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Title: Microbial biosynthesis of lactate esters

Abstract

Abstract Background Green organic solvents such as lactate esters have broad industrial applications and favorable environmental profiles. Thus, manufacturing and use of these biodegradable solvents from renewable feedstocks help benefit the environment. However, to date, the direct microbial biosynthesis of lactate esters from fermentable sugars has not yet been demonstrated. Results In this study, we present a microbial conversion platform for direct biosynthesis of lactate esters from fermentable sugars. First, we designed a pyruvate-to-lactate ester module, consisting of a lactate dehydrogenase ( ldhA ) to convert pyruvate to lactate, a propionate CoA-transferase ( pct ) to convert lactate to lactyl-CoA, and an alcohol acyltransferase ( AAT ) to condense lactyl-CoA and alcohol(s) to make lactate ester(s). By generating a library of five pyruvate-to-lactate ester modules with divergent AATs, we screened for the best module(s) capable of producing a wide range of linear, branched, and aromatic lactate esters with an external alcohol supply. By co-introducing a pyruvate-to-lactate ester module and an alcohol (i.e., ethanol, isobutanol) module into a modular Escherichia coli (chassis) cell, we demonstrated for the first time the microbial biosynthesis of ethyl and isobutyl lactate esters directly from glucose. In an attempt to enhance ethyl lactate production as amore » proof-of-study, we re-modularized the pathway into (1) the upstream module to generate the ethanol and lactate precursors and (2) the downstream module to generate lactyl-CoA and condense it with ethanol to produce the target ethyl lactate. By manipulating the metabolic fluxes of the upstream and downstream modules through plasmid copy numbers, promoters, ribosome binding sites, and environmental perturbation, we were able to probe and alleviate the metabolic bottlenecks by improving ethyl lactate production by 4.96-fold. We found that AAT is the most rate-limiting step in biosynthesis of lactate esters likely due to its low activity and specificity toward the non-natural substrate lactyl-CoA and alcohols. Conclusions We have successfully established the biosynthesis pathway of lactate esters from fermentable sugars and demonstrated for the first time the direct fermentative production of lactate esters from glucose using an E. coli modular cell. This study defines a cornerstone for the microbial production of lactate esters as green solvents from renewable resources with novel industrial applications.« less

Authors:
; ORCiD logo
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1618760
Alternate Identifier(s):
OSTI ID: 1627003
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Published Article
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Name: Biotechnology for Biofuels Journal Volume: 12 Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
Springer Science + Business Media
Country of Publication:
Netherlands
Language:
English
Subject:
09 BIOMASS FUELS; Biotechnology & Applied Microbiology; Energy & Fuels; Ester; Lactate ester; Ethyl lactate; Isobutyl lactate; Acetate ester; Alcohol acyltransferase; Green solvent; Modular cell; Escherichia coli

Citation Formats

Lee, Jong-Won, and Trinh, Cong T. Microbial biosynthesis of lactate esters. Netherlands: N. p., 2019. Web. https://doi.org/10.1186/s13068-019-1563-z.
Lee, Jong-Won, & Trinh, Cong T. Microbial biosynthesis of lactate esters. Netherlands. https://doi.org/10.1186/s13068-019-1563-z
Lee, Jong-Won, and Trinh, Cong T. Fri . "Microbial biosynthesis of lactate esters". Netherlands. https://doi.org/10.1186/s13068-019-1563-z.
@article{osti_1618760,
title = {Microbial biosynthesis of lactate esters},
author = {Lee, Jong-Won and Trinh, Cong T.},
abstractNote = {Abstract Background Green organic solvents such as lactate esters have broad industrial applications and favorable environmental profiles. Thus, manufacturing and use of these biodegradable solvents from renewable feedstocks help benefit the environment. However, to date, the direct microbial biosynthesis of lactate esters from fermentable sugars has not yet been demonstrated. Results In this study, we present a microbial conversion platform for direct biosynthesis of lactate esters from fermentable sugars. First, we designed a pyruvate-to-lactate ester module, consisting of a lactate dehydrogenase ( ldhA ) to convert pyruvate to lactate, a propionate CoA-transferase ( pct ) to convert lactate to lactyl-CoA, and an alcohol acyltransferase ( AAT ) to condense lactyl-CoA and alcohol(s) to make lactate ester(s). By generating a library of five pyruvate-to-lactate ester modules with divergent AATs, we screened for the best module(s) capable of producing a wide range of linear, branched, and aromatic lactate esters with an external alcohol supply. By co-introducing a pyruvate-to-lactate ester module and an alcohol (i.e., ethanol, isobutanol) module into a modular Escherichia coli (chassis) cell, we demonstrated for the first time the microbial biosynthesis of ethyl and isobutyl lactate esters directly from glucose. In an attempt to enhance ethyl lactate production as a proof-of-study, we re-modularized the pathway into (1) the upstream module to generate the ethanol and lactate precursors and (2) the downstream module to generate lactyl-CoA and condense it with ethanol to produce the target ethyl lactate. By manipulating the metabolic fluxes of the upstream and downstream modules through plasmid copy numbers, promoters, ribosome binding sites, and environmental perturbation, we were able to probe and alleviate the metabolic bottlenecks by improving ethyl lactate production by 4.96-fold. We found that AAT is the most rate-limiting step in biosynthesis of lactate esters likely due to its low activity and specificity toward the non-natural substrate lactyl-CoA and alcohols. Conclusions We have successfully established the biosynthesis pathway of lactate esters from fermentable sugars and demonstrated for the first time the direct fermentative production of lactate esters from glucose using an E. coli modular cell. This study defines a cornerstone for the microbial production of lactate esters as green solvents from renewable resources with novel industrial applications.},
doi = {10.1186/s13068-019-1563-z},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 12,
place = {Netherlands},
year = {2019},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1186/s13068-019-1563-z

Citation Metrics:
Cited by: 4 works
Citation information provided by
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Figures / Tables:

Fig. 1 Fig. 1: In vivo characterization of various alcohol acyltransferases for biosynthesis of lactate esters. a Biosynthesis pathways of lactate and acetate esters with external supply of alcohols. b Ester production of EcJW101, EcJW102, EcJW103, EcJW104, and EcJW105 harboring ATF1, ATF2, SAAT , VAAT , and atfA, respectively in high cellmore » density cultures with various alcohol doping. Each error bar represents 1 standard deviation (s.d., n = 3). Symbols: n.d. not detected, n.s. not significant, *p < 0.073, and **p < 0.013 (Student’s t-test). c The library of esters produced. Green check marks indicate the esters produced in this study while red star marks indicate the esters produced for first time in engineered strains« less

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