Title: Improving microbial biogasoline production in Escherichia coli using tolerance engineering

Abstract

Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as Escherichia coli. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in E. coli. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerance phenotypes belonged to diverse functional groups, such as oxidative stress response (soxS, fpr, and nrdH), general stress response (metR, yqhD, and gidB), heat shock-related response (ibpA), and transport (mdlB). To determine if these genes could also improve isopentenol production, we coexpressed the tolerance-enhancing genes individually with an isopentenol production pathway. Our data show that expression of 6 of the 8 candidates improved the production of isopentenol in E. coli, with the methionine biosynthesis regulator MetR improving the titer for isopentenol production by 55%. Additionally, expression of MdlB, an ABC transporter, facilitated a 12% improvement in isopentenol production. To our knowledge,more » MdlB is the first example of a transporter that can be used to improve production of a short-chain alcohol and provides a valuable new avenue for host engineering in biogasoline production.The use of microbial host platforms for the production of bulk commodities, such as chemicals and fuels, is now a focus of many biotechnology efforts. Many of these compounds are inherently toxic to the host microbe, which in turn places a limit on production despite efforts to optimize the bioconversion pathways. In order to achieve economically viable production levels, it is also necessary to engineer production strains with improved tolerance to these compounds. We demonstrate that microbial tolerance engineering using transcriptomics data can also identify targets that improve production. Our results include an exporter and a methionine biosynthesis regulator that improve isopentenol production, providing a starting point to further engineer the host for biogasoline production.« less

Authors:

Foo, Jee Loon ^[1]; Jensen, Heather M. ^[2]; Dahl, Robert H. ^[3]; George, Kevin ^[2]; Keasling, Jay D. ^[4]; Lee, Taek Soon ^[2]; Leong, Susanna ^[5]; Mukhopadhyay, Aindrila ^[2]

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+ Show Author Affiliations

1. Nanyang Technological Univ. (Singapore). School of Chemical and Biomedical Engineering; Joint BioEnergy Inst., Emeryville, CA (United States); National Univ. of Singapore (Singapore). Dept. of Biochemistry.
2. Joint BioEnergy Inst., Emeryville, CA (United States); Lawrence Berkeley National Lab., Berkeley, CA (United States). Physical Biosciences Div.
3. Univ. of California, Berkeley, CA (United States). Dept. of Chemical and Biomolecular Engineering and Dept. of Bioengineering.
4. Joint BioEnergy Inst., Emeryville, CA (United States); Lawrence Berkeley National Lab., Berkeley, CA (United States). Physical Biosciences Div.; Univ. of California, Berkeley, CA (United States). Dept. of Chemical and Biomolecular Engineering and Dept. of Bioengineering.
5. National Univ. of Singapore (Singapore). Dept. of Biochemistry; Singapore Inst. of Technology (Singapore)

Publication Date:

Tue Nov 04 00:00:00 EST 2014

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Org.:

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

OSTI Identifier:

1213053

Alternate Identifier(s):

OSTI ID: 1215645

Grant/Contract Number:  

AC02-05CH11231

Resource Type:

Accepted Manuscript

Journal Name:

mBio (Online)

Additional Journal Information:

Journal Name: mBio (Online); Journal Volume: 5; Journal Issue: 6; Journal ID: ISSN 2150-7511

Publisher:

American Society for Microbiology

Country of Publication:

United States

Language:

English

Subject:

09 BIOMASS FUELS