Functional Integration of Two CYP450 Genes Involved in Biosynthesis of Tanshinones for Improved Diterpenoid Production by Synthetic Biology
Functional Integration of Two CYP450 Genes Involved in Biosynthesis of Tanshinones for Improved Diterpenoid Production by Synthetic Biology
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DOI：
10.1021/acssynbio.0c00136
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发表时间：
2020-07-17
影响因子：
4.7
通讯作者：
Huang, Luqi
中科院分区：
文献类型：
--
作者：
Mao, Yaping;Ma, Ying;Huang, Luqi
细胞色素P450（CYP）是植物次生代谢中的重要酶，在生物工程和合成生物学中发挥着重要作用。CYP 76 AH 1和CYP 76 AH 3具有80%以上的序列同源性，在丹参中丹参酮的生物合成中起着持续的催化作用。同源建模表明，四个网站可能是负责两种酶之间的催化活性的差异。设计了一系列基于模型的CYP 76 AH 1突变体，以整合这两种CYP的功能。突变体CYP 76 AH 1（D301 E，V479 F）整合了CYP 76 AH 1和CYP 76 AH 3的功能，能高效催化米替雷烯底物的C11和C12羟基化和C7氧化。通过合成生物学方法整合和利用CYP 76 AH 1（D301 E，V479 F）允许在酵母中稳健地生产11-羟基Ferruginol、sugiol和11-羟基sugiol。活性位点修饰后的功能整合的p53基因通过减少组分蛋白之间的中间代谢物的转移来提高催化效率。这为提高在微生物中生产植物天然产物的系统的催化效率提供了合成生物学参考。
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 inte_rate the functions of the two CYPs. The mutant CYP76AH1(D301E,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 CYP76AH1(D301E,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.