PMID- 18983675
OWN - NLM
STAT- MEDLINE
DCOM- 20081218
LR  - 20220310
IS  - 1472-6750 (Electronic)
IS  - 1472-6750 (Linking)
VI  - 8
DP  - 2008 Nov 4
TI  - Induction of multiple pleiotropic drug resistance genes in yeast engineered to 
      produce an increased level of anti-malarial drug precursor, artemisinic acid.
PG  - 83
LID - 10.1186/1472-6750-8-83 [doi]
AB  - BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.
FAU - Ro, Dae-Kyun
AU  - Ro DK
AD  - Department of Biological Sciences, University of Calgary, Calgary, T2N 1N4, 
      Canada. daekyun.ro@ucalgary.ca
FAU - Ouellet, Mario
AU  - Ouellet M
FAU - Paradise, Eric M
AU  - Paradise EM
FAU - Burd, Helcio
AU  - Burd H
FAU - Eng, Diana
AU  - Eng D
FAU - Paddon, Chris J
AU  - Paddon CJ
FAU - Newman, Jack D
AU  - Newman JD
FAU - Keasling, Jay D
AU  - Keasling JD
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20081104
PL  - England
TA  - BMC Biotechnol
JT  - BMC biotechnology
JID - 101088663
RN  - 0 (Antimalarials)
RN  - 0 (Artemisinins)
RN  - 0 (Polycyclic Sesquiterpenes)
RN  - 0 (Prodrugs)
RN  - 0 (RNA, Fungal)
RN  - 0 (Sesquiterpenes)
RN  - 0 (amorpha-4,11-diene)
RN  - 53N99527G7 (artemisic acid)
SB  - IM
MH  - Animals
MH  - Antimalarials/*metabolism
MH  - Artemisia annua/chemistry/genetics
MH  - Artemisinins/*metabolism
MH  - Drug Resistance, Multiple, Fungal/genetics
MH  - Fermentation
MH  - Gene Expression Profiling
MH  - Gene Expression Regulation, Fungal
MH  - Genes, Plant
MH  - Genetic Engineering/*methods
MH  - Oligonucleotide Array Sequence Analysis
MH  - Oxidative Stress
MH  - Plasmids
MH  - Point Mutation
MH  - Polycyclic Sesquiterpenes
MH  - Prodrugs/*metabolism
MH  - RNA, Fungal/genetics
MH  - Saccharomyces cerevisiae/genetics/*metabolism
MH  - Sesquiterpenes/metabolism
PMC - PMC2588579
EDAT- 2008/11/06 09:00
MHDA- 2008/12/19 09:00
PMCR- 2008/11/04
CRDT- 2008/11/06 09:00
PHST- 2008/06/05 00:00 [received]
PHST- 2008/11/04 00:00 [accepted]
PHST- 2008/11/06 09:00 [pubmed]
PHST- 2008/12/19 09:00 [medline]
PHST- 2008/11/06 09:00 [entrez]
PHST- 2008/11/04 00:00 [pmc-release]
AID - 1472-6750-8-83 [pii]
AID - 10.1186/1472-6750-8-83 [doi]
PST - epublish
SO  - BMC Biotechnol. 2008 Nov 4;8:83. doi: 10.1186/1472-6750-8-83.