Monday, August 2, 2010
Pacific Concourse (Hyatt Regency San Francisco)
Andrew Main1, Anna Tai1, Patrick Westfall1, Chris Paddon1, Kirsten Benjamin1, Doug Pitera1, Diana Eng1, Hanxiao Jiang1, Tom Treynor1, Michele Fleck1, Tizita Horning1, Jake Lenihan1, Jack Newman1, Keat Teoh2, Devin Polichuk2, Patrick Covello2, Mario Ouellet3, Dae-Kyun Ro3 and Jay Keasling3, (1)Amyris Biotechnologies Inc., Emeryville, CA, (2)National Research Council Canada, Saskatoon, SK, Canada, (3)Lawrence Berkeley National Lab, Berkeley, CA
There are estimated to be 350-500 million clinical episodes of malaria annually, caused by the
Plasmodium parasite, with 1 million deaths, the highest mortality occurring in children under 5 years of age. The World Health Organization recommends the use of Artemisinin Combination Therapies (ACTs) for the treatment of uncomplicated
P. falciparum malaria, but the supply and price of artemisinin (extracted from the plant
Artemisia annua) have fluctuated greatly. An additional semi-synthetic source of artemisinin would be advantageous to stabilize the price and availability of ACTs. To this end, we are developing a process to produce artemisinic acid in
Saccharomyces cerevisiae, with subsequent chemical conversion to artemisinin.
Initial work involved expression of amorphadiene synthase from A. annua in a strain of S. cerevisiae S288C with an upregulated mevalonate pathway, enabling production of amorpha-4,11-diene. Subsequent expression of a cytochrome P450 (CYP71AV1) from A. annua and its cognate reductase (AaCPR1) allowed production of artemisinic acid (Ro et al., 2006).
Significant improvement in amorpha-4,11-diene production was achieved in CEN.PK2 by heterologous expression of the entire mevalonate pathway responsible for farnesyl diphosphate production, but expression of CYP71AV1/AaCPR1 in this strain resulted in production of reactive oxygen species and loss of viability. Decreasing production of AaCPR1 increased viability, but decreased production of artemisinic acid. Expression of a further gene from A. Annua was found to improve activity of CYP71AV1 without decreasing viability. Subsequent expression of dehydrogenases identified from A. annua dramatically increased artemisinic acid production.
