Development of a Vanillate Biosensor for the Vanillin
Biosynthesis Pathway in E. coli
Posted on 2019-08-30 - 21:43
The engineered de novo vanillin biosynthesis pathway
constructed in Escherichia coli is industrially relevant
but limited by the reaction catalyzed by catechol O-methyltransferase, which is intended to catalyze the conversion
of protocatechuate to vanillate. To identify alternative O-methyltransferases, we constructed a vanillate sensor based on the Caulobacter crescentus VanR-VanO system. Using an E. coli promoter library, we achieved greater than 14-fold
dynamic range in our best rationally constructed sensor. We found
that this construct and an evolved variant demonstrate remarkable
substrate selectivity, exhibiting no detectable response to the regioisomer
byproduct isovanillate and minimal response to structurally similar
pathway intermediates. We then harnessed the evolved biosensor to
conduct rapid bioprospecting of natural catechol O-methyltransferases and identified three previously uncharacterized
but active O-methyltransferases. Collectively, these
efforts enrich our knowledge of how biosensing can aid metabolic engineering
and constitute the foundation for future improvements in vanillin
pathway productivity.
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Kunjapur, Aditya M.; Prather, Kristala L. J. (2019). Development of a Vanillate Biosensor for the Vanillin
Biosynthesis Pathway in E. coli. ACS Publications. Collection. https://doi.org/10.1021/acssynbio.9b00071