TY - JOUR
T1 - Removing allosteric feedback inhibition of tomato 4-coumarate:CoA ligase by directed evolution
AU - Alberstein, Moti
AU - Eisenstein, Miriam
AU - Abeliovich, Hagai
PY - 2012/1
Y1 - 2012/1
N2 - 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.
AB - 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.
KW - 4-coumarate:CoA ligase
KW - allosteric regulation
KW - directed evolution
KW - phenylpropanoid metabolism
KW - tomato
UR - http://www.scopus.com/inward/record.url?scp=84355161649&partnerID=8YFLogxK
U2 - 10.1111/j.1365-313X.2011.04770.x
DO - 10.1111/j.1365-313X.2011.04770.x
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C2 - 21883557
AN - SCOPUS:84355161649
SN - 0960-7412
VL - 69
SP - 57
EP - 69
JO - Plant Journal
JF - Plant Journal
IS - 1
ER -