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Functional Integration of Two CYP450 Genes Involved in Biosynthesis of Tanshinones for Improved Diterpenoid Production by Synthetic Biology
journal contribution
posted on 2020-07-03, 12:35 authored by Yaping Mao, Ying Ma, Tong Chen, Xiaohui Ma, Yanqin Xu, Junling Bu, Qishuang Li, Baolong Jin, Yanan Wang, Yong Li, Guanghong Cui, Yujun Zhao, Jinfu Tang, Ye Shen, Changjiangsheng Lai, Wen Zeng, Min Chen, Juan Guo, Luqi HuangCytochrome
P450s (CYPs) are important enzymes in the secondary
metabolism of plants and have been recognized as key players in bioengineering
and synthetic biology. Previously reported CYP76AH1 and CYP76AH3,
having greater than 80% sequence homology, played a continuous catalytic
role in the biosynthesis of tanshinones in Salvia miltiorrhiza. Homology modeling indicates that four sites might be responsible
for differences in catalytic activity between the two enzymes. A series
of modeling-based mutational variants of CYP76AH1 were designed to
integrate the functions of the two CYPs. The mutant CYP76AH1D301E,V479F, which integrated the functions of CYP76AH1 and CYP76AH3, was found
to efficiently catalyze C11 and C12 hydroxylation and C7 oxidation
of miltiradiene substrates. Integration and utilization of CYP76AH1D301E,V479F by synthetic biology methods allowed the robust
production of 11-hydroxy ferruginol, sugiol, and 11-hydroxy sugiol
in yeast. The functionally integrated CYP gene after active site modifications
improves catalytic efficiency by reducing the transfer of intermediate
metabolites between component proteins. This provides a synthetic
biology reference for improving the catalytic efficiencies of systems
that produce plant natural products in microorganisms.