Cobalamin-Dependent C-Methyltransferases From Marine Microbes: Accessibility via Rhizobia Expression

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

Cobalamin-dependent radical S-adenosylmethionine (rSAM) methyltransferases catalyze chemically challenging methylation reactions on diverse natural products at unactivated carbon centers. In vivo reconstitution and biosynthetic studies of natural product gene clusters encoding these enzymes are often severely limited by ineffective heterologous expression hosts, including the otherwise versatile Escherichia coli. In this chapter, we describe the use of rhizobia bacteria as effective expression hosts for cobalamin-dependent rSAM C-methyltransferases. We chose the natural product pathway encoding the heavily modified cytotoxic peptides, the polytheonamides, as our model pathway due to the presence of two methyltransferases responsible for a total of 17 C-methylations. Detailed protocols are given for vector construction, transformation, and heterologous expression in Rhizobium leguminosarum bv. viciae 3841. Additional methods pertaining to analytical separation and mass spectrometric analysis of modified peptides are also entailed. As genomics continues to uncover new enzymes and pathways from unknown and uncultivated microbes, use of metabolically distinct heterologous expression hosts like rhizobia will be a necessary tool to unravel the catalytic and metabolic diversity of marine microbial life.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsBradley S. Moore
PublisherAcademic Press Inc.
Pages259-286
Number of pages28
ISBN (Print)9780128139592
DOIs
StatePublished - 2018

Publication series

NameMethods in Enzymology
Volume604
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Cobalamin
  • Heterologous expression
  • Methyltransferase
  • Natural products
  • Peptides
  • Polytheonamides
  • Radical SAM
  • Rhizobia
  • RiPPs
  • Vitamin B

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