Title: Engineering a Synthetic Escherichia coli Coculture for Compartmentalized de novo Biosynthesis of Isobutyl Butyrate from Mixed Sugars

Abstract

Short-chain esters are versatile chemicals that can be used as flavors, fragrances, solvents, and fuels. The de novo ester biosynthesis consists of diverging and converging pathway submodules, which is challenging to engineer to achieve optimal metabolic fluxes and selective product synthesis. Compartmentalizing the pathway submodules into specialist cells that facilitate pathway modularization and labor division is a promising solution. Here, we engineered a synthetic Escherichia coli coculture with the compartmentalized sugar utilization and ester biosynthesis pathways to produce isobutyl butyrate from a mixture of glucose and xylose. To compartmentalize the sugar-utilizing pathway submodules, we engineered a xylose-utilizing E. coli specialist that selectively consumes xylose over glucose and bypasses carbon catabolite repression (CCR) while leveraging the native CCR machinery to activate a glucose-utilizing E. coli specialist. We found that the compartmentalization of sugar catabolism enabled simultaneous co-utilization of glucose and xylose by a coculture of the two E. coli specialists, improving the stability of the coculture population. Next, we modularized the isobutyl butyrate pathway into the isobutanol, butyl-CoA, and ester condensation submodules, where we distributed the isobutanol submodule to the glucose-utilizing specialist and the other submodules to the xylose-utilizing specialist. Upon compartmentalization of the isobutyl butyrate pathway submodules into these sugar-utilizingmore » specialist cells, a robust synthetic coculture was engineered to selectively produce isobutyl butyrate, reduce the biosynthesis of unwanted ester byproducts, and improve the production titer as compared to the monoculture.« less

Authors:

Seo, Hyeongmin ^[1]; Castro, Gillian ^[2]; 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. Department of Chemical and Biomolecular Engineering, The University of Tennessee, Knoxville, Tennessee 37996, United States, Center of Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830, United States
2. Department of Chemical and Biomolecular Engineering, The University of Tennessee, Knoxville, Tennessee 37996, United States

Publication Date:

Wed Dec 13 00:00:00 EST 2023

Research Org.:

Univ. of Tennessee, Knoxville, TN (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

2229797

Alternate Identifier(s):

OSTI ID: 2283559

Grant/Contract Number:  

SC0022226; AC05-000R22725; AC02-05CH11231

Resource Type:

Published Article

Journal Name:

ACS Synthetic Biology

Additional Journal Information:

Journal Name: ACS Synthetic Biology Journal Volume: 13 Journal Issue: 1; Journal ID: ISSN 2161-5063

Publisher:

American Chemical Society

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; coculture; Escherichia coli; carbon catabolite repression; bioester; isobutyl butyrate; diverging pathway; converging pathway; alcohol acyltransferases