Title: Controlling selectivity of modular microbial biosynthesis of butyryl-CoA-derived designer esters

Abstract

Short-chain esters have broad utility as flavors, fragrances, solvents, and biofuels. Controlling selectivity of ester microbial biosynthesis has been an outstanding metabolic engineering problem. In this study, we enabled the de novo fermentative microbial biosynthesis of butyryl-CoA-derived designer esters (e.g., butyl acetate, ethyl butyrate, butyl butyrate) in Escherichia coli with controllable selectivity. Using the modular design principles, we generated the butyryl-CoA-derived ester pathways as exchangeable production modules compatible with an engineered chassis cell for anaerobic production of designer esters. We designed these modules derived from an acyl-CoA submodule (e.g., acetyl-CoA, butyryl-CoA), an alcohol submodule (e.g., ethanol, butanol), a cofactor regeneration submodule (e.g., NADH), and an alcohol acetyltransferase (AAT) submodule (e.g., ATF1, SAAT) for rapid module construction and optimization by manipulating replication (e.g., plasmid copy number), transcription (e.g., promoters), translation (e.g., codon optimization), pathway enzymes, and pathway induction conditions. To further enhance production of designer esters with high selectivity, we systematically screened various strategies of protein solubilization using protein fusion tags and chaperones to improve the soluble expression of multiple pathway enzymes. Finally, our engineered ester-producing strains could achieve 19-fold increase in butyl acetate production (0.64 g/L, 96% selectivity), 6-fold increase in ethyl butyrate production (0.41 g/L, 86% selectivity), and 13-foldmore » increase in butyl butyrate production (0.45 g/L, 54% selectivity) as compared to the initial strains. Altogether, this study presented a generalizable framework to engineer modular microbial platforms for anaerobic production of butyryl-CoA-derived designer esters from renewable feedstocks.« less

Authors:

Lee, Jong-Won ^[1]; Trinh, Cong T.  ^[1]

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• Search DOE PAGES for ORCID "0000-0002-8362-725X"

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+ Show Author Affiliations

1. Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

Publication Date:

Mon Dec 06 00:00:00 EST 2021

Research Org.:

Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1864486

Alternate Identifier(s):

OSTI ID: 1847495

Grant/Contract Number:  

SC0022226; AC05-00OR22725; AC02-05CH11231

Resource Type:

Accepted Manuscript

Journal Name:

Metabolic Engineering

Additional Journal Information:

Journal Volume: 69; Journal ID: ISSN 1096-7176

Publisher:

Elsevier

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Esters; Butyl acetate; Ethyl butyrate; Butyl butyrate; Alcohol acyltransferase; AAT; ATF1; SAAT; Modular design; Modular cell; Modular pathway design; Codon optimization; Fusion partners; Chaperones; Soluble expression; Enzyme solubilization; Escherichia coli