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Stepwise Metabolic Engineering of Escherichia coli to Produce Triacylglycerol Rich in Medium-Chain Fatty Acids

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Stepwise Metabolic Engineering of Escherichia coli to Produce Triacylglycerol Rich in Medium-Chain Fatty Acids

Lin Xu et al. Biotechnol Biofuels.

Abstract

Background: Triacylglycerols (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.

Results: Here, 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.

Conclusions: We 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.

Keywords: Acyl-ACP thioesterase; Acyltransferase; Escherichia coli; Lipid droplet; Medium-chain fatty acid; Triacylglycerol.

Figures

Fig. 1
Fig. 1
Engineering of E. coli cells for the production of TAG rich in MCFA. Glycolytic pathway, fatty acid biosynthesis II pathway (FASII), fatty acid degradation pathway (β-oxidation), and the reconstructed acyl-CoA-dependent Kennedy pathway are shown in different background colours. Heterologous proteins are highlighted in red. LD lipid droplet, PAP phosphatidic acid phosphatase, MCFAs medium-chain fatty acids, TE acyl-ACP thioesterase, ACS acyl-CoA synthetase, WS/DGAT wax ester synthase/acyl-Coenzyme A:diacylglycerol acyltransferase, AccABCD acetyl-CoA carboxyltransferase, FabA 3-hydroxydecanoyl-ACP dehydrase, FabB 3-ketoacyl-ACP synthase I, FabD malonyl-CoA-ACP transacylase, FabF 3-ketoacyl-ACP synthase II, FabG 3-oxo-acyl-ACP reductase, FabH 3-ketoacyl-ACP synthase III, FabI enoyl-ACP reductase, TesA acyl-CoA thioesterase I, FadA 3-ketoacyl-CoA thiolase, FadB dodecenoyl-CoA delta-isomerase, FadD fatty acyl-CoA synthetase, FadE acyl-CoA dehydrogenase, GlpF glycerol MIP channel, GlpK glycerol kinase, PlsB glycerol-3-phosphate acyltransferase, PlsC 1-acylglycerol-3-phosphate O-acyltransferase, PgpB phosphatidylglycerophosphatase B, DgkA diacylglycerol kinase
Fig. 2
Fig. 2
Evaluation of bacterial WS/DGAT and PAP combinations for the production of TAGs in E. coli. AtfA and RoPAP/RjPAP combinations (a) and six different WS/DGATs and RoPAP combinations (b). Cells were cultured in ZYP-5052 auto-induction medium at 37 °C with shaking at 200 rpm for 48 h. Thin-layer chromatography (TLC) was carried out on lipids extracted from 10 mg of dried cells. tDGAT: WS/DGAT from T. curvata; AtfA: WS/DGAT from A. baylyi ADP1; AtfA_co: AtfA codon-optimized for E. coli; Atf8: WS/DGAT from R. jostii RHA1; Atf1/Atf2: WS/DGATs from R. opacus PD630. RoPAP: PAP from R. opacus PD630, RjPAP: PAP from R. jostii RHA1
Fig. 3
Fig. 3
Coexpression of RoFadD1 and RoTetRs along with tDGAT and RoPAP further increased TAG synthesis. Cells were cultured in ZYP-5052 auto-induction medium at 37 °C with shaking at 200 rpm for 48 h. Thin-layer chromatography (TLC) was carried out on lipids extracted from 5 mg of dried cells. tDGAT, WS/DGAT from T. curvata, RoPAP, PAP from R. opacus PD630. RoFadD1, putative acyl-CoA synthetase from R. opacus PD630. FadR, fatty acid metabolism regulator from E. coli MG1655; RoTetR1/2/3, three putative fatty acid metabolism regulators from R. opacus PD630. All data are the means ± standard deviations from triplicates
Fig. 4
Fig. 4
Assessment of medium-chain specific TE for diverting MCFA flux into TAG backbones. a MCFAs from the TAG fraction extracted from strain 2119 carrying different TEs. b TAG content and titer of strain 2119 carrying different TEs. c Fatty acid profile of the TAG fraction extracted from engineered strain 2119 carrying different TEs. d Time course of TAG production yielded by strain 2119 carrying pACYCDuet::RcFatB. Thin-layer chromatography (TLC) of lipid extracted from dried cells. e TAG profile and the distribution of C14 in the total TAGs from lipids extracted from strain 2119 and strain 2119 plus pACYCDuet::RcFatB, respectively. Strain 2119: E. coli BL21(DE3) carrying plasmid pCDFDuet::tDGAT/RoPAP::RoFadD1/RoTetR2. 48:1 represents 48 carbons with 1 double bond in three acyl chains. All data are the means ± standard deviations from triplicates
Fig. 5
Fig. 5
TAG induction through nutrient level optimization. Contour profiles of TAG content (a) and TAG titer (b) for strain 2119 harboring pACYCDuet::RcFatB cultured in 16 designed media with different ratios of glycerol and N-Z-amine. Floating cells were observed during the collection of cells (c). Increases in the TAG/MCFA content (d, e) and titer (f) realized from nutrient optimization. The detailed information on designed media is shown in Additional file 10: Table S1. Cells were cultured at 37 °C with shaking at 200 rpm for 48 h. All data are the means ± standard deviations from triplicates
Fig. 6
Fig. 6
Observation of LDs in engineered E. coli cells. Cells were observed by transmission electron microscopy (TEM) (a) and N-SIM super-resolution microscope (b). The engineered strain 2119 carrying pACYCDuet::RcFatB cultured in ZYP-5052 auto-induction medium at 37 °C with shaking at 200 rpm for 12, 24, 36 or 48 h. LDs were stained with Nile Red before observing by N-SIM. The scale bar is 1 µm for TEM and 4 µm for N-SIM

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