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Functional Integration of Two CYP450 Genes Involved in Biosynthesis of Tanshinones for Improved Diterpenoid Production by Synthetic Biology.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-06-18 , DOI: 10.1021/acssynbio.0c00136 Yaping Mao 1 , Ying Ma 1 , Tong Chen 1 , Xiaohui Ma 2 , Yanqin Xu 3 , Junling Bu 1 , Qishuang Li 1, 4 , Baolong Jin 1 , Yanan Wang 1 , Yong Li 1, 5 , Guanghong Cui 1 , Yujun Zhao 1 , Jinfu Tang 1 , Ye Shen 1 , Changjiangsheng Lai 1 , Wen Zeng 1 , Min Chen 1 , Juan Guo 1 , Luqi Huang 1
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-06-18 , DOI: 10.1021/acssynbio.0c00136 Yaping Mao 1 , Ying Ma 1 , Tong Chen 1 , Xiaohui Ma 2 , Yanqin Xu 3 , Junling Bu 1 , Qishuang Li 1, 4 , Baolong Jin 1 , Yanan Wang 1 , Yong Li 1, 5 , Guanghong Cui 1 , Yujun Zhao 1 , Jinfu Tang 1 , Ye Shen 1 , Changjiangsheng Lai 1 , Wen Zeng 1 , Min Chen 1 , Juan Guo 1 , Luqi Huang 1
Affiliation
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Cytochrome 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.
中文翻译：
参与丹参酮生物合成的两个CYP450基因的功能整合，以通过合成生物学改善二萜的产生。
细胞色素P450（CYPs）是植物二次代谢中的重要酶，并且被认为是生物工程和合成生物学的关键参与者。先前报道的CYP76AH1和CYP76AH3具有超过80％的序列同源性，在丹参中丹参酮的生物合成中起着持续的催化作用。同源性建模表明，四个位点可能是两种酶之间催化活性差异的原因。CYP76AH1的一系列基于建模的突变变体被设计为整合两个CYP的功能。突变CYP76AH1 D301E，V479FCYP76AH1和CYP76AH3的功能整合在一起，被发现可以有效催化C11和C12羟基化和C7氧化ti丁烯底物。通过合成生物学方法对CYP76AH1 D301E，V479F的整合和利用，使酵母中能够可靠地产生11-羟基铁氨醇，sugiol和11-羟基sugiol。活性位点修饰后功能上整合的CYP基因通过减少中间代谢物在组分蛋白之间的转移而提高了催化效率。这为提高微生物产生植物天然产物的系统的催化效率提供了合成生物学参考。
更新日期：2020-07-17
中文翻译：
参与丹参酮生物合成的两个CYP450基因的功能整合，以通过合成生物学改善二萜的产生。
细胞色素P450（CYPs）是植物二次代谢中的重要酶，并且被认为是生物工程和合成生物学的关键参与者。先前报道的CYP76AH1和CYP76AH3具有超过80％的序列同源性，在丹参中丹参酮的生物合成中起着持续的催化作用。同源性建模表明，四个位点可能是两种酶之间催化活性差异的原因。CYP76AH1的一系列基于建模的突变变体被设计为整合两个CYP的功能。突变CYP76AH1 D301E，V479FCYP76AH1和CYP76AH3的功能整合在一起，被发现可以有效催化C11和C12羟基化和C7氧化ti丁烯底物。通过合成生物学方法对CYP76AH1 D301E，V479F的整合和利用，使酵母中能够可靠地产生11-羟基铁氨醇，sugiol和11-羟基sugiol。活性位点修饰后功能上整合的CYP基因通过减少中间代谢物在组分蛋白之间的转移而提高了催化效率。这为提高微生物产生植物天然产物的系统的催化效率提供了合成生物学参考。
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