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

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

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

11 Citaten (Scopus)
120 Downloads (Pure)

Samenvatting

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.

Originele taal-2English
Pagina's (van-tot)876-886.e4
Aantal pagina's15
TijdschriftCell Chemical Biology
Volume28
Nummer van het tijdschrift6
Vroegere onlinedatum5-mei-2021
DOI's
StatusPublished - 17-jun.-2021

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