PXD066826

PXD066826 is an original dataset announced via ProteomeXchange.

Title	Engineering polyketide stereocenters with ketoreductase domain exchanges
Description	Polyketide synthases (PKSs) are versatile biosynthetic megasynthases capable of producing a diverse range of natural products with many applications, including in pharmaceuticals. The stereochemical precision of PKSs makes them a powerful tool for engineering tailored, unnatural polyketides; however, modifying the stereocenters of a PKS product while maintaining production levels remains a significant challenge. In this study, we systematically tested and evaluated strategies for ketoreductase (KR) domain exchanges, the domain responsible for setting stereocenters of polyketide products. After first optimizing the method for KR exchanges, we then performed 44 KR domain exchanges on three different PKSs to obtain high production of all four stereoisomers in vivo. By testing both one- and two-module PKS systems, we investigated how downstream modules process intermediates with altered stereochemistry and found that the configuration of the α-substituents was critical for gatekeeping by the ketosynthase (KS). To overcome this constraint, we investigated two different strategies for altering the KS domain, including introducing targeted mutations in the downstream KS, and exploring boundaries in exchanging the entire functional unit from the donor PKS. Both strategies successfully modified the KS stereocontrol with distinct tradeoffs; the functional unit exchange resulted in higher improvements, though it was more likely to break the entire PKS. This study demonstrates a comprehensive approach to successfully engineering all four stereochemical configurations in multiple PKS systems, advancing our understanding of and ability to rationally modify polyketide stereochemistry through multiple engineering strategies.
HostingRepository	PRIDE
AnnounceDate	2025-12-01
AnnouncementXML	Submission_2025-11-30_17:17:11.988.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Christopher Petzold
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: NEWT:562;
ModificationList	monohydroxylated residue; deamidated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2025-07-31 15:19:22	ID requested
⏵ 1	2025-11-30 17:17:13	announced

Keiser LS, Gatenil PP, Zhu Y, Deng K, Waldburger L, Gin JW, Chen Y, Baidoo EEK, Petzold CJ, Lanclos N, Northen TR, Englund E, Keasling JD, Engineering Polyketide Stereocenters with Ketoreductase Domain Exchanges. J Am Chem Soc, 147(46):42237-42252(2025) [pubmed]

10.1021/jacs.5c06736;

submitter keyword: Polyketide synthases (PKS);Protein engineering

KR swaps;JBEI

Christopher Petzold
contact affiliation	Staff Scientist Biological Systems & Engineering Division Lawrence Berkeley National Laboratory Berkeley CA 94720
contact email	cjpetzold@lbl.gov
lab head
Christopher Petzold
contact affiliation	Lawrence Berkeley National Laboratory
contact email	cjpetzold@lbl.gov
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/12/PXD066826

PRIDE project URI

• PRIDE
  1. PXD066826
     1. Label: PRIDE project
     2. Name: Engineering polyketide stereocenters with ketoreductase domain exchanges