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...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2019-08-01
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| Series: | mSystems |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mSystems.00187-19 |
| _version_ | 1818345692255485952 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-13T17:06:25Z |
| format | Article |
| id | doaj.art-355e4e0f72ff444ba1219d6689b43338 |
| institution | Directory Open Access Journal |
| issn | 2379-5077 |
| language | English |
| last_indexed | 2024-12-13T17:06:25Z |
| publishDate | 2019-08-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSystems |
| spelling | doaj.art-355e4e0f72ff444ba1219d6689b433382022-12-21T23:37:40ZengAmerican Society for MicrobiologymSystems2379-50772019-08-014410.1128/mSystems.00187-19Systems 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 CitramalateJoseph Webb0Vicki Springthorpe1Luca Rossoni2David-Paul Minde3Swen Langer4Heather Walker5Amias Alstrom-Moore6Tony Larson7Kathryn Lilley8Graham Eastham9Gill Stephens10Gavin H. Thomas11David J. Kelly12Jeffrey Green13Molecular Biology & Biotechnology, University of Sheffield, Sheffield, United KingdomDepartment of Biology, University of York, York, United KingdomBioprocess, Environmental and Chemical Technologies, University of Nottingham, Nottingham, United KingdomCambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, United KingdomDepartment of Biology, University of York, York, United KingdombiOMICS Mass Spectrometry Facility, Department of Animal and Plant Sciences, University of Sheffield, Sheffield, United KingdomMolecular Biology & Biotechnology, University of Sheffield, Sheffield, United KingdomDepartment of Biology, University of York, York, United KingdomCambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, United KingdomLucite International, Wilton, United KingdomBioprocess, Environmental and Chemical Technologies, University of Nottingham, Nottingham, United KingdomDepartment of Biology, University of York, York, United KingdomMolecular Biology & Biotechnology, University of Sheffield, Sheffield, United KingdomMolecular Biology & Biotechnology, University of Sheffield, Sheffield, United KingdomABSTRACT 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.https://journals.asm.org/doi/10.1128/mSystems.00187-19Escherichia colibioproduction of chemicalscitramalatecitramalic acidfed-batch fermentationlipidomics |
| spellingShingle | 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 mSystems Escherichia coli bioproduction of chemicals citramalate citramalic acid fed-batch fermentation lipidomics |
| 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_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_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_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 |
| topic | Escherichia coli bioproduction of chemicals citramalate citramalic acid fed-batch fermentation lipidomics |
| url | https://journals.asm.org/doi/10.1128/mSystems.00187-19 |
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