Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi Rapid discovery and functional characterization of diterpene synthases from basidiomycete fungi by genome mining Xin-Lin Li a,1 , Yu-Xing Xu e,1 , Yuan Li c , Rui Zhang a , Tian-Yuan Hu a , Ping Su c , Man Zhou b , Ting Tang b , Ying Zeng d , Yan-Long Yang b, ⁎ , Wei Gao a,f, ⁎ a School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, People’s Republic of China b State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China c National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, People’s Republic of China d State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China e Key Laboratory of Economic Plants and Biotechnology, Yunnan Key Laboratory for Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China f School of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, People’s Republic of China ARTICLE INFO Keywords: Basidiomycete Ditepene synthase Genome mining Cembrane diterpene (−)-cyatha-3, 12-diene Ent-kauran-16α-ol ABSTRACT Basidiomycete fungi are a rich source of bioactive diterpenoid secondary metabolites. However, compared with the large number of diterpene synthases (di-TPSs) identifed in plants and ascomycete fungi, only three di-TPSs have been described from basidiomycete fungi. Large scale genome sequencing projects combined with the development of synthetic biology techniques now has enabled the rapidly discovery and characterization of di- TPSs from basidiomycete fungi. In this study, we discovered and functionally characterized four di-TPSs from 220 genome sequenced basidiomycete fungi by a combined strategy of genomic data mining, phylogenetic analysis and fast products characterization with synthetic biology techniques. Among them, SteTC1 of Stereum histurum was characterized as the frst fungal cembrane diterpene synthase; PunTC of Punctularia strigosozonata and SerTC of Serpula lacrymans were characterized as ent-kauran-16α-ol synthase and DenTC3 of Dentipellis sp was characterized as a cyathane synthase. Our results provide opportunities for the discovery of new diterpe- noids from basidiomycete fungi by genome mining. 1. Introduction Diterpenoids, derived from the common isoprene precursor ger- anylgeranyl diphosphate (GGPP), are a class of natural products with diverse structures and highly potent bioactivities produced by plants, marine invertebrates, fungi and some prokaryotes (Hanson, 2015; Quin et al., 2014; Berrue and Kerr, 2009; Smanski et al., 2012). The struc- tural diversity of diterpenes largely originates from the frst scafold generating step, which is catalyzed by di-TPSs, a class of terpene cyclase (TC). The di-TPSs are responsible for the cyclization of the linear iso- prenoid precursors, GGPP, to form the complex carbon skeletons of diterpenoids. Depending on the reaction mechanisms and catalytic domains, di-TPSs are usually categorized into two distinct classes (type I and type II). While type II reactions perform a protonation initiated cyclization reaction to generate phosphorylated bicyclic structures, type I reactions are initiated by hydrolysis of the GGPP pyrophosphate moiety that is coordinated by a Mg 2+ -triad thereby generating mono- or poly-cyclic structures (Gong et al., 2014; Dickschat, 2016; Mitsuhashi and Abe, 2018; Minami et al., 2018). Recently, we found some members of the UbiA superfamily proteins, which normally cat- alyze the attachment of the prenyl chain to yield prenylated aromatic compounds, can catalyze the type I terpene cyclization reaction (Yang et al., 2017; Yang et al., 2018). Basidiomycete fungi have been reported to produce a wide array of diterpenes (Schüfer, 2018; Shen et al., 2009). These compounds have attracted much attention from chemists and biologists owing to their unique structures and diverse bioactivities. For example, pleuromutilin (Fig. 1) from Clitopilus passeckerianus is a ribosome-targeting antibiotic, https://doi.org/10.1016/j.fgb.2019.03.007 Received 6 December 2018; Received in revised form 25 February 2019; Accepted 20 March 2019 ⁎ Corresponding authors at: School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, People’s Republic of China (W. Gao). State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China (Y.L. Yang). E-mail addresses: ylyang@lzu.edu.cn (Y.-L. Yang), weigao@ccmu.edu.cn (W. Gao). 1 X.L. Li and Y.X. Xu contributed equally. Fungal Genetics and Biology 128 (2019) 36–42 Available online 21 March 2019 1087-1845/ © 2019 Elsevier Inc. All rights reserved. T