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Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

Rix, Gordon and Watkins-Dulaney, Ella J. and Almhjell, Patrick J. and Boville, Christina E. and Arnold, Frances H. and Liu, Chang C. (2020) Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities. Nature Communications, 11 . Art. No. 5644. ISSN 2041-1723. PMCID PMC7648111. https://resolver.caltech.edu/CaltechAUTHORS:20200605-104245277

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Abstract

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB’s primary activity of synthesizing L-tryptophan from indole and L-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1038/s41467-020-19539-6DOIArticle
https://doi.org/10.1101/2020.06.01.128165DOIDiscussion Paper
http://github.com/palmhjell/bayesian_kineticsRelated ItemCode
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc7648111/PubMed CentralArticle
ORCID:
AuthorORCID
Rix, Gordon0000-0001-7217-8535
Watkins-Dulaney, Ella J.0000-0002-0585-1598
Almhjell, Patrick J.0000-0003-0977-841X
Boville, Christina E.0000-0002-2577-9343
Arnold, Frances H.0000-0002-4027-364X
Liu, Chang C.0000-0002-3290-2880
Additional Information:© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Received 24 June 2020; Accepted 19 October 2020; Published 06 November 2020. This work was funded by NIH NIGMS (1DP2GM119163-01), NSF (MCB 1545158), and the Beckman Young Investigator Award to C.C.L., and the NIH NIGMS (R01GM125887) to F.H.A. The content is solely the responsibility of the authors and does not necessarily represent the official views of funding sources. We thank David Romney, Justin Bois, and other members of the Arnold and Liu groups for thoughtful discussions on experimental design. Data availability: The datasets generated and/or analyzed during the current study are available from the corresponding author upon request. Source data are provided with this paper. Code availability: The code used to generate enzyme kinetic estimates (along with example data) can be found at http://github.com/palmhjell/bayesian_kinetics. Author Contributions: All authors contributed to experimental design and data analysis. G.R. cloned all genetic constructs; set up, performed, and characterized evolution experiments; and carried out yeast growth rate experiments. E.J.W. performed the panel HPLC-MS assay and indole conversion rate measurements on TmTrpBs from variant set 2, and P.J.A. analyzed the results. C.E.B. performed in vitro characterizations of TmTrpBs from variant set 1 and performed the thermal shift assay and substrate scope characterizations for TmTrpB variants WT-003-1-A, Q90*-003-1-A, and Tri-100-2-A. P.J.A. performed enzyme kinetics assays for TmTrpB variants WT-003-1-A, Q90*-003-1-A, and Tri-100-2-A. G.R. and C.C.L. wrote the paper with input and contributions from all authors. Corresponding author: Correspondence to Chang C. Liu. Competing interests: C.E.B and F.H.A. are co-founders of Aralez Bio, focusing on the enzymatic synthesis of unnatural amino acids. All other authors declare no competing interests. Peer review information: Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work.
Funders:
Funding AgencyGrant Number
NIH1DP2GM119163-01
NSFMCB-1545158
Arnold and Mabel Beckman FoundationUNSPECIFIED
NIHR01GM125887
Subject Keywords:Molecular evolution; Protein design; Synthetic biology
PubMed Central ID:PMC7648111
Record Number:CaltechAUTHORS:20200605-104245277
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200605-104245277
Official Citation:Rix, G., Watkins-Dulaney, E.J., Almhjell, P.J. et al. Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities. Nat Commun 11, 5644 (2020). https://doi.org/10.1038/s41467-020-19539-6
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:103732
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:05 Jun 2020 19:08
Last Modified:16 Nov 2020 21:52

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