Cell-Free Production of Pentacyclic Triterpenoid Compound Betulinic Acid from Betulin by the Engineered  Saccharomyces cerevisiae

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Abstract

Betulinic acid is a product of plant secondary metabolism which has shown various bioactivities. Several CYP716A subfamily genes were recently characterized encoding multifunctional oxidases capable of C-28 oxidation. CYP716A12 was identified as betulin C-28 oxidase, capable of modifying betulin. This study aimed to induce the transformation of betulin to betulinic acid by co-expressing enzymes CYP716A12 from Medicago truncatula and ATR1 from Arabidopsis thaliana in Saccharomyces cerevisiae. The microsome protein extracted from the transgenic yeast successfully catalyzed the transformation of betulin to betulinic acid. We also characterized the optimization of cell fragmentation, protein extraction method, and the conversion conditions. Response surface methodology was implemented, and the optimal yield of betulinic acid reached 18.70%. After optimization, the yield and the conversion rate of betulin were increased by 83.97% and 136.39%, respectively. These results may present insights and strategies for the sustainable production of betulinic acid in multifarious transgenic microbes.

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Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

Dae-Kyun Ro, Eric M Paradise, Mario Ouellet … (2006)

Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.

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Box-Behnken design: an alternative for the optimization of analytical methods.

S. L. C. Ferreira, R. E. Bruns, H. S. Ferreira … (2007)

The present paper describes fundamentals, advantages and limitations of the Box-Behnken design (BBD) for the optimization of analytical methods. It establishes also a comparison between this design and composite central, three-level full factorial and Doehlert designs. A detailed study on factors and responses involved during the optimization of analytical systems is also presented. Functions developed for calculation of multiple responses are discussed, including the desirability function, which was proposed by Derringer and Suich in 1980. Concept and evaluation of robustness of analytical methods are also discussed. Finally, descriptions of applications of this technique for optimization of analytical methods are presented.

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Pentacyclic Triterpene Distribution in Various Plants – Rich Sources for a New Group of Multi-Potent Plant Extracts

Sebastian Jäger, Holger Trojan, Thomas Kopp … (2009)

Pentacyclic triterpenes are secondary plant metabolites widespread in fruit peel, leaves and stem bark. In particular the lupane-, oleanane-, and ursane triterpenes display various pharmacological effects while being devoid of prominent toxicity. Therefore, these triterpenes are promising leading compounds for the development of new multi-targeting bioactive agents. Screening of 39 plant materials identified triterpene rich (> 0.1% dry matter) plant parts. Plant materials with high triterpene concentrations were then used to obtain dry extracts by accelerated solvent extraction resulting in a triterpene content of 50 ‑ 90%. Depending on the plant material, betulin (birch bark), betulinic acid (plane bark), oleanolic acid (olive leaves, olive pomace, mistletoe sprouts, clove flowers), ursolic acid (apple pomace) or an equal mixture of the three triterpene acids (rosemary leaves) are the main components of these dry extracts. They are quantitatively characterised plant extracts supplying a high concentration of actives and therefore can be used for development of phytopharmaceutical formulations.

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Journal

Journal ID (nlm-ta): Molecules

Journal ID (iso-abbrev): Molecules

Journal ID (publisher-id): molecules

Title: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Publisher: MDPI

ISSN (Electronic): 1420-3049

Publication date (Electronic): 27 June 2017

Publication date Collection: July 2017

Volume: 22

Issue: 7

Electronic Location Identifier: 1075

Affiliations

Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China; 21613036@ 123456zju.edu.cn (J.W.); 11713002@ 123456zju.edu.cn (Y.N.); bakur888@ 123456yahoo.com (A.B.); lihaoenjoy@ 123456126.com (H.L.)

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[* ]Correspondence: chenqh@ 123456zju.edu.cn ; Tel.: +86-571-8698-4316

Article

Publisher ID: molecules-22-01075

DOI: 10.3390/molecules22071075

PMC ID: 6152010

PubMed ID: 28653998

SO-VID: 828c72c0-19fb-40a2-8615-aee6ccc66251

License:

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

History

Date received : 01 June 2017

Date accepted : 26 June 2017

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Cell-Free Production of Pentacyclic Triterpenoid Compound Betulinic Acid from Betulin by the Engineered Saccharomyces cerevisiae

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Most cited references 34

Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

Box-Behnken design: an alternative for the optimization of analytical methods.

Pentacyclic Triterpene Distribution in Various Plants – Rich Sources for a New Group of Multi-Potent Plant Extracts

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