Characterization of a new fusicoccane-type diterpene synthase and an associated P450 enzyme

Jia-Hua Huang; Jian-Ming Lv; Liang-Yan Xiao; Qian Xu; Fu-Long Lin; Gao-Qian Wang; Guo-Dong Chen; Sheng-Ying Qin; Dan Hu; Hao Gao

doi:10.3762/bjoc.18.144

Characterization of a new fusicoccane-type diterpene synthase and an associated P450 enzyme

Beilstein J Org Chem. 2022 Oct 5:18:1396-1402. doi: 10.3762/bjoc.18.144. eCollection 2022.

Authors

Jia-Hua Huang^#¹, Jian-Ming Lv^#¹, Liang-Yan Xiao¹, Qian Xu¹, Fu-Long Lin¹, Gao-Qian Wang¹, Guo-Dong Chen¹, Sheng-Ying Qin², Dan Hu^{1

3}, Hao Gao¹

Affiliations

¹ Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy / Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research / International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Ministry of Education (MOE) of China, Jinan University, Guangzhou 510632, China.
² Clinical Experimental Center, First Affiliated Hospital of Jinan University, Guangzhou 510630, China.
³ Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.

^# Contributed equally.

Abstract

Fusicoccane-type terpenoids are a subgroup of diterpenoids featured with a unique 5-8-5 ring system. They are widely distributed in nature and possess a variety of biological activities. Up to date, only five fusicoccane-type diterpene synthases have been identified. Here, we identify a two-gene biosynthetic gene cluster containing a new fusicoccane-type diterpene synthase gene tadA and an associated cytochrome P450 gene tadB from Talaromyces wortmannii ATCC 26942. Heterologous expression reveals that TadA catalyzes the formation of a new fusicoccane-type diterpene talaro-7,13-diene. D₂O isotope labeling combined with site-directed mutagenesis indicates that TadA might employ a different C2,6 cyclization strategy from the known fusicoccane-type diterpene synthases, in which a neutral intermediate is firstly formed and then protonated by an environmental proton. In addition, we demonstrate that the associated cytochrome P450 enzyme TadB is able to catalyze multiple oxidation of talaro-7,13-diene to yield talaro-6,13-dien-5,8-dione.

Keywords: cytochrome P450 enzyme; diterpene synthase; gene cluster; genome mining; site-directed mutagenesis.

Grants and funding

This work was financially supported by grants from National Key Research and Development Program of China (2018YFA0903200 / 2018YFA0903201), the National Natural Science Foundation of China (82073046, 81925037, 32170060, 31870032, 22177037), the 111 Project of Ministry of Education of the People’s Republic of China (B13038), National Foreign Experts Project (G2021199001L, China), National High-level Personnel of Special Support Program (2017RA2259, China), the Guangdong Natural Science Funds for Distinguished Young Scholar (2019B151502014, 2022B1515020028, China), Guangdong International Science and Technology Cooperation Base (2021A0505020015, China), Guangdong Science and Technology Planning Project (2020A0505100041, China), Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Y036, China), Innovative and Research Teams Project of Guangdong Higher Education Institution (2021KCXTD001, China), Guangzhou Science and Technology Project (202102010114, China), and Shenzhen Institute of Synthetic Biology Scientific Research Program (DWKF20210002, China).