1. INTRODUCTION
Ganoderic acids (GAs), a group of highly oxygenated lanostane-type triterpenoids, are well recognized as the primary ingredient ofGanoderma lucidum , a traditional famous medicinal basidiomycete, as they possess unique bioactivities such as anti-tumor and anti-metastasis effects (Bishop et al., 2015; Shiao, 2003; Wu et al., 2013; Xiao and Zhong, 2016; Zhong et al., 2009). The GAs exhibit a wide range of structural diversity, and up to now more than 150 kinds of GAs have been reported (Baby et al., 2015; Li et al., 2018; Liang et al., 2019). They were hypothesized to be derived from the key precursor lanosterol undergoing extensive oxidations catalyzed by cytochrome P450 enzymes (CYPs) (Chen et al., 2012; Xiao and Zhong, 2016). We reported its initial key enzyme - lanosterol C-26 oxidase CYP5150L8, which could convert lanosterol to 3-hydroxy-lanosta-8,24-dien-26-oic acid (HLDOA) in a heterologous host - Saccharomyces cerevisiae (Wang et al., 2018). However, it is yet difficult to biosynthesize other GAs in a heterologous host like yeast, because the subsequent biosynthetic steps from HLDOA to other GAs remain unknown.
Mining the catalytic enzymes involved in post-modification of HLDOA is critical to the heterologous synthesis of other GAs. Overall, 197 CYP genes have been annotated from analyses of the G. lucidum genome, some of which were supposed to participate in GA biosynthesis (Chen et al., 2012). Due to the chemical structure difference of various GAs and the catalytic behavior of CYPs, hydroxylation/oxidation of GA skeleton at the C-7, C-12, C-15, C-22 or C-23 position is likely to be achieved by CYPs. Recently, CYP512U6 was reported to be responsible for the hydroxylation of GA-DM and GA-TR at C-23 position (Yang et al., 2018). But, in their work, use of expensive substrates GA-DM and GA-TR was required to synthesize hainanic acid A and GA-Jc, which is not helpful to synthesize GAs from a cheap and simple carbon source such as glucose. Therefore, taking the HLDOA-producing yeast (Wang et al., 2018) as a starting platform to further mine other key enzymes is considered as practical and critical for gradually elucidating the biosynthetic pathway of GAs as well as for their heterologous production.
In this work, we systematically screened candidate CYP genes by using the HLDOA producing yeast, and identified a functional CYP5139G1which could convert HLDOA to a new product. Then, the product was purified and its chemical structure was identified. Finally, we expressed both CYP5150L8 and CYP5139G1 by adopting a tunable expression strategy, and significantly enhanced the new GA production titer. The results will be helpful to further elucidating the synthetic pathway of GAs and constructing heterologous cell factories for efficient GAs production.