Friedelane‐type triterpene cyclase in celastrol biosynthesis from Tripterygium wilfordii and its application for triterpenes biosynthesis in yeast

Abstract

Summary Celastrol is a promising bioactive compound isolated from Tripterygium wilfordii and has been shown to possess many encouraging preclinical applications. However, the celastrol biosynthetic pathway is poorly understood, especially the key oxidosqualene cyclase (OSC) enzyme responsible for cyclisation of the main scaffold. Here, we report on the isolation and characterisation of three OSCs from T. wilfordii: TwOSC1, TwOSC2 and TwOSC3. Both TwOSC1 and TwOSC3 were multiproduct friedelin synthases, while TwOSC2 was a β‐amyrin synthase. We further found that TwOSC1 and TwOSC3 were involved in the biosynthesis of celastrol and that their common product, friedelin, was a precursor of celastrol. We then reconstituted the biosynthetic pathway of friedelin in engineered yeast constructed by the CRISPR/Cas9 system, with protein modification and medium optimisation, leading to heterologous production of friedelin at 37.07 mg l−1 in a shake flask culture. Our study was the first to identify the genes responsible for biosynthesis of the main scaffold of celastrol and other triterpenes in T. wilfordii. As friedelin has been found in many plants, the results and approaches described here have laid a solid foundation for further explaining the biosynthesis of celastrol and related triterpenoids. Moreover, our results provide insights for metabolic engineering of friedelane‐type triterpenes.

Integrating pathway elucidation with yeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol

“…By transient expression in tobacco, we identified one of the four candidates, TwOSC4, to be a friedelin synthase. While the preparation of this manuscript was in progress, three OSCs from T. wilfordii cell suspension cultures (Genbank accession KY885467, KY885468 and KY885469) were published [55]. In agreement with our data, they were found to encode for β-amyrin, α-amyrin and friedelin synthase activities, respectively.…”

“…We interpret this as the result of inherent properties of friedelin itself, such as potential toxicity to yeast, or the mechanism of friedelin production by KdFRS, as friedelin is the last product of a long and complicated cycloisomerization cascade [33]. However, the recently published work of Zhou et al [55], reports a titer of more than 2 mg/L from expressing KY885469 in strain BY4741 [70]. This suggests that culture conditions or extraction method may be responsible for the differences in the reported yields and indicates that there is potential for important further improvement of friedelin production titers.…”

The genome analysis ofTripterygium wilfordiireveals TwCYP712K1 andTwCYP712K2responsible for oxidation of friedelin in celastrol biosynthesis pathway

“…On the other hand, we investigated the expression patterns of the identified 213 CYPs with TwOSC1 and TwOSC3, which were the two committed enzymes involved in precursor of celastrol biosynthesis in T. wilfordii (Zhou et al, 2019). Based on the RNA-Seq data of various tissues, we obtained 20 coexpression profiles of which only profile #3 and #13 showed significance (P-value<0.05) (Supplemental Figure 5).…”

“…Celastrol is pentacyclic triterpenoid synthesized from 2, 3-oxidosqualene, which is the common biosynthetic precursor of triterpenoid derived from the cytosolic mevalonate (MVA) and plastid 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway (Miettinen et al, 2017a;Thimmappa et al, 2014). Two oxidosqualene cyclase (OSC), TwOSC1 and TwOSC3, are identified as key enzymes in cyclization of 2, 3-oxidosqualene to form friedelin, which is the first committed step in celastrol formation (Zhou et al, 2019). The subsequent step in this pathway is consider to be the hydroxylation of C-29 position of friedelin to produce 29-hydroxy-friedelin-3-one and turn into polpunonic acid by carboxylation, which is then undergone a serious of oxidation and rearrangements to produce celastrol (Zhou et al, 2019).…”

“…Two oxidosqualene cyclase (OSC), TwOSC1 and TwOSC3, are identified as key enzymes in cyclization of 2, 3-oxidosqualene to form friedelin, which is the first committed step in celastrol formation (Zhou et al, 2019). The subsequent step in this pathway is consider to be the hydroxylation of C-29 position of friedelin to produce 29-hydroxy-friedelin-3-one and turn into polpunonic acid by carboxylation, which is then undergone a serious of oxidation and rearrangements to produce celastrol (Zhou et al, 2019).…”