Improving the Robustness of Engineered Bacteria to Nutrient Stress Using Programmed Proteolysis

Klara Szydlo; Zoya Ignatova; Thomas E Gorochowski

doi:10.1021/acssynbio.1c00490

Improving the Robustness of Engineered Bacteria to Nutrient Stress Using Programmed Proteolysis

Klara Szydlo
, Zoya Ignatova^*
, Thomas E Gorochowski^*

^*Corresponding author for this work

School of Biological Sciences

Research output: Contribution to journal › Article (Academic Journal) › peer-review

12 Citations (Scopus)

130 Downloads (Pure)

Abstract

The use of short peptide tags in synthetic genetic circuits allows for the tuning of gene expression dynamics and release of amino acid resources through targeted protein degradation. Here, we use elements of the Escherichia coli and Mesoplasma florum transfer-mRNA (tmRNA) ribosome rescue systems to compare endogenous and foreign proteolysis systems in E. coli. We characterize the performance and burden of each and show that, while both greatly shorten the half-life of a tagged protein, the endogenous system is approximately 10 times more efficient. On the basis of these results we then demonstrate using mathematical modeling and experiments how proteolysis can improve cellular robustness through targeted degradation of a reporter protein in auxotrophic strains, providing a limited secondary source of essential amino acids that help partially restore growth when nutrients become scarce. These findings provide avenues for controlling the functional lifetime of engineered cells once deployed and increasing their tolerance to fluctuations in nutrient availability.

Original language	English
Pages (from-to)	1049-1059
Number of pages	11
Journal	ACS Synthetic Biology
Volume	11
Issue number	3
Early online date	17 Feb 2022
DOIs	https://doi.org/10.1021/acssynbio.1c00490
Publication status	Published - 18 Mar 2022

Bibliographical note

Funding Information:
We thank Irem Avcilar-Kucukgoze for creating the original GFP-expressing plasmid, Robert Sauer for providing us with the pBAD33-mf-lon plasmid, and Robert Gennis and Toshio Iwasaki for providing us with the ML17 and RF10 E. coli strains. This work was supported by European Union’s Horizon 2020 research and innovation program as part of the SynCrop ETN under the Marie Skłodowska-Curie Grant 764591 (Z.I.), BrisSynBio, a BBSRC/EPSRC Synthetic Biology Research Centre Grant BB/L01386 X/1 (T.E.G.), a Royal Society University Research Fellowship Grant UF160357 (T.E.G.), and a Turing Fellowship from The Alan Turing Institute under the EPSRC Grant EP/N510129/1 (T.E.G.)

Publisher Copyright:
© 2022 American Chemical Society.

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 12 Responsible Consumption and Production

Research Groups and Themes

BrisSynBio
Bristol BioDesign Institute

Keywords

synthetic biology
burden
protein degradation
genetic circuit
proteolysis
resource recycling

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10.1021/acssynbio.1c00490

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title = "Improving the Robustness of Engineered Bacteria to Nutrient Stress Using Programmed Proteolysis",

abstract = "The use of short peptide tags in synthetic genetic circuits allows for the tuning of gene expression dynamics and release of amino acid resources through targeted protein degradation. Here, we use elements of the Escherichia coli and Mesoplasma florum transfer-mRNA (tmRNA) ribosome rescue systems to compare endogenous and foreign proteolysis systems in E. coli. We characterize the performance and burden of each and show that, while both greatly shorten the half-life of a tagged protein, the endogenous system is approximately 10 times more efficient. On the basis of these results we then demonstrate using mathematical modeling and experiments how proteolysis can improve cellular robustness through targeted degradation of a reporter protein in auxotrophic strains, providing a limited secondary source of essential amino acids that help partially restore growth when nutrients become scarce. These findings provide avenues for controlling the functional lifetime of engineered cells once deployed and increasing their tolerance to fluctuations in nutrient availability.",

keywords = "synthetic biology, burden, protein degradation, genetic circuit, proteolysis, resource recycling",

author = "Klara Szydlo and Zoya Ignatova and Gorochowski, \{Thomas E\}",

note = "Funding Information: We thank Irem Avcilar-Kucukgoze for creating the original GFP-expressing plasmid, Robert Sauer for providing us with the pBAD33-mf-lon plasmid, and Robert Gennis and Toshio Iwasaki for providing us with the ML17 and RF10 E. coli strains. This work was supported by European Union{\textquoteright}s Horizon 2020 research and innovation program as part of the SynCrop ETN under the Marie Sk{\l}odowska-Curie Grant 764591 (Z.I.), BrisSynBio, a BBSRC/EPSRC Synthetic Biology Research Centre Grant BB/L01386 X/1 (T.E.G.), a Royal Society University Research Fellowship Grant UF160357 (T.E.G.), and a Turing Fellowship from The Alan Turing Institute under the EPSRC Grant EP/N510129/1 (T.E.G.) Publisher Copyright: {\textcopyright} 2022 American Chemical Society.",

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doi = "10.1021/acssynbio.1c00490",

language = "English",

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N1 - Funding Information: We thank Irem Avcilar-Kucukgoze for creating the original GFP-expressing plasmid, Robert Sauer for providing us with the pBAD33-mf-lon plasmid, and Robert Gennis and Toshio Iwasaki for providing us with the ML17 and RF10 E. coli strains. This work was supported by European Union’s Horizon 2020 research and innovation program as part of the SynCrop ETN under the Marie Skłodowska-Curie Grant 764591 (Z.I.), BrisSynBio, a BBSRC/EPSRC Synthetic Biology Research Centre Grant BB/L01386 X/1 (T.E.G.), a Royal Society University Research Fellowship Grant UF160357 (T.E.G.), and a Turing Fellowship from The Alan Turing Institute under the EPSRC Grant EP/N510129/1 (T.E.G.) Publisher Copyright: © 2022 American Chemical Society.

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N2 - The use of short peptide tags in synthetic genetic circuits allows for the tuning of gene expression dynamics and release of amino acid resources through targeted protein degradation. Here, we use elements of the Escherichia coli and Mesoplasma florum transfer-mRNA (tmRNA) ribosome rescue systems to compare endogenous and foreign proteolysis systems in E. coli. We characterize the performance and burden of each and show that, while both greatly shorten the half-life of a tagged protein, the endogenous system is approximately 10 times more efficient. On the basis of these results we then demonstrate using mathematical modeling and experiments how proteolysis can improve cellular robustness through targeted degradation of a reporter protein in auxotrophic strains, providing a limited secondary source of essential amino acids that help partially restore growth when nutrients become scarce. These findings provide avenues for controlling the functional lifetime of engineered cells once deployed and increasing their tolerance to fluctuations in nutrient availability.

AB - The use of short peptide tags in synthetic genetic circuits allows for the tuning of gene expression dynamics and release of amino acid resources through targeted protein degradation. Here, we use elements of the Escherichia coli and Mesoplasma florum transfer-mRNA (tmRNA) ribosome rescue systems to compare endogenous and foreign proteolysis systems in E. coli. We characterize the performance and burden of each and show that, while both greatly shorten the half-life of a tagged protein, the endogenous system is approximately 10 times more efficient. On the basis of these results we then demonstrate using mathematical modeling and experiments how proteolysis can improve cellular robustness through targeted degradation of a reporter protein in auxotrophic strains, providing a limited secondary source of essential amino acids that help partially restore growth when nutrients become scarce. These findings provide avenues for controlling the functional lifetime of engineered cells once deployed and increasing their tolerance to fluctuations in nutrient availability.

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KW - burden

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ER -

Improving the Robustness of Engineered Bacteria to Nutrient Stress Using Programmed Proteolysis

Abstract

Bibliographical note

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Keywords

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