Synthetic Production of Artemisinin Patrick J. Westfall, Ph.D. Senior Scientist, Amyris Inc.
Synthetic Production of Artemisinin Patrick J. Westfall, Ph.D. Senior Scientist, Amyris Inc. CONFIDENTIAL [1] Introduction to Amyris, Inc. – World wide locations CONFIDENTIAL [2] Introduction to Amyris, Inc. - Founders • • • • Jack Newman – CSO Kinkead Reiling Jay Keasling – UC Berkeley Neil Renninger -CTO CONFIDENTIAL [3] Introduction to Amyris, Inc. – The Power of Synthetic Biology Isolate Gene Scale Up Engineer Host Identify Single Gene Product CONFIDENTIAL [4] Introduction to Amyris, Inc. – The Power of Synthetic Biology Optimize Pathway Ergosterol Identify Entire Metabolic Pathway CONFIDENTIAL [5] Malaria Female Anopheles Mosquito • • • • Plasmodium falciparum Infected Red Blood Cells Affects 109 Countries Worldwide 216 Million cases in 2010 ~665,000 deaths in 2010 1 child dies every minute of Malaria in Africa • Source: 2010 WHO malaria fact sheet CONFIDENTIAL [6] A Brief History of Artemisinin 168 B.C Recipes For 52 Kinds Of Diseases found in the Mawangdui Han Dynasty tomb Hemorrhoids 340 A.D. Zhou Hou Bei Ji Fang (Handbook of Prescriptions for Emergency Treatments) Fevers (malaria) 1967 China sets up top-secret “Project 523” to find malaria medicines to help Vietnam during the war with the U.S. 1972 Active ingredient (artemisinin) isolated. 2004 World Health Organization (WHO) recommends artemisinin-based combination therapy (ACT) for uncomplicated malaria in areas experiencing chloroquine resistance CONFIDENTIAL [7] Treating Malaria : The Challenges Treating malaria would require: 189 to 327 million ACT treatments per year Artemisinin needed: 150 to 200 tons of artemisinin per year • 2-3X increase in production • Decrease/stabilization in price • Elimination of stock-outs CONFIDENTIAL [8] The Artemisinin Project: Significant lives saved due to scalable supply Artemisinin extraction A. annua cultivation (10-12 month growing cycle) Fermentation production (2 week cycle) Artemisinic Acid 3-Step Chemical Synthesis Steps CONFIDENTIAL [9] Primer to Important Compounds Oxidation/ Reduction Oxidation/ Reduction Oxidation/ Reduction H H H H H H H H H HO Amorphadiene Artemisinic Alcohol HO O O Artemisinic Aldehyde Artemisinic Acid H Chemistry O O H Artemisinin H O H O CONFIDENTIAL [ 10 ] The Mevalonate Metabolic Pathway in S. cerevisiae O H 3C SCoA ERG 10 E RG13 HM G1 HO C 2 Acetyl-CoA OH OH Mevalonate OPP E RG20 FPP HO H C 3 H IDI1 E RG19 E RG8 E RG12 H Ergosterol Ergosterol Identify Metabolic Pathway CONFIDENTIAL [ 11 ] Engineering the Mevalonate Pathway in Yeast tHMGR O H3C ERG10 S CoA Acetyl-CoA ERG13 ERG13 thiolase HMG1 synthase reductase tHMGR H3 C OH H O2C OH Mevalonate FPP OPP UPC2-1 ERG20 ERG20 FPP synthase IDI1 ERG19 isomerase decarboxylase ERG8 P-mev kinase ERG12 ERG12 mev kinase H ADS ERG9 squalene synthase H H HO Amorphadiene H Ergosterol CONFIDENTIAL [ 12 ] 160 140 120 100 80 60 40 20 0 S FP P R M G 2 x tH PC U P (M E 21 G 9 R T3 )-E tH M 1x A G R Amorphadiene concentration D S Amorphadiene (mg/l) Amorphadiene Production in S. cerevisiae Genetic modification From Ro et al Nature Biotech. 2006 CONFIDENTIAL [ 13 ] Engineering the Mevalonate Pathway in Yeast tHMGR O H3C ERG10 S CoA Acetyl-CoA thiolase H3 C ERG13 OH H O2C synthase reductase tHMGR OH Mevalonate FPP OPP ERG20 FPP synthase IDI1 ERG19 isomerase decarboxylase ERG8 P-mev kinase ERG12 mev kinase UPC2-1 H H CYP71AV1 CYP71AV1 CYP71AV1 AaCPR1 AaCPR1 AaCPR1 ADS H H Amorphadiene HO H H H H HO O Art. Acid H O CONFIDENTIAL [ 14 ] Production of Artemisinic Acid in S. cerevisiae 30 Target Product [g/L] 25 20 15 S. cerevisiae Art. Acid S. cerevisiae Amorph. 10 5 0 0 50 100 150 200 250 Time (hrs) CONFIDENTIAL [ 15 ] Heterologous Expression of the Entire S. cerevisiae Mev Pathway tHMGR O H3C ERG10 S CoA Acetyl-CoA thiolase H3 C ERG13 OH H O2C synthase reductase tHMGR OH Mevalonate FPP OPP ERG20 FPP synthase IDI1 ERG19 ERG8 ERG12 isomerase decarboxylase P-mev kinase mev kinase UPC2-1 H H CYP71AV1 CYP71AV1 CYP71AV1 AaCPR1 AaCPR1 AaCPR1 ADS H H Amorphadiene HO H H H H HO O Art. Acid H O CONFIDENTIAL [ 16 ] Heterologous Expression of the Entire S. cerevisiae Mev Pathway Yeast 1.0 CONFIDENTIAL [ 17 ] Heterologous Expression of the Entire S. cerevisiae Mev Pathway tHMGR O H3 C H3C ERG10 S CoA Acetyl-CoA thiolase tHMGR ERG13 OH H O2C synthase OH Mevalonate tHMGR FPP OPP ERG20 FPP synthase IDI1 ERG8 ERG19 isomerase P-mev kinase decarboxylase ERG12 mev kinase H H CYP71AV1 CYP71AV1 CYP71AV1 AaCPR1 AaCPR1 AaCPR1 ADS H H H H H H HO O Amorphadiene HO Art. Acid H O CONFIDENTIAL [ 18 ] Yeast 2.0 Amorphadiene Production CONFIDENTIAL [ 19 ] Large “Gap” in Production Between Amorphadiene and Art. Acid CONFIDENTIAL [ 20 ] Chemical Conversion of Amorphadiene to Artemisinin (Plan B) H H H c,d a,b 76% H H 76.4% H O HO H H H e,f O O H O 77.7% H H O H H O Westfall et al. PNAS USA 2012 Jan 17;109(3):E111-8 CONFIDENTIAL [ 21 ] Possible Sources of Toxicity and Poor P450 Activity 2. Product (AA) toxicity 1. Protein expression toxicity H H H HO ER O ER H ? NADPH CPR NADP 5. Poor coupling leads to excessive NADPH oxidation P450 ?-OH b5 H2O2 3. Improper oxidation toxicity 4. Oxidative stress 6. Poor coupling due to lack of CYT b5 22 CONFIDENTIAL & PROPRIETARY - Do Not Distribute Without Authorization CONFIDENTIAL [ 22 ] Artemisinic Acid Production in Shakeflasks CONFIDENTIAL [ 23 ] Heterologous Expression of the Entire S. cerevisiae Mev Pathway tHMGR H3 C O H3C ERG10 S CoA thiolase Acetyl-CoA tHMGR ERG13 OH H O2C OH Mevalonate synthase tHMGR FPP OPP ERG20 IDI1 FPP synthase isomerase ERG8 ERG19 P-mev kinase decarboxylase ERG12 mev kinase H CYP71AV1 ADS H CYP71AV1 H CYP71AV1 AaCYB5 AaCYB5 AaCYB5 AaCPR1 AaCPR1 AaCPR1 H HO H H O Amorphadiene H HO H H Art. Acid O CONFIDENTIAL [ 24 ] Product Formation in Shakeflasks CONFIDENTIAL [ 25 ] Product Formation in Shakeflasks CONFIDENTIAL [ 26 ] Conversion of Amorphadiene to Artemisinic Acid P450 P450 P450 H H H H H H H H HO Amorphadiene Artemisinic Alcohol H HO O O Artemisinic Aldehyde Artemisinic Acid CONFIDENTIAL [ 27 ] Intermediate Build Up CONFIDENTIAL [ 28 ] Collaboration with Dr. Pat Covello, Canadian Natural Resource Council H H HO AaADH1 H H H Pat Covello CNRC-PBI AaALDH1 H HO H O O CONFIDENTIAL [ 29 ] Conversion of Amorphadiene to Artemisinic Acid P450 AaALDH1 P450 AaADH1 P450 H H H H H H H H HO Amorphadiene Artemisinic Alcohol H HO O O Artemisinic Aldehyde Artemisinic Acid CONFIDENTIAL [ 30 ] Product Formation in Shakeflasks CONFIDENTIAL [ 31 ] Heterologous Expression of the Entire S. cerevisiae Mev Pathway tHMGR H3 C O H3C ERG10 S CoA thiolase Acetyl-CoA tHMGR ERG13 OH H O2C OH Mevalonate synthase tHMGR FPP OPP ERG20 IDI1 FPP synthase isomerase ERG8 ERG19 P-mev kinase decarboxylase ERG12 mev kinase H ADS H CYP71AV1 CYP71AV1 CYP71AV1 AaCYB5 AaCYB5 AaADH1 AaCYB5 AaALDH1 AaCPR1 AaCPR1 AaCPR1 H H Amorphadiene H HO H H HO H H O Art. Acid O CONFIDENTIAL [ 32 ] Product Formation in Bioreactors CONFIDENTIAL [ 33 ] Summary Heterologous expression of the entire mevalonate pathway in yeast allowed us to exceed the target goal for Amorphadiene production.. High level expression of the P450 and the AaCPR1 causes oxidative stress in cells resulting in decreased product formation and lower cell viability. Although the P450/CPR were capable of performing all three oxidation steps to convert Amorphadiene to Artemisinic Acid, discovery of additional pathway genes led reduced buildup of potentially toxic pathway intermediates. CONFIDENTIAL [ 34 ] The Opportunity for Impact CONFIDENTIAL [ 35 ] Predicted artemisinin supply by The Boston Consulting Group (November 2009) 2009 2010 2011 2012 2013 2014 2015 2010 2011 2012-2015 Supply of Artemisinin likely to be sufficient to meet ACT demand Artemisinin shortage probable unless the supply situation improves Significant increase in supply needed to meet steady state demand Some sources have predicted shortage or tight supply will be tight • We feel a shortage is unlikely given the most probable set of supply data and demand assumptions • Wild leafs would be able to make any potential small gap Three critical factors: (1) Amount of safety stock held in the supply chain today (2) Amount of Artemisia currently cultivated (3) AMFm uptake Phase I countries Companies will deplete majority of safety stock to meet 2010 demand Even with delayed AMFm demand will outstrip growing projected growing capacity available in 2011 Significantly increase in area under cultivation needed in 2010 to meet expected demand Cultivated Artemisia needs to increase to 23,000 hectares (vs 7,000 today) • Assuming that AMFm ramps to cover 50% of the private antimalarial market in 38 highest burden countries Historically, prices needed to reach >$1,000 / kg to attract this many farmers/ extractors to the market • Unclear what price point would be required to stimulate this level of growing in future New technologies may begin to affect artemisinin supply in this timeframe CONFIDENTIAL [ 36 ] July 7, 2010 press release “intent to commercialize ACTs containing the semisynthetic artemisinin in 2012” CONFIDENTIAL [ 37 ] Acknowledgements UC Berkeley Eric Paradise Dae-Kyun Ro Mario Oulett Jim Kirby Michelle Chang Syd Withers Jay Keasling Institute for OneWorld Health Tue Nguyen Sanofi-Aventis Corinne Masson-Brocard Denis Thibaut Ronan Guevel Marc Ferron Bruno Dumas Paul Baduel Henri Farret Amyris Biotechnologies Chris Paddon Hanxiao Jiang Kirsten Benjamin Tom Treynor Diana Eng Anna Tai Rika Regentin Andrew Main Hiroko Tsuruta Dan Dengrove Doug Pitera Michele Fleck Jake Lenihan Gordon Dang Jack Newman Stephanie Secrest Neil Renninger Tizita Horning Kinkead Reiling Karl Fischer Zygaya Kay Monroe Derek McPhee Consultants Jasper Rine (UCB) Hans van Dijken (Bird Engineering) Paul Ortiz de Montellano (UCSF) Pat Covello (CNRC) CONFIDENTIAL [ 38 ] Synthetic Biology in Northern California Institute for OneWorld Health Amyris JBEI (Jay Keasling) & SynBERC Amyris CONFIDENTIAL [ 39 ]
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