Stepwise metabolic engineering of Escherichia coli to produce triacylglycerol rich in medium-chain fatty acids

Abstract

BackgroundTriacylglycerols (TAGs) rich in medium-chain fatty acids (MCFAs, C10–14 fatty acids) are valuable feedstocks for biofuels and chemicals. Natural sources of TAGs rich in MCFAs are restricted to a limited number of plant species, which are unsuitable for mass agronomic production. Instead, the modification of seed or non-seed tissue oils to increase MCFA content has been investigated. In addition, microbial oils are considered as promising sustainable feedstocks for providing TAGs, although little has been done to tailor the fatty acids in microbial TAGs.ResultsHere, we first assessed various wax synthase/acyl-coenzyme A:diacylglycerol acyltransferases, phosphatidic acid phosphatases, acyl-CoA synthetases as well as putative fatty acid metabolism regulators for producing high levels of TAGs in Escherichia coli. Activation of endogenous free fatty acids with tailored chain length via overexpression of the castor thioesterase RcFatB and the subsequent incorporation of such fatty acids into glycerol backbones shifted the TAG profile in the desired way. Metabolic and nutrient optimization of the engineered bacterial cells resulted in greatly elevated TAG levels (399.4 mg/L) with 43.8% MCFAs, representing the highest TAG levels in E. coli under shake flask conditions. Engineered cells were observed to contain membrane-bound yet robust lipid droplets.ConclusionsWe introduced a complete Kennedy pathway into non-oleaginous E. coli towards developing a bacterial platform for the sustainable production of TAGs rich in MCFAs. Strategies reported here illustrate the possibility of prokaryotic cell factories for the efficient production of TAGs rich in MCFAs.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1177-x) contains supplementary material, which is available to authorized users.

The Phospholipid:Diacylglycerol Acyltransferase-Mediated Acyl-Coenzyme A-Independent Pathway Efficiently Diverts Fatty Acid Flux from Phospholipid into Triacylglycerol in Escherichia coli

“…PDAT and DGAT play overlapping roles in TAG synthesis in algae and plants (10,14). In addition, we previously constructed TAG-producing E. coli by overexpressing the bacterial WS/DGAT-and PAP-encoding genes atfA (from Acinetobacter baylyi) and RoPAP (from Rhodococcus opacus) (21). Therefore, we investigated if expressing CrPDAT would further increase TAG levels.…”

“…E. coli does not produce any TAGs naturally (18), mainly due to the lack of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT), which catalyzes the last and rate-limiting step of the acyl-CoA-dependent TAG synthesis pathway (19). The WS/DGAT-mediated acyl-CoA-dependent pathway has been successfully introduced into E. coli for the production of regular TAGs or TAGs rich in medium-chain fatty acids (20,21). Early strategies attempting the accumulation of TAGs were focused on the enhancement of fatty acid and/or acyl-CoA synthesis or on blocking the degradation of fatty acid and/or TAGs (20,(22)(23)(24).…”

Molecular and biochemical analysis of the castor caruncle reveals a set of unique genes involved in oil accumulation in non-seed tissues

“…The expression of RcFATB in Escherichia coli showed high catalytic efficiency with both oleoyl-ACP and palmitoyl-ACP [32]. Our recent work also showed the preference of RcFATB towards C14:0, and increased TAGs due to RcFATB activity in E. coli [33]. Overexpression of RcFATB alone in leaves resulted in significant increases in TAG accumulation, with strong preference for C16:0 accumulation among the TAGs.…”

Lipid Droplet Is an Ancient and Inheritable Organelle in Bacteria

“…Rothia dentocariosa (ATCC® 14189™), Bifidobacterium longum (ATCC® 15707™), [41]. 25% glutaraldehyde solution, 8% paraformaldehyde solution (EM grade), Embed 812 kit, uranyl acetate and lead citrate were all purchased from Electron Microscopy Sciences (Hatfield, USA).…”