Heterologous expression of a thermophilic diacylglycerol acyltransferase triggers triglyceride accumulation in Escherichia coli

Abstract

Triglycerides (TAGs), the major storage molecules of metabolic energy and source of fatty acids, are produced as single cell oil by some oleogenic microorganisms. However, these microorganisms require strict culture conditions, show low carbon source flexibilities, lack efficient genetic modification tools and in some cases pose safety concerns. TAGs have essential applications such as behaving as a source for added-value fatty acids or giving rise to the production of biodiesel. Hence, new alternative methods are urgently required for obtaining these oils. In this work we describe TAG accumulation in the industrially appropriate microorganism Escherichia coli expressing the heterologous enzyme tDGAT, a wax ester synthase/triacylglycerol:acylCoA acyltranferase (WS/DGAT). With this purpose, we introduce a codon-optimized gene from the thermophilic actinomycete Thermomonospora curvata coding for a WS/DGAT into different E. coli strains, describe the metabolic effects associated to the expression of this protein and evaluate neutral lipid accumulation. We observe a direct relation between the expression of this WS/DGAT and TAG production within a wide range of culture conditions. More than 30% TAGs were detected within the bacterial neutral lipids in 90 minutes after induction. TAGs were observed to be associated with the hydrophobic enzyme while forming round intracytoplasmic bodies, which could represent a bottleneck for lipid accumulation in E. coli. We detected an increase of almost 3-fold in the monounsaturated fatty acids (MUFA) occurring in the recombinant strains. These MUFA were predominant in the accumulated TAGs achieving 46% of the TAG fatty acids. These results set the basis for further research on the achievement of a suitable method towards the sustainable production of these neutral lipids.

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

“…4 d). This was consistent with the previous results, where TAGs were detected after induction of tDGAT expression for only 30 min [ 18 ]. Compared to oleaginous bacteria and yeasts, a shorter fermentation time was observed for TAG accumulation in engineered E .…”

“…Unlike AtfA or Sco0958 (WS/DGAT from S . coelicolor ), overexpression of a solo tDGAT was enough to accumulate considerable TAGs with a threefold increase in monounsaturated fatty acids in E. coli C41(DE3) [ 18 , 21 , 24 ]. Combined with the data here, tDGAT was highly active in E. coli, and it has the potential for use in the production of functional lipids.…”

“…The predicted three-dimensional structure of tDGAT revealed that it had a longer helical linker between the N- and C-terminal domains. In addition, it has been predicted as the most hydrophobic protein among existing representative WS/DGATs, which led to relatively difficult purification [ 18 ].…”

“…We also observed that lowering the culturing temperature from 37 to 30 °C caused drastically decreased TAGs in strain 2119 (Additional file 7 : Figure S7). Because tDGAT was isolated from a thermophilic actinobacterium with an optimal growing temperature of 50 °C [ 18 ]. Thus, it was likely that tDGAT exhibited higher activity at 37 °C compared to 30 °C.…”

“…In addition, a thermophilic tDGAT from Thermomonospora curvata was shown to yield over 30% of TAGs to neutral lipids in 30 min in E . coli [ 18 ]. It would be interesting to compare the abilities of these newly isolated WS/DGATs to atfA.…”