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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.

SUBMITTER: Hansen NL

PROVIDER: S-EPMC6988343 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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

Hansen Nikolaj L NL Miettinen Karel K Zhao Yong Y Ignea Codruta C Andreadelli Aggeliki A Raadam Morten H MH Makris Antonios M AM Møller Birger L BL Stærk Dan D Bak Søren S Kampranis Sotirios C SC

Microbial cell factories 20200128 1

<h4>Background</h4>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.<h4>Results</h4>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 candid ...[more]

PMID: 31992268

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Project description:Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required.Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 microg mL(-1) in shake-flask cultures and 1 g L(-1) in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast.The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast.

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Dataset Information

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

Publications

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

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