Norditerpenoids biosynthesized by variediene synthase-associated P450 machinery along with modifications by the host cell Aspergillus oryzae

Lan Jiang; Kangjie Lv; Guoliang Zhu; Zhi Lin; Xue Zhang; Cuiping Xing; Huanting Yang; Weiyan Zhang; Zhixin Wang; Chengwei Liu; Xudong Qu; Tom Hsiang; Lixin Zhang; Xueting Liu

doi:10.1016/j.synbio.2022.08.002

Norditerpenoids biosynthesized by variediene synthase-associated P450 machinery along with modifications by the host cell Aspergillus oryzae

Synth Syst Biotechnol. 2022 Aug 23;7(4):1142-1147. doi: 10.1016/j.synbio.2022.08.002. eCollection 2022 Dec.

Authors

Lan Jiang¹, Kangjie Lv¹, Guoliang Zhu¹, Zhi Lin², Xue Zhang¹, Cuiping Xing¹, Huanting Yang¹, Weiyan Zhang¹, Zhixin Wang¹, Chengwei Liu³, Xudong Qu², Tom Hsiang⁴, Lixin Zhang¹, Xueting Liu¹

Affiliations

¹ State Key Laboratory of Bioreactor Engineering, East China University of Science of Technology, Shanghai, China.
² State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
³ Key Laboratory of Saline-alkali Vegetation Ecology Restoration, Ministry of Education, College of Life Sciences, Northeast Forestry University, Harbin, China.
⁴ School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada.

Abstract

The chemical diversity of terpenoids is typically established by terpene synthase-catalyzed cyclization and diversified by post-tailoring modifications. Fungal bifunctional terpene synthase (BFTS) associated P450 enzymes have shown significant catalytic potentials through the development of various new terpenoids with different biological activities. This study discovered the BFTS and its related gene cluster from the plant endophytic fungus Didymosphaeria variabile 17020. Heterologous expression of the BFTS in Saccharomyces cerevisiae resulted in the characterization of a major product diterpene variediene (1), along with two new minor products neovariediene and neoflexibilene. Further heterologous expression of the BFTS and one cytochrome P450 enzyme VndE (CYP6138B1) in Aspergillus oryzae NSAR1 led to the identification of seven norditerpenoids (19 carbons) with a structurally unique 5/5 bicyclic ring system. Interestingly, in vivo experiments suggested that the cyclized terpene variediene (1) was modified by VndE along with the endogenous enzymes from the host cell A. oryzae through serial chemical conversions, followed by multi-site hydroxylation via A. oryzae endogenous enzymes. Our work revealed that the two-enzymes biosynthetic system and host cell machinery could produce structurally unique terpenoids.

Keywords: Aspergillus oryzae; Bifunctional terpene synthases; Cytochrome P450; Heterologous expression; Norditerpenoids.