Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acidReport as inadecuate




Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid - Download this document for free, or read online. Document in PDF available to download.

BMC Biotechnology

, 8:83

First Online: 04 November 2008Received: 05 June 2008Accepted: 04 November 2008

Abstract

BackgroundDue 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.

ResultsThree 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 μg mL in shake-flask cultures and 1 g L 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.

ConclusionThe 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.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-8-83 contains supplementary material, which is available to authorized users.

Dae-Kyun Ro, Mario Ouellet contributed equally to this work.

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Author: Dae-Kyun Ro - Mario Ouellet - Eric M Paradise - Helcio Burd - Diana Eng - Chris J Paddon - Jack D Newman - Jay D Keasl

Source: https://link.springer.com/article/10.1186/1472-6750-8-83



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