CYP76AH1 Catalyzes Turnover of Miltiradiene in Tanshinones Biosynthesis and Enables Heterologous Production of Ferruginol in Yeasts
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CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts Juan Guoa,1, Yongjin J. Zhoub,1, Matthew L. Hillwigc, Ye Shena, Lei Yangd, Yajun Wanga, Xianan Zhanga, Wujun Liub, Reuben J. Petersc,2, Xiaoya Chend, Zongbao K. Zhaob,2, and Luqi Huanga,2 aNational Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, People’s Republic of China; bDivision of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, People’s Republic of China; cDepartment of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011; and dNational Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, and Plant Science Research Center of Shanghai Chenshan Botanical Garden, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People’s Republic of China Edited by Joseph P. Noel, Howard Hughes Medical Institute and The Salk Institute for Biological Studies, La Jolla, CA, and accepted by the Editorial Board May 22, 2013 (received for review October 16, 2012) Cytochrome P450 enzymes (CYPs) play major roles in generating sequence homology (2), and the encoding genes generally are not highly functionalized terpenoids, but identifying the exact bio- physically clustered together with genes responsible for the up- transformation step(s) catalyzed by plant CYP in terpenoid biosyn- stream enzymatic reactions as found in microbial genomes (3). thesis is extremely challenging. Tanshinones are abietane-type For example, despite tremendous efforts during the past two norditerpenoid naphthoquinones that are the main lipophilic bio- decades to delineate the biosynthetic pathway for the most im- active components of the Chinese medicinal herb danshen (Salvia portant diterpenoid, taxol, there are still unresolved steps likely miltiorrhiza). Whereas the diterpene synthases responsible for the involving transformations catalyzed by CYP (4). Nonetheless, fi conversion of (E,E,E)-geranylgeranyl diphosphate into the abie- research continues, and has even intensi ed, because un- tane miltiradiene, a potential precursor to tanshinones, have been derstanding the molecular basis of terpenoid biosynthesis recently described, molecular characterization of further transfor- holds great promise to engineer the native-producing organ- isms, as well as surrogate microbes, to produce the desired mation of miltiradiene remains unavailable. Here we report stable- fi isotope labeling results that demonstrate the intermediacy of miltira- molecules in suf cient quantities for commercial purposes. Our recent efforts have been focused on the biosynthesis of a diene in tanshinone biosynthesis. We further use a next-generation group of abietane-type norditerpenoids, tanshinones (Fig. 1), the sequencing approach to identify six candidate CYP genes being cor- major lipophilic bioactive components of the rhizome of the egulated with the diterpene synthase genes in both the rhizome Chinese medicinal herb, Salvia miltiorrhiza Bunge (Lamiaceae), and danshen hairy roots, and demonstrate that one of these, known as tanshen or danshen (5). Danshen has been prominently CYP76AH1, catalyzes a unique four-electron oxidation cascade on mentioned in discussions of how traditional Chinese medicine miltiradiene to produce ferruginol both in vitro and in vivo. We then might be coupled to a more modern approach (6). More impor- build upon the previous establishment of miltiradiene production in tantly, tanshinones have been shown to exhibit a variety of bi- Saccharomyces cerevisiae, with incorporation of CYP76AH1 and ological activities, which includes antibiotic, anti-inflammatory, phyto-CYP reductase genes leading to heterologous production of and antioxidant effects, as well as activity against various aspects of ferruginol at 10.5 mg/L. As ferruginol has been found in many plants heart disease. Indeed, danshen preparations are in phase II clinical including danshen, the results and the approaches that were de- trials against cardiovascular disease (7). Although the production scribed here provide a solid foundation to further elucidate the bio- of tanshinones is inducible in S. miltiorrhiza hairy roots (8), the synthesis of tanshinones and related diterpenoids. Moreover, these current supply is dependent on field-grown plants, which are results should facilitate the construction of microbial cell factories subject to variable yields, and genetic modification offers poten- for the production of phytoterpenoids. tially increased yields in either system. However, this requires some understanding of the underlying biosynthetic pathway. Our phytoterpenoids biosynthesis | gene discovery | synthetic pathway | initial investigations were carried out using a functional genomics metabolic engineering approach based on a cDNA microarray using ∼4,400 expressed sequence tags (ESTs), generated with traditional sequencing tech- fi erpenoids represent a diverse class of secondary metabolites nology (9). This led to functional identi cation of two sequentially acting diterpene cyclases, as required for the formation of labdane- Tattracting commercial interest due to their use as drugs, fra- fi grances, and alternative fuels. In plants, terpenoids are synthe- related diterpenoids such as the tanshinones (10, 11): speci cally, a class II diterpene cyclase, which produces copalyl diphosphate sized from two C precursors, isopentenyl diphosphate (IPP) and 5 from GGPP, termed CPP synthase (SmCPS), and a subsequently dimethylallyl diphosphate (DMAPP), which are derived via the 1-deoxyxylulose-5-phosphate pathway or the mevalonate pathway. Prenyltransferases then combine DMAPP and IPP to form other Author contributions: J.G., Y.J.Z., R.J.P., X.C., Z.K.Z., and L.H. designed research; J.G., Y.J.Z., building blocks, (E)-geranyl pyrophosphate (GPP; C10), (E,E)- E,E,E M.L.H., Y.S., Y.W., X.Z., and W.L. performed research; X.C. contributed new reagents/ana- farnesyl pyrophosphate (FPP; C15), and ( )-geranylgeranyl lytic tools; J.G., Y.J.Z., M.L.H., Y.S., L.Y., R.J.P., Z.K.Z., and L.H. analyzed data; and J.G., Y.J.Z., pyrophosphate (GGPP; C20). These acyclic precursors are trans- R.J.P., Z.K.Z., and L.H. wrote the paper. formed by terpene synthases/cyclases, followed by tailoring The authors declare no conflict of interest. enzymes to generate compounds with tremendous structure This article is a PNAS Direct Submission. J.P.N. is a guest editor invited by the Editorial Board. diversities. Thus, over 50,000 terpenoid compounds have been Data deposition: The sequences reported in this paper have been deposited in the GenBank identified. The most important tailoring process in terpenoid database (accession nos. SRX224100, JX422213–JX422218,andCBX24555). biosynthesis is oxidation/oxygenation, which is largely catalyzed by 1J.G. and Y.J.Z. contributed equally to this work. cytochrome P450 (CYP) enzymes (1). However, functionally char- 2To whom correspondence may be addressed. E-mail: [email protected], rjpeters@iastate. acterizing the role of plant CYP in particular biotransformation edu, or [email protected]. step(s) in terpenoid biosynthesis is extremely challenging, because This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. these CYPs normally have high substrate specificity, share low 1073/pnas.1218061110/-/DCSupplemental. 12108–12113 | PNAS | July 16, 2013 | vol. 110 | no. 29 www.pnas.org/cgi/doi/10.1073/pnas.1218061110 Downloaded by guest on October 2, 2021 serve as a precursor to compounds found in the late stage of the proposed tanshinone biosynthetic pathway (Fig. 1). CYP Candidate Gene Discovery. From our previous work with SmCPS and SmKSL, transcription of the encoding genes was clearly increased by induction of hairy root cultures (11). Such inducible transcription has been shown to extend to the sub- sequently acting CYPs in other terpenoid biosynthetic pathways (3, 18–24). Accordingly, to find candidate CYPs for tanshinone biosynthesis we moved beyond our previously reported EST da- tabase (9) and used next-generation sequencing of mRNA from induced hairy roots to generate an extensive transcriptome (ac- cession no. SRX224100), resulting in 25,793 isotigs ranging from Fig. 1. Partial pathways for tanshinones biosynthesis and structures for 100 to 1,100 nucleotides in length. Analysis of this dataset revealed some representative tanshinones. Solid arrows indicate the established the presence of ∼300 CYP isotigs, close to the numbers of CYPs relationships, and dashed arrows indicate hypothetical relationships. found in other plant species (22). Moreover, using an RNA-seq approach to examine the change in transcriptome upon induction + ent with Ag , 14 CYP genes were selected as initial candidates for acting class I diterpene cyclase, termed -kaurene synthase-like investigation on the basis of their significant increase in tran- (SmKSL), which produces the abietane miltiradiene (11). script levels. Miltiradiene contains a cyclohexa-1,4-diene moiety that impo- fi Given the accumulation of tanshinones in the rhizome (8), and ses a planar con guration on the distal ring, which is suggestively previous tissue-specific transcription demonstrated for other poised for aromatization, as required for the production of tan- diterpenoid metabolism (18, 19, 25, 26), we hypothesized that shinones. Thus, miltiradiene is a potential intermediate in tan- tanshinone