Developing Commercial Production of Semi-Synthetic Artemisinin, and of β-Farnesene, an Isoprenoid Produced by Fermentation of Brazilian Sugar

The antimalarial drug artemisinin and the specialty chemical β-farnesene are examples of natural product isoprenoids that can help solve global challenges, but whose usage has previously been limited by supply and cost impediments. This review describes the path to commercial production of these compounds utilizing fermentation of engineered yeast. Development of commercially viable yeast strains was a substantial challenge that was addressed by creation and implementation of an industrial synthetic biology pipeline. Using the engineered strains, production of β-farnesene from Brazilian sugarcane offers several environmental advantages. Among the many commercial applications of β-farnesene, its use as a feedstock for making biodegradable lubricants is highlighted. This example, along with others, highlight a powerful new suite of technologies that will become increasingly important for production of chemicals, spanning from pharmaceuticals through commodity chemicals.

Keywords:
artemisinin; farnesene; fermentation; natural product; lubricant

Authorship

Kirsten R. Benjamin

Amyris, Inc., 5885 Hollis Street, Suite 100, CA 94608 Emeryville, USAAmyris, IncUSAEmeryville, USAAmyris, Inc., 5885 Hollis Street, Suite 100, CA 94608 Emeryville, USA

Kirsten Benjamin is a Senior Research Fellow at Amyris, Inc. in Emeryville, CA. She built and tested improved microbial strains for the Semi-Synthetic Artemisinin project and was director of the Farnesene (BiofeneTM) Strain Improvement project. She received her Bachelor's degree in Molecular and Cell Biology from the University of Michigan (USA), and her doctorate in Biochemistry from the University of California at Berkeley (USA). Following postdoctoral work at the University of California at San Francisco, she performed systems biology research at the Molecular Sciences Institute (Berkeley, CA, USA) before joining Amyris.

Iris R. Silva

Amyris Brasil Ltda, Rua John Dalton 301-Bloco B-Edificio 3, Condominio Techno Plaza, 13069-330 Campinas-SP, BrazilAmyris Brasil LtdaBrazilCampinas, SP, BrazilAmyris Brasil Ltda, Rua John Dalton 301-Bloco B-Edificio 3, Condominio Techno Plaza, 13069-330 Campinas-SP, Brazil

Iris R. Silva is Process Development Manager of Brazil at Amyris Brazil. She received her BSc degree in Chemical Engineering from University of São Carlos (Brazil) and has an MBA in Project Management and Business. She has worked at Amyris with fermentation and downstream processing for the last 8 years, having previously worked in production processes and currently in process development areas.

João P. Cherubim

Amyris Brasil Ltda, Rodovia Brotas/Torrinha-km 7.5, 17380-000 Brotas-SP, BrazilAmyris Brasil LtdaBrazilBrotas, SP, BrazilAmyris Brasil Ltda, Rodovia Brotas/Torrinha-km 7.5, 17380-000 Brotas-SP, Brazil

João Paulo Cherubim is Manufacturing and Process Development Director at Amyris Brazil. He has a BSc degree in Chemical Engineering and Master in Brewery Process, Sugar and Ethanol Production and in Business Managment. In the last 22 years, he was responsible for manufacturing activities at ABInbev, Imerys, Zilor and Amyris, with more than 15 years of fermentation processes expertise.

Derek McPhee

Amyris, Inc., 5885 Hollis Street, Suite 100, CA 94608 Emeryville, USAAmyris, IncUSAEmeryville, USAAmyris, Inc., 5885 Hollis Street, Suite 100, CA 94608 Emeryville, USA

Derek McPhee is the Senior Director of Technology Strategy at Amyris Inc., where he was previously the Director/Sr. Director of Chemistry. He has a LicC in Applied Chemistry from the Universidad de Málaga (Spain) and a PhD in Organic Chemistry from the University of Calgary (Canada). After a NSERC post-doctoral fellowship at the Canadian NRC, the remainder of his career has been in industry, first in the crop protection, specialty and rubber chemicals divisions of Uniroyal Chemical (now Chemtura Corp.), then the generic pharmaceutical industry at Brantford Chemicals (now Apotex Pharmachem), and as Director of Chemistry/VP of AstaTech Canada.

Chris J. Paddon ^**e-mail: paddon@amyris.com

Amyris, Inc., 5885 Hollis Street, Suite 100, CA 94608 Emeryville, USAAmyris, IncUSAEmeryville, USAAmyris, Inc., 5885 Hollis Street, Suite 100, CA 94608 Emeryville, USA

Chris Paddon is a Principal Scientist at Amyris, Inc. in Emeryville, CA. He was project leader for the Semi-Synthetic Artemisinin project, and subsequently led a number of projects at Amyris using synthetic biology for the production of natural products. He received his Bachelor's degree in Microbiology from The University of Surrey (UK), and doctorate in Biochemistry from Imperial College (London, UK). Following postdoctoral work at The National Institutes for Health (Bethesda, MD) he joined the pharmaceutical industry, working for GSK (London, UK). He subsequently worked for Affymax (Palo Alto, CA) and Xenoport (Santa Clara, CA) before joining Amyris.

^*e-mail: paddon@amyris.com

Chris Paddon is a Principal Scientist at Amyris, Inc. in Emeryville, CA. He was project leader for the Semi-Synthetic Artemisinin project, and subsequently led a number of projects at Amyris using synthetic biology for the production of natural products. He received his Bachelor's degree in Microbiology from The University of Surrey (UK), and doctorate in Biochemistry from Imperial College (London, UK). Following postdoctoral work at The National Institutes for Health (Bethesda, MD) he joined the pharmaceutical industry, working for GSK (London, UK). He subsequently worked for Affymax (Palo Alto, CA) and Xenoport (Santa Clara, CA) before joining Amyris.

Derek McPhee is the Senior Director of Technology Strategy at Amyris Inc., where he was previously the Director/Sr. Director of Chemistry. He has a LicC in Applied Chemistry from the Universidad de Málaga (Spain) and a PhD in Organic Chemistry from the University of Calgary (Canada). After a NSERC post-doctoral fellowship at the Canadian NRC, the remainder of his career has been in industry, first in the crop protection, specialty and rubber chemicals divisions of Uniroyal Chemical (now Chemtura Corp.), then the generic pharmaceutical industry at Brantford Chemicals (now Apotex Pharmachem), and as Director of Chemistry/VP of AstaTech Canada.

Kirsten Benjamin is a Senior Research Fellow at Amyris, Inc. in Emeryville, CA. She built and tested improved microbial strains for the Semi-Synthetic Artemisinin project and was director of the Farnesene (Biofene^TM) Strain Improvement project. She received her Bachelor's degree in Molecular and Cell Biology from the University of Michigan (USA), and her doctorate in Biochemistry from the University of California at Berkeley (USA). Following postdoctoral work at the University of California at San Francisco, she performed systems biology research at the Molecular Sciences Institute (Berkeley, CA, USA) before joining Amyris.

João Paulo Cherubim is Manufacturing and Process Development Director at Amyris Brazil. He has a BSc degree in Chemical Engineering and Master in Brewery Process, Sugar and Ethanol Production and in Business Managment. In the last 22 years, he was responsible for manufacturing activities at ABInbev, Imerys, Zilor and Amyris, with more than 15 years of fermentation processes expertise.

Iris R. Silva is Process Development Manager of Brazil at Amyris Brazil. She received her BSc degree in Chemical Engineering from University of São Carlos (Brazil) and has an MBA in Project Management and Business. She has worked at Amyris with fermentation and downstream processing for the last 8 years, having previously worked in production processes and currently in process development areas.

SCIMAGO INSTITUTIONS RANKINGS

Amyris, Inc., 5885 Hollis Street, Suite 100, CA 94608 Emeryville, USAAmyris, IncUSAEmeryville, USAAmyris, Inc., 5885 Hollis Street, Suite 100, CA 94608 Emeryville, USA

Figures

Figures (8)

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Figure 1
Production of artemisinic acid or β-farnesene by engineered yeast. The sesquiterpene alkenes β-farnesene and amorphadiene are both derived from FPP (farnesyl diphosphate) by the action of specific enzymes introduced from plants: amorphadiene synthase (ADS) generates amorphadiene and β-farnesene synthase (FS) generates β-farnesene. Production strains express either ADS or FS, not both. Oxidation of amorphadiene to artemisinic acid is accomplished by the action of five plant enzymes expressed in the engineered yeast.¹⁷17 Paddon, C. J.; Westfall, P. J.; Pitera, D. J.; Benjamin, K.; Fisher, K.; McPhee, D.; Leavell, M. D.; Tai, A.; Main, A.; Eng, D.; Polichuk, D. R.; Teoh, K. H.; Reed, D. W.; Treynor, T.; Lenihan, J.; Jiang, H.; Fleck, M.; Bajad, S.; Dang, G.; Dengrove, D.; Diola, D.; Dorin, G.; Ellens, K. W.; Fickes, S.; Galazzo, J.; Gaucher, S. P.; Geistlinger, T.; Henry, R.; Hepp, M.; Horning, T.; Iqbal, T.; Kizer, L.; Lieu, B.; Melis, D.; Moss, N.; Regentin, R.; Secrest, S.; Tsuruta, H.; Vazquez, R.; Westblade, L. F.; Xu, L.; Yu, M.; Zhang, Y.; Zhao, L.; Lievense, J.; Covello, P. S.; Keasling, J. D.; Reiling, K. K.; Renninger, N. S.; Newman, J. D.; Nature 2013, 496, 528. Conversion of purified artemisinic acid to artemisinin is accomplished by in vitro organic chemistry. Isoprenoid production strains make little ethanol.

Thumbnail

Figure 2
Sanofi industrial semi-synthesis of artemisinin. The process starts with a moderate pressure catalytic diastereoselective hydrogenation of artemisinic acid to produce a high (95:5) ratio of the desired (R)-isomer. To avoid formation of a lactone byproduct, dihydro-epi-deoxyarteannuin B, during the photooxidation, the carboxylic acid is protected as a mixed anhydride. The final step combines formation of the intermediate hydroperoxide via photoxidation using a Hg vapor lamp and commercially available tetraphenylporphyin (TPP) as sensitizer with a Hock cleavage and rearrangement catalyzed by trifluoroacetic acid to give, after workup, the best yield reported to date of pure isolated artemisinin (55%).

Thumbnail

Figure 3
Amyris fermentation plant located in the city of Brotas, SP, Brazil. The 200,000 liter fermentation tanks are shown at the right of the picture.

Figure 1 Production of artemisinic acid or β-farnesene by engineered yeast. The sesquiterpene alkenes β-farnesene and amorphadiene are both derived from FPP (farnesyl diphosphate) by the action of specific enzymes introduced from plants: amorphadiene synthase (ADS) generates amorphadiene and β-farnesene synthase (FS) generates β-farnesene. Production strains express either ADS or FS, not both. Oxidation of amorphadiene to artemisinic acid is accomplished by the action of five plant enzymes expressed in the engineered yeast.¹⁷17 Paddon, C. J.; Westfall, P. J.; Pitera, D. J.; Benjamin, K.; Fisher, K.; McPhee, D.; Leavell, M. D.; Tai, A.; Main, A.; Eng, D.; Polichuk, D. R.; Teoh, K. H.; Reed, D. W.; Treynor, T.; Lenihan, J.; Jiang, H.; Fleck, M.; Bajad, S.; Dang, G.; Dengrove, D.; Diola, D.; Dorin, G.; Ellens, K. W.; Fickes, S.; Galazzo, J.; Gaucher, S. P.; Geistlinger, T.; Henry, R.; Hepp, M.; Horning, T.; Iqbal, T.; Kizer, L.; Lieu, B.; Melis, D.; Moss, N.; Regentin, R.; Secrest, S.; Tsuruta, H.; Vazquez, R.; Westblade, L. F.; Xu, L.; Yu, M.; Zhang, Y.; Zhao, L.; Lievense, J.; Covello, P. S.; Keasling, J. D.; Reiling, K. K.; Renninger, N. S.; Newman, J. D.; Nature 2013, 496, 528. Conversion of purified artemisinic acid to artemisinin is accomplished by in vitro organic chemistry. Isoprenoid production strains make little ethanol.

Figure 2 Sanofi industrial semi-synthesis of artemisinin. The process starts with a moderate pressure catalytic diastereoselective hydrogenation of artemisinic acid to produce a high (95:5) ratio of the desired (R)-isomer. To avoid formation of a lactone byproduct, dihydro-epi-deoxyarteannuin B, during the photooxidation, the carboxylic acid is protected as a mixed anhydride. The final step combines formation of the intermediate hydroperoxide via photoxidation using a Hg vapor lamp and commercially available tetraphenylporphyin (TPP) as sensitizer with a Hock cleavage and rearrangement catalyzed by trifluoroacetic acid to give, after workup, the best yield reported to date of pure isolated artemisinin (55%).

Figure 3 Amyris fermentation plant located in the city of Brotas, SP, Brazil. The 200,000 liter fermentation tanks are shown at the right of the picture.

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E-mail: office@jbcs.sbq.org.br

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Developing Commercial Production of Semi-Synthetic Artemisinin, and of β-Farnesene, an Isoprenoid Produced by Fermentation of Brazilian Sugar

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[1] ^*e-mail: paddon@amyris.com