Rapid in vitro prototyping of O-methyltransferases for pathway applications in Escherichia coli

Kristina Haslinger*, Thomas Hackl, Kristala L J Prather*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

1 Citation (Scopus)
64 Downloads (Pure)

Abstract

O-Methyltransferases are ubiquitous enzymes involved in biosynthetic pathways for secondary metabolites such as bacterial antibiotics, human catecholamine neurotransmitters, and plant phenylpropanoids. While thousands of putative O-methyltransferases are found in sequence databases, few examples are functionally characterized. From a pathway engineering perspective, however, it is crucial to know the substrate and product ranges of the respective enzymes to fully exploit their catalytic power. In this study, we developed an in vitro prototyping workflow that allowed us to screen ∼30 enzymes against five substrates in 3 days with high reproducibility. We combined in vitro transcription/translation of the genes of interest with a microliter-scale enzymatic assay in 96-well plates. The substrate conversion was indirectly measured by quantifying the consumption of the S-adenosyl-L-methionine co-factor by time-resolved fluorescence resonance energy transfer rather than time-consuming product analysis by chromatography. This workflow allowed us to rapidly prototype thus far uncharacterized O-methyltransferases for future use as biocatalysts.

Original languageEnglish
Pages (from-to)876-886.e4
Number of pages15
JournalCell Chemical Biology
Volume28
Issue number6
Early online date5-May-2021
DOIs
Publication statusPublished - 17-Jun-2021

Keywords

  • CAFFEIC ACID
  • STRUCTURAL-CHARACTERIZATION
  • CHLOROGENIC ACID
  • CELL-FREE
  • METHYLATION
  • BIOSYNTHESIS
  • FLAVONOIDS
  • TYROSINE
  • SYSTEMS

Cite this