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

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-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.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.

Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response

“…Interestingly, Ro et al (2008) found similar effects studying the synthesis of artemisinic acid in S. cerevisiae. Production of this isoprenoid led to a larger number of genes being up-and downregulated genes compared to this study, but shared some of the pleiotropic drug resistance genes that were also found in our study.…”

“…The ergosterol biosynthetic pathway, in both S. cerevisiae and X. dendrorhous, provides precursors for carotenoids. Solid arrow, direct conversion; dashed arrow, indirect conversion; IPP, isopentenyl pyrophosphate; DMAP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate impact on yeast physiology have been reported recently (Ro et al, 2008). Most transcriptome studies with S. cerevisiae have been performed with cells grown in shake-flask cultures.…”

Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin

“…Any process involving fermentation to economically produce precursors for the semisynthesis of artemisinin for the developing world would be unable to use galactose as a sole carbon source as it is too expensive. Strains of S. cerevisiae lacking the gal1 gene are unable to metabolize galactose, but galactose still acts as a gratuitous inducer (16) for all galactose-regulated genes in the artemisinic acid-producing strain EPY330 (15). Fig.…”

“…This strain was used for functional expression of CYP71AV1 [also known as amorphadiene oxidase (AMO)], the cytochrome P450 responsible for the three-step oxidation of amorpha-4,11-diene, along with its cognate reductase, AaCPR (A. annua cytochrome P450 reduc-tase), producing >100 mg∕L artemisinic acid (13). Incorporation of all A. annua-derived genes (ADS, CYP71AV1, and AaCPR) on a single expression plasmid allowed production of 2.5 g∕L of artemisinic acid by development of a galactose-based fermentation process (14), though plasmid stability was shown to be lower in a strain producing artemisinic acid compared to a strain producing amorpha-4,11-diene, and production of artemisinic acid led to induction of pleiotropic drug resistance genes (15). We now describe the creation of new S. cerevisiae strains and processes for the production of amorpha-4,11-diene, culminating in a 250-fold increase in production of amorpha-4,11-diene to 40 g∕L concentration, and a process for its efficient chemical conversion to the artemisinin precursor dihydroartemisinic acid.…”

Two Herbivore-Induced Cytochrome P450 Enzymes CYP79D6 and CYP79D7 Catalyze the Formation of Volatile Aldoximes Involved in Poplar Defense

“…The complete open reading frames of CYP79D6v3 and CYP79D7v2 were cloned into the pESC-Leu2d vector (Ro et al, 2008) as NotI-SacI fragments. For expression, the resulting constructs were transferred into the S. cerevisiae strain WAT11 (Pompon et al, 1996).…”