Removing allosteric feedback inhibition of tomato 4-coumarate:CoA ligase by directed evolution

Moti Alberstein, Miriam Eisenstein, Hagai Abeliovich*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

Abstract

Plant secondary metabolites, such as those derived from the phenylpropanoid pathway, have a beneficial effect on human health. Manipulation of metabolic flux in the phenylpropanoid pathway is important for achieving enhanced production of compounds such as anthocyanins, flavonoids and isoflavonoids. Here, we describe the development of a high-throughput molecular evolution approach that can be used for catalytic improvement of at least four key phenylpropanoid pathway enzymes, within the context of the metabolic pathway. This method uses yeast cells that express plant phenylpropanoid pathway enzymes, leading to formation of a colored intermediate that can be used as a readout in high-throughput screening. Here we report the identification of improved tomato peel 4-coumarate:CoA ligase variants using this approach. We found that the wild-type enzyme is strongly allosterically inhibited by naringenin, a downstream product of the pathway. Surprisingly, at least two of the improved variants are completely insensitive to feedback inhibition by naringenin. We suggest that this inhibition is exerted through a unique and previously unrecognized allosteric domain.

Original languageAmerican English
Pages (from-to)57-69
Number of pages13
JournalPlant Journal
Volume69
Issue number1
DOIs
StatePublished - Jan 2012

Keywords

  • 4-coumarate:CoA ligase
  • allosteric regulation
  • directed evolution
  • phenylpropanoid metabolism
  • tomato

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