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

Abstract

Background: Celastrol is a promising anti-obesity agent that acts as a sensitizer of the protein hormone leptin. Despite its potent activity, a sustainable source of celastrol and celastrol derivatives for further pharmacological studies is lacking. Results:To elucidate the celastrol biosynthetic pathway and reconstruct it in Saccharomyces cerevisiae, we mined a root-transcriptome of Tripterygium wilfordii and identified four oxidosqualene cyclases and 49 cytochrome P450s as candidates to be involved in the early steps of celastrol biosynthesis. Using functional screening of the candidate genes in Nicotiana benthamiana, TwOSC4 was characterized as a novel oxidosqualene cyclase that produces friedelin, the presumed triterpenoid backbone of celastrol. In addition, three P450s (CYP712K1, CYP712K2, and CYP712K3) that act downstream of TwOSC4 were found to effectively oxidize friedelin and form the likely celastrol biosynthesis intermediates 29-hydroxy-friedelin and polpunonic acid. To facilitate production of friedelin, the yeast strain AM254 was constructed by deleting UBC7, which afforded a fivefold increase in friedelin titer. This platform was further expanded with CYP712K1 to produce polpunonic acid and a method for the facile extraction of products from the yeast culture medium, resulting in polpunonic acid titers of 1.4 mg/L. Conclusion:Our study elucidates the early steps of celastrol biosynthesis and paves the way for future biotechnological production of this pharmacologically promising compound in engineered yeast strains.

Biosynthesis, total synthesis, structural modifications, bioactivity, and mechanism of action of the quinone‐methide triterpenoid celastrol

“…This valuable study deepened the understanding of P450s and prompted further studies on screening and functional verification of enzymes involved in the following steps. Later, Hansen et al 22 reported another three P450s (CYP712K1–3) from the roots of T. wilfordii ; their functions are identical to that of CYP712K4, as described previously. Subsequently, they reconstructed the biosynthetic pathway in Saccharomyces cerevisiae and achieved a yield of 1.4 mg/L polpunonic acid.…”

“…Researchers have made significant efforts to explore key enzymes in its biosynthesis pathway and characterized sequential synthetic steps catalyzed by nearly 20 different functional enzymes, especially including 2,3‐oxidosqualene cyclases 20 and cytochrome P450s 21 . Hansen et al 22 constructed the yeast chassis cells that can produce the celastrol precursor polpunonic acid with a yield of 1.4 mg/L. Research is getting closer to the complete biosynthesis of celastrol.…”

Engineered butyrate-producing bacteria prevents high fat diet-induced obesity in mice

“…Bacillus subtilis (B. subtilis) is a food-grade probiotic with a clear genetic background and has also been used as an animal feed additive to improve growth performance and immunity [23]. Genetically modified probiotics are increasingly being used as therapeutics for the treatment of obesity [24,25]. In the present study, B. subtilis SCK6 (SCK6) was genetically modified to enhance its production of BA.…”

Introducing climate change into the biochemistry and molecular biology curriculum

“…They found, for example, that TwOSC1‐L486V produces a 4:1 product ratio of β‐amyrin to friedelin and that TwOSC3‐L482V yields a 1:0 ratio (Figure 11b). Finally, in a separate study, another group has reported the discovery of several cytochrome P450 enzymes that catalyze additional oxidation steps on the pathway to celastrol 108 ; Figure 11a).…”