Integrating pathway elucidation with yeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol.
Biosynthetic pathway
P450
Terpenoid
Tobacco
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
28 Jan 2020
28 Jan 2020
Historique:
received:
20
09
2019
accepted:
14
01
2020
entrez:
30
1
2020
pubmed:
30
1
2020
medline:
23
9
2020
Statut:
epublish
Résumé
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. 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. 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.
Sections du résumé
BACKGROUND
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
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
CONCLUSIONS
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.
Identifiants
pubmed: 31992268
doi: 10.1186/s12934-020-1284-9
pii: 10.1186/s12934-020-1284-9
pmc: PMC6988343
doi:
Substances chimiques
Anti-Obesity Agents
0
Pentacyclic Triterpenes
0
Terpenes
0
Triterpenes
0
maytenonic acid
33600-93-0
Cytochrome P-450 Enzyme System
9035-51-2
celastrol
L8GG98663L
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
15Subventions
Organisme : Novo Nordisk Fonden
ID : NNF18OC0031872
Organisme : Novo Nordisk Fonden
ID : NNF16OC0021616
Organisme : Novo Nordisk Fonden
ID : NNF17OC0027646
Organisme : Novo Nordisk Fonden
ID : NNF16OC0021760
Organisme : Seventh Framework Programme
ID : 613692
Organisme : General Secretariat for Research and Technology
ID : 11ΣΥΝ_3_770
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