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Engineering isoflavone metabolism with an artificial bifunctional enzyme

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Abstract

Plant secondary metabolism has been a focus of research in recent years due to its significant roles in plant defense and in human medicine and nutrition. A protein engineering strategy was designed to more effectively manipulate plant secondary metabolite (isoflavonoid) biosynthesis. A bifunctional isoflavone synthase/chalcone isomerase (IFS/CHI) enzyme was constructed by in-frame gene fusion, and expressed in yeast and tobacco. The fusion protein was targeted to the endoplasmic reticulum (ER) membrane and the individual enzymatic functions of its component fragments were retained when assayed in yeast. Petals and young leaves of IFS/CHI transgenic tobacco plants produced higher levels of the isoflavone genistein and genistein glycosides as a ratio of total flavonoids produced than did plants transformed with IFS alone. Thus, through a combined molecular modeling, in vitro protein engineering and in planta metabolic engineering approach, it was possible to increase the potential for accumulation of isoflavonoid compounds in non-legume plants. Construction of bifunctional enzymes will simplify the transformation of plants with multiple pathway genes, and such enzymes may find broad uses for enzyme (e.g., cytochrome P450 family) and biochemical pathway engineering.

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Abbreviations

CHI:

Chalcone isomerase

CHS:

Chalcone synthase

EGFP:

Enhanced green fluorescent protein

IFS:

Isoflavone synthase

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Acknowledgements

We thank Dr. Xiaoqiang Wang for molecular modeling of IFS, Dr. Elison Blancaflor for assistance with confocal microscopy, Dr. Lahoucine Achnine for providing pRTL2-C4H MA-EGFP, Dr. Philip Urban for providing the WAT11 strain and pYeDP60 vector, and Drs. Xiaoqiang Wang and Gregory Peel for critical reading of the manuscript. This work was supported by the Samuel Roberts Noble Foundation.

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  1. Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73401, USA

    L. Tian & R.A. Dixon

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  1. L. Tian
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Correspondence to R.A. Dixon.

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Tian, L., Dixon, R. Engineering isoflavone metabolism with an artificial bifunctional enzyme. Planta 224, 496–507 (2006). https://doi.org/10.1007/s00425-006-0233-0

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