Systems Analyses Reveal the Resilience of <named-content content-type="genus-species">Escherichia coli</named-content> Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate

ABSTRACT Productivity of bacterial cell factories is frequently compromised by stresses imposed by recombinant protein synthesis and carbon-to-product conversion, but little is known about these bioprocesses at a systems level. Production of the unnatural metabolite citramalate in Escherichia coli r...

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Autores principales:	Joseph Webb, Vicki Springthorpe, Luca Rossoni, David-Paul Minde, Swen Langer, Heather Walker, Amias Alstrom-Moore, Tony Larson, Kathryn Lilley, Graham Eastham, Gill Stephens, Gavin H. Thomas, David J. Kelly, Jeffrey Green
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Escherichia coli bioproduction of chemicals citramalate citramalic acid fed-batch fermentation lipidomics Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/355e4e0f72ff444ba1219d6689b43338
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spelling	oai:doaj.org-article:355e4e0f72ff444ba1219d6689b433382021-12-02T18:39:47ZSystems Analyses Reveal the Resilience of <named-content content-type="genus-species">Escherichia coli</named-content> Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate10.1128/mSystems.00187-192379-5077https://doaj.org/article/355e4e0f72ff444ba1219d6689b433382019-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00187-19https://doaj.org/toc/2379-5077ABSTRACT Productivity of bacterial cell factories is frequently compromised by stresses imposed by recombinant protein synthesis and carbon-to-product conversion, but little is known about these bioprocesses at a systems level. Production of the unnatural metabolite citramalate in Escherichia coli requires the expression of a single gene coding for citramalate synthase. Multiomic analyses of a fermentation producing 25 g liter−1 citramalate were undertaken to uncover the reasons for its productivity. Metabolite, transcript, protein, and lipid profiles of high-cell-density, fed-batch fermentations of E. coli expressing either citramalate synthase or an inactivated enzyme were similar. Both fermentations showed downregulation of flagellar genes and upregulation of chaperones IbpA and IbpB, indicating that these responses were due to recombinant protein synthesis and not citramalate production. Citramalate production did not perturb metabolite pools, except for an increased intracellular pyruvate pool. Gene expression changes in response to citramalate were limited; none of the general stress response regulons were activated. Modeling of transcription factor activities suggested that citramalate invoked a GadW-mediated acid response, and changes in GadY and RprA regulatory small RNA (sRNA) expression supported this. Although changes in membrane lipid composition were observed, none were unique to citramalate production. This systems analysis of the citramalate fermentation shows that E. coli has capacity to readily adjust to the redirection of resources toward recombinant protein and citramalate production, suggesting that it is an excellent chassis choice for manufacturing organic acids. IMPORTANCE Citramalate is an attractive biotechnology target because it is a precursor of methylmethacrylate, which is used to manufacture Perspex and other high-value products. Engineered E. coli strains are able to produce high titers of citramalate, despite having to express a foreign enzyme and tolerate the presence of a nonnative biochemical. A systems analysis of the citramalate fermentation was undertaken to uncover the reasons underpinning its productivity. This showed that E. coli readily adjusts to the redirection of metabolic resources toward recombinant protein and citramalate production and suggests that E. coli is an excellent chassis for manufacturing similar small, polar, foreign molecules.Joseph WebbVicki SpringthorpeLuca RossoniDavid-Paul MindeSwen LangerHeather WalkerAmias Alstrom-MooreTony LarsonKathryn LilleyGraham EasthamGill StephensGavin H. ThomasDavid J. KellyJeffrey GreenAmerican Society for MicrobiologyarticleEscherichia colibioproduction of chemicalscitramalatecitramalic acidfed-batch fermentationlipidomicsMicrobiologyQR1-502ENmSystems, Vol 4, Iss 4 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Escherichia coli bioproduction of chemicals citramalate citramalic acid fed-batch fermentation lipidomics Microbiology QR1-502
spellingShingle	Escherichia coli bioproduction of chemicals citramalate citramalic acid fed-batch fermentation lipidomics Microbiology QR1-502 Joseph Webb Vicki Springthorpe Luca Rossoni David-Paul Minde Swen Langer Heather Walker Amias Alstrom-Moore Tony Larson Kathryn Lilley Graham Eastham Gill Stephens Gavin H. Thomas David J. Kelly Jeffrey Green Systems Analyses Reveal the Resilience of <named-content content-type="genus-species">Escherichia coli</named-content> Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate
description	ABSTRACT Productivity of bacterial cell factories is frequently compromised by stresses imposed by recombinant protein synthesis and carbon-to-product conversion, but little is known about these bioprocesses at a systems level. Production of the unnatural metabolite citramalate in Escherichia coli requires the expression of a single gene coding for citramalate synthase. Multiomic analyses of a fermentation producing 25 g liter−1 citramalate were undertaken to uncover the reasons for its productivity. Metabolite, transcript, protein, and lipid profiles of high-cell-density, fed-batch fermentations of E. coli expressing either citramalate synthase or an inactivated enzyme were similar. Both fermentations showed downregulation of flagellar genes and upregulation of chaperones IbpA and IbpB, indicating that these responses were due to recombinant protein synthesis and not citramalate production. Citramalate production did not perturb metabolite pools, except for an increased intracellular pyruvate pool. Gene expression changes in response to citramalate were limited; none of the general stress response regulons were activated. Modeling of transcription factor activities suggested that citramalate invoked a GadW-mediated acid response, and changes in GadY and RprA regulatory small RNA (sRNA) expression supported this. Although changes in membrane lipid composition were observed, none were unique to citramalate production. This systems analysis of the citramalate fermentation shows that E. coli has capacity to readily adjust to the redirection of resources toward recombinant protein and citramalate production, suggesting that it is an excellent chassis choice for manufacturing organic acids. IMPORTANCE Citramalate is an attractive biotechnology target because it is a precursor of methylmethacrylate, which is used to manufacture Perspex and other high-value products. Engineered E. coli strains are able to produce high titers of citramalate, despite having to express a foreign enzyme and tolerate the presence of a nonnative biochemical. A systems analysis of the citramalate fermentation was undertaken to uncover the reasons underpinning its productivity. This showed that E. coli readily adjusts to the redirection of metabolic resources toward recombinant protein and citramalate production and suggests that E. coli is an excellent chassis for manufacturing similar small, polar, foreign molecules.
format	article
author	Joseph Webb Vicki Springthorpe Luca Rossoni David-Paul Minde Swen Langer Heather Walker Amias Alstrom-Moore Tony Larson Kathryn Lilley Graham Eastham Gill Stephens Gavin H. Thomas David J. Kelly Jeffrey Green
author_facet	Joseph Webb Vicki Springthorpe Luca Rossoni David-Paul Minde Swen Langer Heather Walker Amias Alstrom-Moore Tony Larson Kathryn Lilley Graham Eastham Gill Stephens Gavin H. Thomas David J. Kelly Jeffrey Green
author_sort	Joseph Webb
title	Systems Analyses Reveal the Resilience of <named-content content-type="genus-species">Escherichia coli</named-content> Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate
title_short	Systems Analyses Reveal the Resilience of <named-content content-type="genus-species">Escherichia coli</named-content> Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate
title_full	Systems Analyses Reveal the Resilience of <named-content content-type="genus-species">Escherichia coli</named-content> Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate
title_fullStr	Systems Analyses Reveal the Resilience of <named-content content-type="genus-species">Escherichia coli</named-content> Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate
title_full_unstemmed	Systems Analyses Reveal the Resilience of <named-content content-type="genus-species">Escherichia coli</named-content> Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate
title_sort	systems analyses reveal the resilience of <named-content content-type="genus-species">escherichia coli</named-content> physiology during accumulation and export of the nonnative organic acid citramalate
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/355e4e0f72ff444ba1219d6689b43338
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Systems Analyses Reveal the Resilience of <named-content content-type="genus-species">Escherichia coli</named-content> Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate

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