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

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 three 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.

• Publication year

  2020

• Venue

  bioRxiv

• Publication date

  2020-08-27

• Fields of study

  Biology, Medicine, Chemistry, Engineering

• Identifiers
  DOI 10.1101/2020.08.27.258715PMID 33957079
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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