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Evolving a mitigation of the stress response pathway to change the basic chemistry of life

Isabella Tolle, Stefan Oehm, Michael Georg Hoesl, Christin Treiber-Kleinke, View ORCID ProfileLauri Peil, View ORCID ProfileAbdul-Rahman Adamu Bukari, View ORCID ProfileTorsten Semmler, View ORCID ProfileJuri Rappsilber, View ORCID ProfileAleeza Gerstein, View ORCID ProfileNediljko Budisa
doi: https://doi.org/10.1101/2021.09.23.461486
Isabella Tolle
1Technische Universität Berlin, Institut für Chemie, Müller-Breslau Str. 10, 10623 Berlin, Germany
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Stefan Oehm
2Pulcra Chemicals GmbH, Isardamm 79-83, 82538 Geretsried, Germany
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Michael Georg Hoesl
3Clariant Produkte Deutschland GmbH., Semmelweisstraße 1, 82152 Planegg, Germany
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Christin Treiber-Kleinke
4Freie Universität Berlin, Institut für Chemie und Biochemie, Arnimallee 20, 14195 Berlin, Germany
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Lauri Peil
5Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK
6Icosagen Cell Factory OÜ, Eerika tee 1, Õssu, 61713 Tartumaa, Estonia
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Abdul-Rahman Adamu Bukari
7Department of Microbiology, University of Manitoba, R3T 2N2 Winnipeg, MB, Canada
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Torsten Semmler
8Robert Koch Institut, Nordufer 20, 13353 Berlin (Germany)
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Juri Rappsilber
5Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK
9Technische Universität Berlin, Institut für Biotechnologie, Gustav-Meyer-Allee 25, 13355 Berlin (Germany)
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Aleeza Gerstein
7Department of Microbiology, University of Manitoba, R3T 2N2 Winnipeg, MB, Canada
10Department of Statistics, University of Manitoba, R3T 2N2 Winnipeg, MB, Canada
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  • For correspondence: aleeza.gerstein@umanitoba.ca nediljko.budisa@tu-berlin.de nediljko.budisa@umanitoba.ca
Nediljko Budisa
1Technische Universität Berlin, Institut für Chemie, Müller-Breslau Str. 10, 10623 Berlin, Germany
11Department of Chemistry, University of Manitoba, R3T 2N2 Winnipeg, MB, Canada
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  • For correspondence: aleeza.gerstein@umanitoba.ca nediljko.budisa@tu-berlin.de nediljko.budisa@umanitoba.ca
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ABSTRACT

Billions of years of evolution have produced only slight variations in the standard genetic code, and the number and identity of proteinogenic amino acids have remained mostly consistent throughout all three domains of life. These observations suggest a certain rigidity of the genetic code and prompt musings as to the origin and evolution of the code. Here we conducted an adaptive laboratory evolution (ALE) to push the limits of the code restriction, by evolving Escherichia coli to fully replace tryptophan, thought to be the latest addition to the genetic code, with the analog L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa). We identified an overshooting of the stress response system to be the main inhibiting factor for limiting ancestral growth upon exposure to β-(thieno[3,2-b]pyrrole ([3,2]Tp), a metabolic precursor of [3,2]Tpa, and Trp limitation. During the ALE, E. coli was able to “calm down” its stress response machinery, thereby restoring growth. In particular, the inactivation of RpoS itself, the master regulon of the general stress response, was a key event during the adaptation. Knocking out the rpoS gene in the ancestral background independent of other changes conferred growth on [3,2]Tp. Our results add additional evidence that frozen regulatory constraints rather than a rigid protein translation apparatus are Life’s gatekeepers of the canonical amino acid repertoire. This information will not only enable us to design enhanced synthetic amino acid incorporation systems but may also shed light on a general biological mechanism trapping organismal configurations in a status quo.

SIGNIFICANCE STATEMENT The (apparent) rigidity of the genetic code, as well as its universality, have long since ushered explorations into expanding the code with synthetic, new-to-nature building blocks and testing its boundaries. While nowadays even proteome-wide incorporation of synthetic amino acids has been reported on several occasions1–3, little is known about the underlying mechanisms.

We here report ALE with auxotrophic E. coli that yielded successful proteome-wide replacement of Trp by its synthetic analog [3,2]Tpa accompanied with the selection for loss of RpoS4 function. Such laboratory domestication of bacteria by the acquisition of rpoS mitigation mutations is beneficial not only to overcome the stress of nutrient (Trp) starvation but also to evolve the paths to use environmental xenobiotics (e.g. [3,2]Tp) as essential nutrients for growth.

We pose that regulatory constraints rather than a rigid and conserved protein translation apparatus are Life’s gatekeepers of the canonical amino acid repertoire (at least where close structural analogs are concerned). Our findings contribute a step towards understanding possible environmental causes of genetic changes and their relationship to evolution.

Our evolved strain affords a platform for homogenous protein labeling with [3,2]Tpa as well as for the production of biomolecules5, which are challenging to synthesize chemically. Top-down synthetic biology will also benefit greatly from breaking through the boundaries of the frozen bacterial genetic code, as this will enable us to begin creating synthetic cells capable to utilize an expanded range of substrates essential for life.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted September 23, 2021.
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Evolving a mitigation of the stress response pathway to change the basic chemistry of life
Isabella Tolle, Stefan Oehm, Michael Georg Hoesl, Christin Treiber-Kleinke, Lauri Peil, Abdul-Rahman Adamu Bukari, Torsten Semmler, Juri Rappsilber, Aleeza Gerstein, Nediljko Budisa
bioRxiv 2021.09.23.461486; doi: https://doi.org/10.1101/2021.09.23.461486
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Evolving a mitigation of the stress response pathway to change the basic chemistry of life
Isabella Tolle, Stefan Oehm, Michael Georg Hoesl, Christin Treiber-Kleinke, Lauri Peil, Abdul-Rahman Adamu Bukari, Torsten Semmler, Juri Rappsilber, Aleeza Gerstein, Nediljko Budisa
bioRxiv 2021.09.23.461486; doi: https://doi.org/10.1101/2021.09.23.461486

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