Directed Evolution of an Adenylation Domain Alters Substrate Specificity and Generates a New Catechol Siderophore in Escherichia coli

Conley, Erin; Wadler, Caryn S.; Bell, Bailey A.; Lucier, Ivy; Haynie, Caroline; Eldred, Sophie; Nguyen, Valerie; Bugni, Tim S.; Thomas, Michael G.

doi:10.1021/acs.biochem.4c00499

Title: Directed Evolution of an Adenylation Domain Alters Substrate Specificity and Generates a New Catechol Siderophore in Escherichia coli

Journal Article · 20 November 2024 · Biochemistry

DOI: https://doi.org/10.1021/acs.biochem.4c00499 · OSTI ID:2506699

^[1]; Wadler, Caryn S. ^[1]; Bell, Bailey A. ^[1]; Lucier, Ivy ^[1]; Haynie, Caroline ^[1]; Eldred, Sophie ^[1]; Nguyen, Valerie ^[1]; Bugni, Tim S. ^[1];

^[1]

University of Wisconsin-Madison, WI (United States)

Nonribosomal peptide synthetases (NRPS) biosynthesize numerous natural products with therapeutic, agricultural, and industrial significance. Reliably altering substrate selection in these enzymes has been a longstanding goal, as this would enable the production of tailor-made peptides with desired activities. In this study, the NRPS EntF and the associated biosynthesis of the siderophore enterobactin (ENT) were used as a model system to interrogate substrate selection by an adenylation (A) domain. We employed a directed evolution pipeline that harnesses an in vivo genetic selection for siderophore production to alter A domain substrate selection. Surprisingly, this led to the formation of a new, physiologically active catechol siderophore in Escherichia coli. We characterized the enzyme variants in vitro and demonstrated transferability of our findings to the well-studied TycC and GrsB NRPSs. Furthermore, this work identifies critical binding pocket residues that allow for altered substrate selection in our model system and expands upon our understanding of iron acquisition in E. coli.

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Research Organization:: Great Lakes Bioenergy Research Center (GLBRC), Madison, WI (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: SC0018409

OSTI ID:: 2506699

Journal Information:: Biochemistry, Journal Name: Biochemistry Journal Issue: 23 Vol. 63; ISSN 0006-2960

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Title: Directed Evolution of an Adenylation Domain Alters Substrate Specificity and Generates a New Catechol Siderophore in Escherichia coli

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