(175x) De Novo Production of a Biodegradable Solvent in Escherichia coli via Metabolic Engineering

With the advent of environmental safety policies and development of sustainable industrialization practices, global efforts are being made towards the use of biodegradable organic solvents like lactate esters. However, chemical esterification process for their production is thermodynamically unfavourable. Thus, their synthesis using genetically modified microbial cell factories becomes a viable alternative.

Our study presents metabolic engineering of Escherichia coli for production of ethyl lactate through heterologous expression of genes encoding for acyl-alcohol transferase (AcAlT). Brettanomyces bruxellensis AWRI1499 was selected to mine for enzymes based on previous literature that suggested the formation of ethyl lactate. The study encompasses three step substantiation; in silico analysis to discover homologous enzyme sequences; cloning, purification and in vitro catalysis to determine enzyme functionality; and in vivo metabolic modulations for recombinant synthesis of ethyl lactate.

On the basis of sequence homology with fatty acyl-alcohol transferase that catalyses esterification of fatty acids with alcohols, we discovered four enzymes from the yeast viz. diacylglycerol acyltransferase (602 bp), ethanol-o-acyltransferase (1120 bp), acetyl-xylan esterase (984 bp), and carbohydrate esterase family-9 (1289 bp) using blastp analysis. In vitro catalysis reactions using purified enzymes from recombinant strains and LC-MS data analysis yielded significant ethyl lactate formation, ensuring effective enzyme functionality. Therefore, we cloned these sequences in E.coli along with lactate dehydrogenases (glda101 for D-Lactate production from Bacillus coagulans or ldh for L-Lactate production from E.coli BL21) and alcohol dehydrogenase (adh) genes that can improve in vivo substrate flux towards AcAlT catalysis for de novo ethyl lactate production. We further determined effect of extracellular supplementation of the substrates on the product synthesis.

Hence, hereby we demonstrate E.coli as efficient platform for recombinant synthesis of green solvents like ethyl lactate. Owing to faster growth, 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.