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

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

Biology of triacylglycerol accumulation by Rhodococcus

Members of the genus Rhodococcus are specialist in the accumulation of triacylglycerols (TAG). Some of them can be considered oleaginous microorganisms since they are able to produce significant amounts of those lipids under certain conditions. In this context, R. opacus strain PD630 and R. jostii RHA1 became models among prokaryotes in this research area. The basic knowledge generated for rhodococci could be also extrapolated to related microorganisms with clinical importance, such as mycobacteria. The biosynthesis and accumulation of TAG by species of the genus Rhodococcus and other actinomycetes seems to be a process linked to the stationary growth phase or as a response to stress. The chemical structure of rhodococcal TAG can be controlled by the composition of the carbon source used. The biosynthesis and accumulation of novel TAG containing unusual components, such as aromatic and isoprenoid fatty acids, by members of Rhodococcus and related genera has been reported. The low specificity of wax ester synthase/diacylglycerol acyltransferase enzymes (WS/DGAT), which catalyze TAG biosynthesis in prokaryotes, may contribute to the high variability of TAG composition. The occurrence of genes coding for WS/DGAT enzymes is highly redundant in rhodococcal genomes. The enrichment of genes and enzymes involved in TAG metabolism in rhodococci suggest an important role of these lipids in the physiology of these microorganisms. Genomic, transcriptomic and proteomic data from TAG-accumulating rhodococci are now available and some genes coding for enzymes of the central metabolism, the Kennedy pathway, lipid transporter proteins, structural lipid inclusion bodies associated proteins, and transcriptional regulatory proteins have been identified and characterized. This article aims to summarize the most relevant achievements of basic research in this field, including the most recent knowledge emerged from studies on TAG accumulation by rhodococci.Fil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia "San Juan Bosco". Instituto de Biociencias de la Patagonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biociencias de la Patagonia; ArgentinaFil: Steinbüchel, Alexander. Westfälische Wilhelms Universität; Alemania. King Abdulaziz University; Arabia Saudit