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

Haslinger, K., Hackl, T., & Prather, K. L. (2020). Rapid in vitro prototyping of O-methyltransferases for pathway applications in Escherichia coli. bioRxiv, 258715, pp. 1-31. doi:10.1101/2020.08.27.258715.

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bioRxiv_epub_2020_715.pdf (Preprint), 2MB
 
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Haslinger, Kristina, Author
Hackl, Thomas1, Author              
Prather, Kristala L.J., Author
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1Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              

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 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 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.

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Language(s): eng - English
 Dates: 2020-08-27
 Publication Status: Published online
 Pages: 31
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 Identifiers: DOI: 10.1101/2020.08.27.258715
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Title: bioRxiv
Source Genre: Collected Edition
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Pages: - Volume / Issue: - Sequence Number: 258715 Start / End Page: 1 - 31 Identifier: -