Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG

Patrick Beller, Phillipp Fink, Felix Wolf, Daniel Männle, Irina Helmle, Wolfgang Kuttenlochner, Daniel Unterfrauner, Alicia Engelbrecht, Nicole D. Staudt, Andreas Kulik, Michael Groll, Harald Gross, Leonard Kaysser

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a β-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin β-lactone warheads.

Original languageEnglish
Article number105507
JournalJournal of Biological Chemistry
Volume300
Issue number1
DOIs
StatePublished - Jan 2024

Keywords

  • biosynthesis
  • gene knockout
  • heterologous expression
  • inhibitor
  • lactone
  • natural product
  • proteasome

Fingerprint

Dive into the research topics of 'Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG'. Together they form a unique fingerprint.

Cite this