Bioproduction Of C16and C18Higher Fatty Alcohols By Engineered E. Coli

Oxyethylene higher aliphatic alcohols (OHAA) are new fresh-keeping materials with many advantages, such as harmless to humans, easy to use and effective in improving the overall quality and extended shelf life. The C16and C18fatty alcohols are the main feedstock used for OHAA production. Now the fatty alcohol market is dominated by natural alcohol and synthetic alcohol products. The natural alcohols are prepared from natural oil such as coconut oil and palm oil and the synthetic alcohols are produced from petrochemical feedstocks. However, these processes either need harsh production environments, or bring harmful materials to the environment. Besides, they are both now facing the huge challenge from the increasingly higher price of the raw materials. Therefore, increasing attentions have been paid to the microbial production of fatty alcohols from renewable resources.This study mainly employs the de novo fatty acid synthetic pathway of microbes for the production of fatty alcohols. To better improve the production of fatty alcohols, we firstly carried out an investigation on the biosynthesis of free fatty acids (FFAs), which are important intermediates in the fatty alcohol production. To enhance the production of FFAs, seven engineered E. coli strains were successfully constructed by overexpressing seven different thioesterases, respectively. Next, taking one engineered strain as an example, an optimization of the production conditions was carried out to improve the production of FFAs. The optimized conditions are:37.8g/L Na2HPO4-12H2O and7.5g/L KH2PO4were used as the concentration of the phosphates; the engineered strains were induced with0.25mM IPTG at30℃. Under this condition, the FFAs production of seven engineered strains was determined by gas chromatography (GC). The strains LL1, LL2and LL4, which overexpress BTE, AtFatA and ’tesA, respectively, achieved346.4mg/L,137.2mg/L and288.5mg/L FFAs, respectively. But the other engineered strains did not obviously enhance the production of FFAs. In addition, BTE, AtFatA and’TesA was found to have different substrate specificities by analyzing the composition of FFAs they achieved. BTE prefers the C12acyl-ACPs, AtFatA prefers the C16and C18acyl-ACPs, and’TesA possesses a much broader substrate preference towards C14-C18. Based on the analysis of substrate specificities and FFA production of BTE, AtFatA and’TesA,’tesA was finally chosen as the thioesterase gene for the production of C16and C18fatty alcohols.Having obtained the proper thioesterase and improved the production of FFAs, investigations on the acyl-CoA synthase and fatty acyl-CoA reductase in the fatty alcohol biosynthetic pathway were preformed. The fatty acyl-CoA reductase FAR was found to have a substrate preference towards C16and C18acyl-CoAs by determining its substrate specificity in vivo. Therefore, it can be used for the production of C16and C18higher fatty alcohols. In addition, compared with the acyl-CoA synthases Yngl and FAA2, acyl-CoA synthase FadD exhibited a much higher catalytic activity. The engineered strain overexpressing fadD gene achieved the highest fatty alcohol production. Finally, an optimal engineered strain ZhO54was obtained by doing optimization of higher fatty alcohol biosynthetic pathway. ZhO54produced C16and C18higher fatty alcohols with high specificity, C16and C18alcohols accounting for89.2%of the total fatty alcohols. In addition, ZhO54achieved101.5mg/L C16and C18higher fatty alcohols in the fed-batch fermentation, representing the highest titer reported so far.This study presents a green and environment-friendly production process of higher fatty alcohol, open up a new relm for higher fatty alcohol production, and provides the groundwork for microbial production of a surfactant range of fatty alcohols from the rich lignocellulose sources.