With the advent of environmental safety policies and development of sustainable industrialization practices, global research has been geared towards the use of biodegradable solvents like esters from renewable feedstocks. While majority of the biological esterification studies involve the use of acyltransferase enzymes, exploring esterases for esterification would be advantageous in two ways; their precursors can be synthesized in relatively high titers and is one step short as compared to the acyltransferase pathway. To verify our approach, E. coli strains were constructed for the heterologous expression of four enzymes diacylglycerol transferase, ethanol-o-acyltransferase, acetylxylan esterase, carbohydrate esterase) from Brettanomyces bruxellensis AWRI1499, yeast well known for its role in wine fermentation. However, the gene sequences for the selected protein candidates were not completely curated in the GenBank. Bioinformatics analysis was performed to curate distinct gene/peptide sequences to replace inappropriate nucleotides based on polynucleotide and/or polypeptide sequence homologies, followed by their codon optimization. Esterase A (EstA) from Pseudomonas aeruginosa was selected as a bacterial esterase. All the five sequences were cloned in E. coli for ethyl lactate and ethyl acetate analysis. Based on preliminary high-cell density fermentation (lab-scale) of these five strains, two were selected for bioreactor optimizations, SSL74 (possessing CE, Carbohydrate esterase) and SSL76 (possessing EstA). Fed-batch fermentation at pH 7.0 exhibited 10 mg/L ethyl lactate and 81 mg/L ethyl acetate by both SSL76 and SSL74 strains, denoting comparable functionality of both the esterases. Fed-batch fermentation at pH 6.0 with SSL76 showed promising increasing in titers, 18.2 mg/L ethyl lactate and 225 mg/L ethyl acetate. These are the highest reported ethyl lactate titers from E. coli. Importantly, the study reveals commercial potential of these underexplored esterases for industrial esterification reactions. They can be foreseen as efficient candidates for in vivo as well as in vitro enzyme catalysis at commercial scales.

Esterase catalyzed biosynthesis of short-chain esters from engineered E. coli

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ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.