Functional expression of diverse post-translational peptide-modifying enzymes in Escherichia coli under uniform expression and purification conditions
Name
journal.pone.0266488.pdf
Description
Published version
Size
1.54 MB
Format
Adobe PDF
Checksum (MD5)
a1bcd7062eb641b506bb1eda0c0000f4
Author(s)
Glassey, Emerson
King, Andrew M
Anderson, Daniel A
Zhang, Zhengan
Voigt, Christopher A
Date Issued
2022
Journal
PLoS ONE
Publisher
Public Library of Science (PLoS)
Citation
Glassey, Emerson, King, Andrew M, Anderson, Daniel A, Zhang, Zhengan and Voigt, Christopher A. 2022. "Functional expression of diverse post-translational peptide-modifying enzymes in Escherichia coli under uniform expression and purification conditions." PLoS ONE, 17 (9).
Version
Final published version
Abstract
RiPPs (ribosomally-synthesized and post-translationally modified peptides) are a class of pharmaceutically-relevant natural products expressed as precursor peptides before being enzymatically processed into their final functional forms. Bioinformatic methods have illuminated hundreds of thousands of RiPP enzymes in sequence databases and the number of characterized chemical modifications is growing rapidly; however, it remains difficult to functionally express them in a heterologous host. One challenge is peptide stability, which we addressed by designing a RiPP stabilization tag (RST) based on a small ubiquitin-like modifier (SUMO) domain that can be fused to the N- or C-terminus of the precursor peptide and proteolytically removed after modification. This is demonstrated to stabilize expression of eight RiPPs representative of diverse phyla. Further, using Escherichia coli for heterologous expression, we identify a common set of media and growth conditions where 24 modifying enzymes, representative of diverse chemistries, are functional. The high success rate and broad applicability of this system facilitates: (i) RiPP discovery through high-throughput “mining” and (ii) artificial combination of enzymes from different pathways to create a desired peptide.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Terms of Use
Creative Commons Attribution 4.0 International license
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1371/JOURNAL.PONE.0266488
