Semi-synthe+c

artemisinin produc+on

Technology for biobased products

Chris Paddon, Ph.D., Principal Scien4st, Amyris Inc.

Semi-Synthe+c Artemisinin

Developing produc7on of the worlds most important

an7-malarial drug using synthe7c biology and
fermenta7on technology

Copyright 2014 Amyris, Inc. All rights reserved

Semi-Synthe+c Artemisinin

Proven technology, mul1ple molecules

Opera1ng at industrial manufacturing scale
Dieren1ated & proven business model

BIOLDING
investment

Copyright 2014 Amyris, Inc. All rights reserved

 Semi-Synthe+c Artemisinin

Key Company Highlights

Founded in 2003 by post-doctoral fellows from the

University of California, Berkeley.
Headquartered in the San Francisco Bay Area and
with opera1ons in Brazil
395 full-1me employees (27% of US employees are
PhDs)
Amyris HQ California
Over 270 issued patents and >300 pending
applica1ons
BIOLDING
investment

Copyright 2014 Amyris, Inc. All rights reserved

Malaria

Aects 109 Countries Worldwide
~207 Million cases in 2012 Female Anopheles Mosquito
~627,000 deaths in 2012
1 child dies every minute of Malaria
Source: 2013 WHO World Malaria report
Plasmodium falciparum

Infected Red Blood Cells

Copyright 2014 Amyris, Inc. All rights reserved
Malaria: The old drugs are failing
Chloroquines most widely-used an1 malaria drugs
Plasmodium parasite is largely resistant to chloroquine
Successor drugs (sulfadoxine/ pyrimethamine) largely ineec1ve

Copyright 2014 Amyris, Inc. All rights reserved

Malaria: A highly eec+ve drug
was iden+ed in 1974
Artemisia Annua Artemisinin
(Sweet wormwood)
H

O
O O
H H
O

O
2004 WHO recommends
Artemisinin-based Combi-
na1on Therapy (ACT)
Source: WHO

Copyright 2014 Amyris, Inc. All rights reserved

The price and supply of plant-
derived Artemisinin has uctuated greatly
Plant-derived Artemisinin Price (US $/Kg)
1200
Artemisinin price ($/Kg)

1000

800

600

400

200

0
2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014
Year
Periodic supply shortages (e.g. 2009)
Hard for ACT manufacturers to plan produc1on
Copyright 2014 Amyris, Inc. All rights reserved
Semi-Synthe+c Artemisinin produc+on uses
fermenta+on and chemistry to stabilize supply

Semisynthe1c method
Fermenta1on & Photochemistry

About 3 months from lab to ACT

Agricultural method
Cul1va1on and extrac1on

About 10 months from seed to ACT

Copyright 2014 Amyris, Inc. All rights reserved

So whats a terpenoid?

~50,000 terpenoids exist, all made from the ve-carbon

isoprene unit

Isoprene

FLAVORS DRUGS PIGMENTS RUBBER

(Hops) (Taxol) (Carotenoids) Copyright 2014 Amyris, Inc. All rights reserved
Amyris origins lie in academic research on
engineering microbes to produce terpenes

Nature Biotechnology 2003

Copyright 2014 Amyris, Inc. All rights reserved

Amyris origins lie in academic research on
engineering microbes to produce terpenes

The Semi-Synthe+c Artemisinin Project

Nature Biotechnology 2003
Not-for-prot project funded by the Bill and Melinda Gates Founda1on

Collabora1on between partners

University of California, Berkeley
Amyris, Inc.
PATH
Sano

Copyright 2014 Amyris, Inc. All rights reserved

E. coli engineering: Impor+ng the
Isoprenoid Pathway from Yeast and Artemisia annua
Glucose

G6P

FDP

G3P

PEP
DXP
idi
PYR IPP DMAP
ispA
AcCoA Amorphadiene
Mevalonate FPP
OAA Pathway
TCA
Cycle CIT Amorphadiene
MAL Synthase (ADS)
Copyright 2014 Amyris, Inc. All rights reserved
Produc+on of 25g/L Amorphadiene
in E. coli
30
run1 run2
25 g/L target
Amorphadiene (g/l)

20 run3

10

0
0 50 100 150 200
Time (h)

Copyright 2014 Amyris, Inc. All rights reserved

Important compounds in semi-
synthe+c artemisinin (bio)synthesis
H

Amorphadiene
H Biological oxida1on

Synthe1c chemistry
Artemisinic Acid H
HO
O
H
O
O
H
Artemisinin H O

O
H

Copyright 2014 Amyris, Inc. All rights reserved

An alterna+ve host: E. coli vs. yeast

Yeast
Eukaryo1c system
Simple to engineer
Industrially scalable (Beer, wine, alcohol)
Can express cytochrome P450 enzymes

Copyright 2014 Amyris, Inc. All rights reserved

 Amorphadiene and Artemisinic
Acid produc+on in yeast
160
Amorphadiene (mg/l)

80

0
ADS 1xtHMG P(MET3)-ERG UPC2-1 2x tHMG FPPS
Gene+c modica+on
From Ro et al Nature (2006)

Yeast made small amounts of amorphadiene (150 mg/L)

Expressing a plant enzyme enabled produc1on of Artemisinic acid (100 mg/L)
Copyright 2014 Amyris, Inc. All rights reserved
An alterna+ve host: E. coli vs. yeast

Aim to produce 25 g/L artemisinic acid by fermenta+on

Amorphadiene produc1on H
E. coli produced over 100x higher
concentra1on than yeast H

H

Artemisinic acid produc1on
H
Yeast had produced a low concentra1on HO
E. coli had produced none O

Copyright 2014 Amyris, Inc. All rights reserved
Which way should the project team go?

Resources were limited

Engineer E. coli to make more artemisinic acid?
Engineer yeast to make more artemisinic acid?
Use E. coli to mWhat would you adnd
ake amorphadiene o? develop the chemistry to

convert it to artemisinin?

Copyright 2014 Amyris, Inc. All rights reserved
See you next unit