(160l) Metabolic Engineering of E. coli for the Sustainable Production of Short-Chain Esters

Teaching interest: Biochemistry, Biotechnology, Genetic engineering, Microbiology

Biological production of industrially valuable chemicals from renewable feedstocks imparts sustainability to the manufacturing sector. With the advent of environmental safety policies and development of sustainable industrialization practices, efforts are being made towards the use of biodegradable organic solvents like lactate esters. In this work, we have constructed engineered E. coli to produce ethyl lactate from renewable substrates. As a first step, bioprospecting of ester producing enzymes was done and the following seven were selected: diacylglycerol-transferase, ethanol-o-acyltransferase, acetylxylan-esterase, carbohydrate-esterase; acyl-coenzymeA:ethanol O-acyltransferase, ethyl-ester-synthase-1; and esterase-A. In vitro assays were conducted to identify the optimal pH and temperature for these novel enzymes. Recombinant E. coli strains were constructed by expressing the esterase and acyl-alcohol transferase in E. coli strains engineered for overproducing ethanol and lactic acid. High-cell density fermentations were conducted under microaerobic conditions and the strain expressing esterase-A produced the highest ethyl lactate (3.5±1.5 mg/L) and ethyl acetate (16±8 mg/L) from glucose as a carbon source. Further work is underway, to increase the production rate and titer.

Finally, owing to the faster growth of the microbial host, minimal nutritional requirement of the organism, and extracellular release of ethyl lactate, continuous fermentation and product extraction strategy could be successfully implemented for industrial scale-up using these strains.