Engineering E. coli for triglyceride accumulation through native and heterologous metabolic reactions

Abstract

Triglycerides, traditionally sourced from plant oils, are heavily used in both industrial and healthcare applications. Commercially significant products produced from triglycerides include biodiesel, lubricants, moisturizers, and oils for cooking and dietary supplements. The need to rely upon plant-based production, however, raises concerns of increasing demand and sustainability. The reliance on crop yields and a strong demand for triglycerides provides motivation to engineer production from a robust microbial platform. In this study, Escherichia coli was engineered to synthesize and accumulate triglycerides. Triglycerides were produced from cell wall phospholipid precursors through engineered expression of two enzymes, phosphatidic acid phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). A liquid chromatography-mass spectrometry (LC-MS) method was developed to analyze the production of triglycerides by the engineered E. coli strains. This proof-of-concept study demonstrated a yield of 1.1 mg/L triglycerides (2 g/L dry cell weight) in lysogeny broth medium containing 5 g/L glucose at 8 h following induction of PAP and DGAT expression. LC-MS results also demonstrated that the intracellular triglyceride composition of E. coli was highly conserved. Triglycerides containing the fatty acid distributions 16:0/16:0/16:1, 16:0/16:0/18:1, and 18:1/16:0/16:1 were found in highest concentrations and represent ∼70 % of triglycerides observed.

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

“…coli strains [12,18–22], little attention has been paid to culture times required for TAG production through these processes. Analysis of neutral lipid fractions of recombinant bacteria at different times after the induction of the expression of tDGAT showed the capacity of the engineered strain of producing TAGs in a very short period of time (Fig 2B).…”

“…In fact, mono unsaturated fatty acids or MUFA, have been reported to be the best components for biodiesel. Hence, the great demand of new bio-resources for the production of all triglyceride-derived products and, specifically, the need of optimizing a method for producing the optimal FAEEs that could be employed for diesel engines substituting the traditional plant-based oil production model, provides motivation to engineer production from a robust microbial platform [11,12]. However, an economic process for microbial production of TAGs at an industrial level has not been established yet.…”

“…coli strains with deleted genes [17–20] and in wild-type (WT) E . coli strains overexpressing additional proteins [12,19,21,22]. However, recombinant oil production through the heterologous expression of a WS/DGAT has not been achieved in WT E .…”

New Frontiers in the Production of Biodiesel: Biodiesel Derived from Macro and Microorganisms

“…For these reasons, genetic engineering is supporting this biotechnology to be considered a viable alternative for the biodiesel industry. Rucker et al (2013) demonstrated the feasibility of the lipid metabolism of E. coli for TAG accumulation, but the yield achieved was below the threshold to be considered a viable source for biodiesel production. Authors propose two metabolic engineering steps, to increase either the supply of phosphatidic acid during late exponential and stationary phase growth or the supply of acyl-CoA.…”

Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

“…[181] Metabolomics were recentlyu sedt oi dentify the successful metabolic engineering of E. coli for triglyceride accumulation; within this study ah ighc onservation of triglyceride composition was confirmed. [182] Another recent example for the success of metabolomics as ac ontrol strategy wasd isclosed by metabolicf lux redirection towards as ynthetic pathway with am etabolict oggle switch for changing the E. coli metabolism to isopropyl alcohol production. Metabolomics were usedt o follow the changes in the intracellular fluxes.…”