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Improving microbial biogasoline production in Escherichia coli using tolerance engineering.

Foo JL, Jensen HM, Dahl RH, George K, Keasling JD, Lee TS, Leong S, Mukhopadhyay A - MBio (2014)

Bottom Line: Overexpression of several of these candidates improved tolerance to exogenously added isopentenol.In order to achieve economically viable production levels, it is also necessary to engineer production strains with improved tolerance to these compounds.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical & Biomolecular Engineering and Department of Bioengineering, University of California, Berkeley, California, USA.

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Related in: MedlinePlus

Scheme for screening tolerance and increasing titer of isopentenol. (A) First, transcriptomics of E. coli DH1 expressing an inactive mevalonate pathway (MevT*; see Materials and Methods) in the presence of exogenous isopentenol was used to determine what genes are upregulated in response to isopentenol stress. (B) A library of those genes was overexpressed in the presence of exogenous isopentenol. Growth curves were used to measure enhanced tolerance. (C) Finally, genes that conferred tolerance were tested for their ability to increase the isopentenol titer in a production strain.
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fig1: Scheme for screening tolerance and increasing titer of isopentenol. (A) First, transcriptomics of E. coli DH1 expressing an inactive mevalonate pathway (MevT*; see Materials and Methods) in the presence of exogenous isopentenol was used to determine what genes are upregulated in response to isopentenol stress. (B) A library of those genes was overexpressed in the presence of exogenous isopentenol. Growth curves were used to measure enhanced tolerance. (C) Finally, genes that conferred tolerance were tested for their ability to increase the isopentenol titer in a production strain.

Mentions: Functional genomics measurements reveal a large number of responses to toxic compounds, including many other modes of alleviating solvent stress (13, 14). Due to the promise of utilizing systems biology to engineer alcohol-tolerant strains (15), we used transcriptomics data to identify genes that may serve as candidates for tolerance engineering for isopentenol. Whole-genome transcriptomics revealed several genes that were responsive to exogenous isopentenol (Fig. 1A). We then tested the efficacies of the most highly induced candidates to improve tolerance to exogenous isopentenol (Fig. 1B). The candidates that successfully alleviated growth lag when overexpressed in the presence of isopentenol were then integrated with an isopentenol production strain (Fig. 1C) (6). Several candidate genes improved isopentenol tolerance, and one candidate, MetR, improved the isopentenol titer by 55%. The putative ATP binding cassette (ABC) transporter MdlB increased the titer by 12% and is, to our knowledge, the first example of an exporter that has improved the titer of a short-chain alcohol. The knowledge of even one such transporter for a desirable final product can lead to considerable additional discoveries, such as bioprospecting for homologs (7) and utilizing directed evolution (12, 16). These results demonstrate the importance of utilizing transcriptomics data for improving short-chain alcohol production as a guide for host engineering.


Improving microbial biogasoline production in Escherichia coli using tolerance engineering.

Foo JL, Jensen HM, Dahl RH, George K, Keasling JD, Lee TS, Leong S, Mukhopadhyay A - MBio (2014)

Scheme for screening tolerance and increasing titer of isopentenol. (A) First, transcriptomics of E. coli DH1 expressing an inactive mevalonate pathway (MevT*; see Materials and Methods) in the presence of exogenous isopentenol was used to determine what genes are upregulated in response to isopentenol stress. (B) A library of those genes was overexpressed in the presence of exogenous isopentenol. Growth curves were used to measure enhanced tolerance. (C) Finally, genes that conferred tolerance were tested for their ability to increase the isopentenol titer in a production strain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222104&req=5

fig1: Scheme for screening tolerance and increasing titer of isopentenol. (A) First, transcriptomics of E. coli DH1 expressing an inactive mevalonate pathway (MevT*; see Materials and Methods) in the presence of exogenous isopentenol was used to determine what genes are upregulated in response to isopentenol stress. (B) A library of those genes was overexpressed in the presence of exogenous isopentenol. Growth curves were used to measure enhanced tolerance. (C) Finally, genes that conferred tolerance were tested for their ability to increase the isopentenol titer in a production strain.
Mentions: Functional genomics measurements reveal a large number of responses to toxic compounds, including many other modes of alleviating solvent stress (13, 14). Due to the promise of utilizing systems biology to engineer alcohol-tolerant strains (15), we used transcriptomics data to identify genes that may serve as candidates for tolerance engineering for isopentenol. Whole-genome transcriptomics revealed several genes that were responsive to exogenous isopentenol (Fig. 1A). We then tested the efficacies of the most highly induced candidates to improve tolerance to exogenous isopentenol (Fig. 1B). The candidates that successfully alleviated growth lag when overexpressed in the presence of isopentenol were then integrated with an isopentenol production strain (Fig. 1C) (6). Several candidate genes improved isopentenol tolerance, and one candidate, MetR, improved the isopentenol titer by 55%. The putative ATP binding cassette (ABC) transporter MdlB increased the titer by 12% and is, to our knowledge, the first example of an exporter that has improved the titer of a short-chain alcohol. The knowledge of even one such transporter for a desirable final product can lead to considerable additional discoveries, such as bioprospecting for homologs (7) and utilizing directed evolution (12, 16). These results demonstrate the importance of utilizing transcriptomics data for improving short-chain alcohol production as a guide for host engineering.

Bottom Line: Overexpression of several of these candidates improved tolerance to exogenously added isopentenol.In order to achieve economically viable production levels, it is also necessary to engineer production strains with improved tolerance to these compounds.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical & Biomolecular Engineering and Department of Bioengineering, University of California, Berkeley, California, USA.

Show MeSH
Related in: MedlinePlus