesterase has substrate specificity and is related to fatty acid composition of strain.Thioesterase may be a key gene for improving lipids accumulation in R. opacus PD630.Making research on thioesterase of this strain will bring a feasible way to produce biofuel. So far, the thioesterase gene for fatty acid synthesis in R. opacus PD630 has not been studied. In this paper, four thioesterases(TE1 to TE4) from R. opacus PD630 were overexpressed respectively in Escherichia coli and R. opacus PD630 to investigate their effect on fatty acid biosynthesis. The specific contents of researches are as follows:Four thioesterases were overexpressed in E. coli BL21(DE3) respectively, resulting recombinant strains BLTE1 to BLTE4. Those strains were cultured under the same condition,four thioesterses proteins overexpressed in cells of them were mostly soluble. Then the content and composition of total fatty acid in those strains were analyzed. In comparison to the control strain BL1, the content of total fatty acid in strains BLTE2 and BLTE4 increased34.6% and 21.3%, respectively, while the total fatty acid production of strains BLTE1 and BLTE3 didn’t increase. Overexpression of four thioesterases in recombinant strains led to huge changes in fatty acid composition. Four recombinants had considerable increases in the contents of 16:1 and 18:1 by 1- to 2-fold and 5- to 7-fold, respectively, and led to a 45% to60% decrease in the content of 14:0, 20% to 50% decrease in the content of 16:0, more than80% decrease in the content of 17:0 and 18:0 can’t be detected. Thus, it suggested that the four thioesterases all have biological activity and substrate preference towards C16:1- and C18:1-ACPs in cells of E. coli.Then the four thioesterases were overexpressed in R. opacus PD630 to construct recombinant strains PDTE1 to PDTE4. We analysed the growth condition, total lipid content and fatty acid composition of those strains which were cultured under the same condition. We found that recombinant strains grew more slow than the control strain PDPJAM2, the former entered exponential phase after 24 h and stationary phase after 72 h, while the later entered exponential phase after 18 h and stationary phase after 60 h. The content of total fatty acid in strains PDTE2 and PDTE4 reached 44-45%(w/w) of cell dry weight, which showed an increase of the lipid by 28% in comparison to strain PDPJAM2. Meanwhile, the contents of16:0 and 17:0 fatty acids decreased by 24% and 55-65% respectively in strains PDTE2 and PDTE4, the contents of 16:1, 17:1 and 18:1 all increased 35% in PDTE2, the contents of 17:1and 18:1 increased 55% and 34% respectively in PDTE4. However, strains PDTE1 and PDTE3 showed no obvious changes in the content of lipid and fatty acid composition.This work demonstrated that TE2 and TE4 related to the fatty acid synthesis of R. opacus PD630 could improve the content of total fatty acid and also regulated composition of them in R. opacus PD630. This work also provided a feasible strategy for enhancing biodieselproduction in engineered R. opacus PD630 strain.