Eudesmane-type sesquiterpene diols directly synthesized by a sesquiterpene cyclase in Tripterygium wilfordii

Author:

Tong Yu-ru1,Su Ping2,Guan Hong-yu3,Hu Tian-yuan3,Chen Jin-long2,Zhang Yi-feng3,Zhao Yu-jun2,Gao Lin-hui3,Zhang Xia-nan3,Huang Lu-qi12,Gao Wei3

Affiliation:

1. School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China

2. National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China

3. School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, P.R. China

Abstract

Cryptomeridiol, a typical eudesmane diol, is the active principle component of the antispasmodic Proximol. Although it has been used for many years, the biosynthesis pathway of cryptomeridiol has remained blur. Among terpenoid natural products, terpenoid cyclases are responsible for cyclization and generation of hydrocarbon backbones. The cyclization is mediated by carbocationic cascades and ultimately terminated via deprotonation or nucleophilic capture. Isoprene precursors are, respectively, converted into hydrocarbons or hydroxylated backbones. A sesquiterpene cyclase in Tripterygium wilfordii (TwCS) was determined to directly catalyze (E,E)-farnesyl pyrophosphate (FPP) to unexpected eudesmane diols, primarily cryptomeridiol. The function of TwCS was characterized by a modular pathway engineering system in Saccharomyces cerevisiae. The major product determined by NMR spectroscopy turned out to be cryptomeridiol. This unprecedented production was further investigated in vitro, which verified that TwCS can directly produce eudesmane diols from FPP. Some key residues for TwCS catalysis were screened depending on the molecular model of TwCS and mutagenesis studies. As cryptomeridiol showed a small amount of volatile and medicinal properties, the biosynthesis of cryptomeridiol was reconstructed in S. cerevisiae. Optimized assays including modular pathway engineering and the CRISPR–cas9 system were successfully used to improve the yield of cryptomeridiol in the S. cerevisiae. The best engineered strain TE9 (BY4741 erg9::Δ-200-176 rox1::mut/pYX212-IDI + TwCS/p424-tHMG1) ultimately produced 19.73 mg/l cryptomeridiol in a shake flask culture.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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