Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin

Article abstract of DOI:10.1073/pnas.1110740109

Malaria, caused by Plasmodium sp, results in almost one million deaths and over 200 million new infections annually. The World Health Organization has recommended that artemisinin-based combination therapies be used for treatment of malaria. Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua. However, the supply and price of artemisinin fluctuate greatly, and an alternative production method would be valuable to increase availability. We describe progress toward the goal of developing a supply of semisynthetic artemisinin based on production of the artemisinin precursor amorpha-4,11-diene by fermentation from engineered Saccharomyces cerevisiae, and its chemical conversion to dihydroartemisinic acid, which can be subsequently converted to artemisinin. Previous efforts to produce artemisinin precursors used S. cerevisiae S288C overexpressing selected genes of the mevalonate pathway [Ro et al. (2006) Nature 440:940-943]. We have now overexpressed every enzyme of the mevalonate pathway to ERG20 in S. cerevisiae CEN.PK2, and compared production to CEN.PK2 engineered identically to the previously engineered S288C strain. Overexpressing every enzyme of the mevalonate pathway doubled artemisinic acid production, however, amorpha-4,11-diene production was 10-fold higher than artemisinic acid. We therefore focused on amorpha-4,11-diene production. Development of fermentation processes for the reengineered CEN.PK2 amorpha-4,11-diene strain led to production of >40 g/L product. A chemical process was developed to convert amorpha- 4,11-diene to dihydroartemisinic acid, which could subsequently be converted to artemisinin. The strains and procedures described represent a complete process for production of semisynthetic artemisinin.

Full text of DOI:10.1073/pnas.1110740109

Production of amorphadiene in yeast, and its
conversion to dihydroartemisinic acid, precursor
to the antimalarial agent artemisinin
Patrick J. Westfalla, Douglas J. Piteraa, Jacob R. Lenihana, Diana Enga, Frank X. Woolarda, Rika Regentina,
Tizita Horninga, Hiroko Tsurutaa, David J. Melisa, Andrew Owensa, Scott Fickesa, Don Diolaa,
Kirsten R. Benjamina, Jay D. Keaslingb,c,d,e, Michael D. Leavella, Derek J. McPheea,
Neil S. Renningera, Jack D. Newmana,1, and Chris J. Paddona,1
aAmyris, Inc., 5885 Hollis Street, Suite 100, Emeryville, CA 94608; bDepartment of Chemical and Biomolecular Engineering, University of California,
c
Berkeley, CA 94720; Department of Bioengineering, University of California, Berkeley, CA 94720; dPhysical Biosciences Division, Lawrence Berkeley
National Laboratory, Berkeley, CA 94720; and eJoint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608
Edited by Lonnie O. Ingram, University of Florida, Gainesville, FL, and approved November 16, 2011 (received for review July 7, 2011)
Malaria, caused by Plasmodium sp, results in almost one million
deaths and over 200 million new infections annually. The World
Health Organization has recommended that artemisinin-based
combination therapies be used for treatment of malaria. Artemisi-
nin is a sesquiterpene lactone isolated from the plant Artemisia
annua. However, the supply and price of artemisinin fluctuate
greatly, and an alternative production method would be valuable
to increase availability. We describe progress toward the goal of
developing a supply of semisynthetic artemisinin based on produc-
tion of the artemisinin precursor amorpha-4,11-diene by fermenta-
tion from engineered Saccharomyces cerevisiae, and its chemical
conversion to dihydroartemisinic acid, which can be subsequently
converted to artemisinin. Previous efforts to produce artemisinin
precursors used S. cerevisiae S288C overexpressing selected genes
of the mevalonate pathway [Ro et al. (2006) Nature 440:940–943].
We have now overexpressed every enzyme of the mevalonate
pathway to ERG20 in S. cerevisiae CEN.PK2, and compared pro-
duction to CEN.PK2 engineered identically to the previously engi-
neered S288C strain. Overexpressing every enzyme of the mevalo-
nate pathway doubled artemisinic acid production, however,
amorpha-4,11-diene production was 10-fold higher than artemisi-
nic acid. We therefore focused on amorpha-4,11-diene production.
Development of fermentation processes for the reengineered
CEN.PK2 amorpha-4,11-diene strain led to production of >40 g∕L
product. A chemical process was developed to convert amorpha-
4,11-diene to dihydroartemisinic acid, which could subsequently
be converted to artemisinin. The strains and procedures described
represent a complete process for production of semisynthetic
artemisinin.
developing world, but a significant increase in the cultivation
of A. annua would be needed to satisfy projected global demand
(6). Chemical synthesis of artemisinin is not economically feasible
because of the complexity and low yield of the process (7), how-
ever semisynthesis of artemisinin following microbial production
of a precursor molecule may be feasible (6). This report describes
progress toward the production of semisynthetic artemisinin.
Heterologous production of amorpha-4,11-diene (Fig. 5, com-
pound 1) was first described from Escherichia coli which had been
engineered to express the mevalonate pathway from Saccharo-
myces cerevisiae and amorpha-4,11-diene synthase (ADS) from
A. annua (8). Development of a two-phase partitioning bioreac-
tor allowed production of 0.5 g∕L of amorpha-4,11-diene (9).
Analysis of transcriptional responses and pathway metabolites
showed that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-
CoA) reductase limited production, leading to perturbation of
fatty acid biosynthesis and generalized membrane stress (10, 11).
Subsequent replacement of the S. cerevisiae HMG-CoA reduc-
tase and HMG-CoA synthase with equivalent enzymes from
Staphylococcus aureus, and concomitant fermentation develop-
ment, allowed production of 27 g∕L of amorpha-4,11-diene (12).
Production of amorpha-4,11-diene from S. cerevisiae was initi-
ally developed as a tool to aid the cloning of the cytochrome P450
believed to be responsible for the production of artemisinic acid,
a late-stage oxidized precursor of artemisinin (13). A series of
engineering steps, namely high-level expression of ADS, over-
expression of farnesyl diphosphate synthase and the catalytic
domain of HMG-CoA reductase, reduced expression of squalene
synthase, and generalized increased expression of the mevalonate
pathway by overexpression of an activated allele of the UPC2
transcription factor, UPC2-1, allowed production of 153 mg∕L of
amorpha-4,11-diene in shake-flasks. This strain was used for
functional expression of CYP71AV1 [also known as amorpha-
diene oxidase (AMO)], the cytochrome P450 responsible for
the three-step oxidation of amorpha-4,11-diene, along with its
cognate reductase, AaCPR (A. annua cytochrome P450 reduc-
alaria, caused primarily by the Plasmodium falciparum and
M
Plasmodium vivax parasites, is a disease overwhelmingly
affecting areas of poverty in the developing world. There were
over 200 million new infections and an estimated 781,000 deaths
in 2009, predominantly affecting children under five years of age
(1). P. falciparum, which causes the most virulent form of malaria,
has become resistant to all inexpensive antimalarial drugs readily
available in the developing world (2, 3). An antimalarial agent
to which widespread resistance has not been observed is artemi-
sinin, a sesquiterpene lactone peroxide extracted from the shrub
Artemisia annua. The World Health Organization (WHO) recom-
mends the use of artemisinin derivatives in combination with
another effective schizontocidal drug, a treatment known as ar-
temisinin-based combination therapy (ACT), for the first-line
treatment of malaria (3). In recent years there have been large
fluctuations in the price and availability of ACTs (4), and in
2007–2008 less than 15% of children under five years of age with
fever received ACTs in 11 out of 13 countries surveyed (5). There
is an urgent need to increase the supply of artemisinin to both
stabilize the price and increase the availability of ACTs in the
Author contributions: P.J.W., D.J.P., J.R.L., F.X.W., R.R., S.F., K.R.B., M.D.L., D.J. McPhee,
N.S.R., J.D.N., and C.J.P. designed research; P.J.W., D.J.P., J.R.L., D.E., F.X.W., R.R., T.H.,
H.T., D.J. Melis, A.O., S.F., D.D., and K.R.B. performed research; J.D.K. contributed new
reagents/analytic tools; P.J.W., D.J.P., J.R.L., D.E., F.X.W., R.R., T.H., H.T., D.J. Melis, A.O.,
S.F., D.D., K.R.B., M.D.L., D.J. McPhee, N.S.R., J.D.N., and C.J.P. analyzed data; and C.J.P.
wrote the paper.
Conflict of interest statement: All authors hold stock options or shares in Amyris, Inc.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: newman@amyris.com or paddon@
amyris.com.
See Author Summary on page 655.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1110740109/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1110740109
PNAS ∣ January 17, 2012 ∣ vol. 109 ∣ no. 3 ∣ E111–E118

tase), producing >100 mg∕L artemisinic acid (13). Incorporation
of all A. annua-derived genes (ADS, CYP71AV1, and AaCPR) on
a single expression plasmid allowed production of 2.5 g∕L of ar-
temisinic acid by development of a galactose-based fermentation
process (14), though plasmid stability was shown to be lower in a
strain producing artemisinic acid compared to a strain producing
amorpha-4,11-diene, and production of artemisinic acid led to
induction of pleiotropic drug resistance genes (15). We now de-
scribe the creation of new S. cerevisiae strains and processes for
the production of amorpha-4,11-diene, culminating in a 250-fold
increase in production of amorpha-4,11-diene to 40 g∕L concen-
tration, and a process for its efficient chemical conversion to the
artemisinin precursor dihydroartemisinic acid.
Results
Eliminating Utilization of Galactose. Any process involving fermen-
tation to economically produce precursors for the semisynthesis
of artemisinin for the developing world would be unable to use
galactose as a sole carbon source as it is too expensive. Strains of
S. cerevisiae lacking the gal1 gene are unable to metabolize galac-
tose, but galactose still acts as a gratuitous inducer (16) for all
galactose-regulated genes in the artemisinic acid-producing strain
EPY330 (15). Fig. 1A shows that deleting GAL1 in EPY330
(¼Y87; Table 1) allows production of artemisinic acid using glu-
cose as a carbon source and galactose as a gratuitous inducer.
Transcript levels were not analyzed, but given that the GAL reg-
ulon is still subject to glucose repression, it is likely that induction
occurred following the diauxic shift and ethanol was used for
artemisinic acid biosynthesis. Specific production of artemisinic
acid by the gal1Δ strain grown in a high concentration of glucose
(2.0%) is similar to the GAL1 strain grown in 1.8% galactose
(Fig. 1A).
Codon Optimization of Heterologous Genes. We investigated
whether S. cerevisiae codon optimization of heterologous genes
would increase production of amorpha-4,11-diene or artemisinic
acid. For production of artemisinic acid the plasmid containing
all three A. annua genes, pESC-Leu2d∷ADS/AMO/CPR (15)
was resynthesized with S. cerevisiae codon-optimized A. annua
genes, and the orientation of the PGAL1-ADS-TPGK1 expression
cassette reversed. The reversal of the GAL1 promoters ensures
that recombination cannot occur [which would lead to elimina-
tion of CYP71AV1 (AMO) and ADS in pESC-Leu2d∷ADS/
AMO/CPR] to generate pAM322 (Fig. S1). We compared pro-
duction of artemisinic acid by strains containing either pESC-
Leu2d∷ADS/AMO/CPR (strain Y87) or pAM322 (strain Y137).
Y137 grew (Fig. 1B) and produced artemisinic acid (Fig. 1C)
to similar levels as Y87, suggesting that codon optimization did
not improve production. However, given the elimination of the
possibility for intergenic recombination to occur, all subsequent
strains producing artemisinic acid contained pAM322. A plasmid
expressing only ADS (pAM426) was generated by recirculariza-
tion of pAM322 following digestion by PvuII, eliminating the
AMO/AaCPR expression cassette (Fig. S1). To allow fair compar-
ison between artemisinic acid and amorpha-4,11-diene produc-
tion, pAM426 was used in all subsequent amorpha-4,11-diene-
producing strains.
Fig. 1. Production of artemisinic acid in Gen 1.0 yeast strains. (A) Production
of artemisinic acid in shake-flask cultures of EPY330 (GAL1) and Y87 (gal1Δ)
in defined media containing either low or high concentrations of glucose (ꢀ1
SD of three flasks). Numbers above the bars show specific production
(mg∕L∕OD600). (B) Growth and (C) artemisinic acid production in fed-batch
glucose-limited fermentors of Y87 containing a high-copy plasmid expressing
E. coli-optimized ADS/CYP71AV1/AaCPR and Y137 containing pAM322, ex-
pressing S. cerevisiae-optimized ADS/CYP71AV1/AaCPR.
as ERG20 is transcribed from high-expression galactose-regu-
lated promoters (PGAL1 or PGAL10). We elected to construct the
new strains in S. cerevisiae CEN.PK2 rather than S288C. Whereas
S288C has been much used for genetic investigation, and was the
first strain to have its genome sequenced (19), little physiological
information exists. CEN.PK2 has better characterized physiology,
and its significantly higher sporulation efficiency (20, 21) simpli-
fies strain construction using Mendelian genetics. CEN.PK2 was
engineered in two ways: (i) the engineering scheme of the earlier
S288C strains (13) was recapitulated to produce strains desig-
nated as Gen 1.0, and (ii) every enzyme of the mevalonate path-
Reconstruction of S. cerevisiae Production Strains. All prior strains,
constructed in S. cerevisiae S288C, overexpressed selected meva-
lonate pathway genes from the galactose-regulated GAL1 pro-
moter. Production of amorpha-4,11-diene and artemisinic acid
were further increased by galactose-regulated (PGAL1) expression
of the UPC2-1 transcription factor (13). UPC2-1 encodes an
activated allele of UPC2 (17, 18), which increases transcription of
most ergosterol pathway biosynthetic genes and is also a regulator
of hypoxia-expressed genes. We decided to reconstruct the pro-
duction strain so that every mevalonate pathway enzyme as far
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Fig. 2. Construction of CEN.PK2 Gen 2.0 and comparison of production by Gen 1.0 and 2.0 strains. (A) Schematic of the mevalonate pathway showing genes
overexpressed in Gen 2.0 strains from GAL1 or GAL10 promoters in block arrows. Dashed arrow represents the ergosterol pathway transcriptionally restricted
at ERG9. (B) Production of amorpha-4,11-diene by Y151 (Gen 1.0) and Y227 (Gen 2.0), and artemisinic acid by Y135 (Gen 1.0) and Y224 (Gen 2.0) after 72 h
growth in shake-flasks. Error bars show ꢀ1 SD of three flasks. Numbers above the bars show specific production (mg∕L∕OD600).
way as far as ERG20 was transcribed from the galactose-regu-
lated divergent GAL1/ GAL10 promoters to produce Gen 2.0
strains (Fig. 2A, Fig. S2). Three copies of tHMG1 were inte-
grated, as HMG-CoA reductase expression has been shown to
be limiting (13). Table 1 and Table S1 summarize Gen 1.0 and
Gen 2.0 strains that were constructed. Two Gen 2.0 strains were
initially generated, Y224 containing pAM322 for production of
artemisinic acid, and Y227 containing pAM426 (expressing only
ADS; Fig. S1), for production of amorpha-4,11-diene. Production
by Y224 and Y227 was compared to equivalent CEN.PK2 Gen
1.0 strains in galactose shake-flask cultures. Production of amor-
pha-4,11-diene by Y227 was approximately fivefold that of Y151,
its Gen 1.0 equivalent, whereas production of artemisinic acid by
Y224 was only approximately twofold that of Y135, its Gen 1.0
equivalent strain (Fig. 2B). Production of both amorpha-4,11-
diene and artemisinic acid were, therefore, significantly increased
by overexpression of the entire mevalonate pathway to ERG20 in
Gen 2.0 strains, but the increase in production of amorpha-4,11-
diene was more than double that of the increase in production of
artemisinic acid. Most notably, production of amorpha-4,11-
diene by Y227, the Gen 2.0 ADS-expressing strain, was over
10-fold higher than production of artemisinic acid by Y224, its
equivalent Gen 2.0 artemisinic acid-producing strain. Interest-
ingly, pAM322 was found to be very stable in Y224 [100% stable
after 72 h growth in flasks, assessed by retention of the LEU2
plasmid selectable marker (15)], in contrast to the artemisinic
acid-producing S288C Gen 1.0 strains containing pESC-Leu2-
d∷ADS/AMO/CPR (15), and CEN.PK2 Gen 1.0 strain bearing
pAM322 (Y135; 70% stable after 72 h growth in flasks). Given
the dramatically higher production of amorpha-4,11-diene com-
pared to artemisinic acid in Gen 2.0 strains, or amorpha-4,11-
diene in Gen 1.0 strains (Fig. 2B), we decided to pursue produc-
tion of amorpha-4,11-diene in Gen 2.0 strains.
Fermentation Testing and Process Development. Utilization of galac-
tose was eliminated prior to testing production of amorpha-4,
11-diene in fermentation by deleting the GAL1 GAL10 GAL7
gene cluster. The resulting strain, Y337 (¼Y227 gal1Δ gal7Δ
gal10Δ; Table 1), is unable to metabolize galactose, but still re-
quires a low concentration of galactose for induction of the
mevalonate pathway and ADS genes transcribed by galactose-
regulated promoters. Y337 was grown in glucose-limited fed-
batch fermentation with induction by galactose occurring at the
end of the batch growth phase (SI Materials and Methods,
Figs. S3–S5); under this fermentation condition Y337 produced
2.0 g∕L amorpha-4,11-diene (Fig. 3B) with a yield of 1.11 Cmol
% (molar percentage of substrate carbon added to the fermenta-
tion incorporated into amorpha-4,11-diene). We tested the effect
of phosphate restriction on production in glucose-limited fed-
batch fermentations (Fig. 3 A and B). A range of lower concen-
trations of phosphate in the feed, batch, or both were tested to
identify phosphate delivery that would capture the increase in per
cell productivity without disrupting carbon delivery in the fed-
batch process to the extent the run would be less productive.
Fig. S6 and Fig. S7 show that decreasing phosphate concentra-
tions in the batch or feed media can indeed lead to higher pro-
duction of amorph-4,11-diene. Highest production was achieved
with 8 g∕L KH2PO4 in the batch but no KH2PO4 in the feed,
Table 1. Strain genotypes and products
Strain
Base strain
S288C
Genotype and [plasmid]
Source Produces
Gen 1.0
EPY330
MATα his3Δ1 leu2Δ0 PGAL1-tHMG1∷δ1 PGAL1-UPC2-1∷δ2 erg9∷PMET3-ERG9∷HIS3 PGAL1-ERG20∷δ3
PGAL1-tHMG1∷δ4 [pESC-leu2d-ADS/AMO/CPR]
(15)
AA
Y87
Y135
S288C
CEN.PK2
EPY330 gal1Δ∷K. lactis URA3 lys2Δ0
This work
This work
AA
AA
MATα erg9∷Kanr-PMET3-ERG9 trp1∷TRP1-PGAL1-ERG20 his3∷HIS-PGAL1-tHMG1
leu2∷HIS-PGAL1-tHMG1 ura3∷URA3-PGAL1-upc2-1 [pAM322: 2μ-leu2d PGAL1-ADS
PGAL10-CYP71AV1 PGAL1-AaCPR (codon opt)]
Y137
S288C
As Y87, but plasmid is pAM322
This work
AA
Y151
CEN.PK2
As Y135, but plasmid is pAM426 [2μ-leu2d PGAL1-ADS (codon opt)]
This work Amorph
Gen 2.0
Y224
CEN.PK2
MATa erg9Δ∷kanr PMET3-ERG9, leu2-3,112∷HIS PGAL1-MVD1 PGAL10-ERG8
his3Δ1∷HIS PGAL1-ERG12 PGAL10-ERG10ade1Δ∷PGAL1-tHMG1 PGAL10-IDI1 ADE1
ura3-52∷PGAL1-tHMG1 PGAL10-ERG13 URA3trp1-289∷PGAL1-tHMG1 PGAL10-ERG20 TRP1
[pAM322]
This work AA
Y227
Y293
Y337
CEN.PK2
CEN.PK2
CEN.PK2
As Y224, but plasmid is pAM426
This work Amorph
This work Amorph
This work Amorph
Y227 gal80Δ∷natr
Y227 gal1Δ gal7Δ gal10Δ∷HphA
Amorph, amorpha-4,11-diene; AA, artemisinic acid. A complete list of strains, including progenitor strains, is listed in Table S1.
Westfall et al.
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Fig. 3. Development of fed-batch glucose-limited fermentations for the production of amorpha-4,11-diene. (A) Growth and (B) amorpha-4,11-diene produc-
tion by Y337 in glucose-limited fermentations with and without phosphate limitation, and Y293 in glucose-limited fermentation. Production of amorpha-4,
11-diene was induced in Y337 fermentations by addition of galactose (see SI Materials and Methods).
compared to 8 g∕L KH2PO4 in the batch and 9 g∕L KH2PO4 in
the unrestricted process. Growth was somewhat restricted
(Fig. 3A), but production of amorpha-4,11-diene was more than
doubled, attaining 5.5 g∕L after 95 h with a yield of 3.23 Cmol %
(Fig. 3B). The use of ethanol has been described as a carbon
source for the production of hydrocortisone (derived from the
mevalonate pathway) in yeast (22). We also wished to see whether
production could be increased when ethanol is provided as a car-
bon source, based on the notion that ethanol could increase the
supply of cytosolic acetyl-CoA. To this end we tested mixed glu-
cose/ethanol feeds following the glucose batch phase of growth.
The mixed-feed strategy resulted in similar growth (Fig. 4A), but
also gave a significant increase in production of amorpha-4,11-
diene, attaining 16.5 g∕L after 118 h with a yield of 6.03 Cmol
% (Fig. 4B). It is also notable that production continued after
maximum cell density had been achieved. Attempts to increase
production of amorpha-4,11-diene by the use of phosphate re-
striction with mixed glucose/ethanol feeds gave variable results
and were not pursued (Fig. S8, Fig. S9).
tageous to eliminate the use of galactose altogether to further
decrease the cost of amorpha-4,11-diene production. To this end,
we deleted the GAL80 gene from Y227, the galactose-utilizing
amorpha-4,11-diene producing Gen 2.0 strain. Gal80p is a nega-
tive regulator of the galactose regulon which acts by binding to
the C-terminal transcriptional activation domain of Gal4p in non-
galactose carbon sources; it is released from Gal4p upon addition
of galactose, and in the absence of a repressing carbon source
such as glucose the release of Gal80p from Gal4p allows induc-
tion of galactose-regulated genes (23). The amorpha-4,11-diene
production strain with deletion of GAL80 was designated Y293
(¼Y227 gal80Δ; Table 1). In glucose-limited fed-batch fermenta-
tion without addition of galactose, Y293 performed similarly to
Y337, which required addition of galactose to induce production
(Fig. 3). Given the similar behavior of Y293 and Y337 we elected
to continue fermentation development with Y293, thus obviating
the use of galactose altogether.
We wished to test whether production could be increased with
the use of pure ethanol feeds. Two different feed strategies were
tested, an ethanol pulse feed without ethanol restriction, and a
restricted ethanol feed with lower oxygen uptake rate (OUR).
The cell density achieved was appreciably lower than that
Y337 (¼Y227 gal1Δ gal7Δ gal10Δ) does not metabolize galac-
tose, but still requires addition of a low concentration of galactose
to initiate production of amorpha-4,11-diene. It would be advan-
Fig. 4. Development of fed-batch glucose/ethanol and ethanol feed fermentations. (A) Growth and (B) amorpha-4,11-diene production by Y337 with re-
stricted glucose feed and mixed glucose and ethanol feed, and Y293 with ethanol pulse feed (replicate fermentations) and restricted ethanol feed. (C) Oxygen
uptake rate of Y293 ethanol pulse feed and restricted ethanol feed fermentors.
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achieved with either glucose or glucose/ethanol mixed feed
(Fig. 4A). Production of amorpha-4,11-diene, however, was sig-
nificantly higher than all earlier processes, attaining 41 g∕L for
the unrestricted ethanol pulse feed after 116 h (yield of 16.98
Cmol %); a replicate fermentation attained 36 g∕L. A restricted
ethanol feed produced 37 g∕L amorpha-4,11-diene (yield of
18.67 Cmol %), albeit after a longer run time of 187 h (Fig. 4B).
An important advantage of using a restricted ethanol feed pro-
cess is that significantly lower OURs are required for production
in comparison to the ethanol pulse feed process (Fig. 4C).
regulated GAL1,10 promoters to overexpress the mevalonate
pathway genes allowed repression of expression during strain
construction, thus avoiding buildup of any potentially toxic inter-
mediates (8) which may have resulted in strain instability during
construction. Intermediate buildup was not assayed in the final
strains, and we did not investigate whether overexpression of all
the mevalonate pathway genes was necessary. Regulated overex-
pression of all relevant mevalonate pathway genes in Gen 2.0
strains led to a fivefold increase in production of amorpha-
4,11-diene in shake-flasks, but only a twofold increased produc-
tion of artemisinic acid. Production of amorpha-4,11-diene by
Y227 was over 10-fold higher than production of artemisinic acid
by Y224, its equivalent Gen 2.0 artemisinic acid-producing strain
(Fig. 2). This very significantly higher production led us to con-
centrate on the development of amorpha-4,11-diene production
by fermentation. Low plasmid stability was not the reason for the
poor production of artemisinic acid, as plasmid pAM322, expres-
sing ADS, CYP71AV1, and AaCPR, was found to be 100% stable
in Gen 2.0 strains, in contrast to Gen 1.0 strains where marked
instability was observed (15). Presumably there is strong selective
pressure for maintenance of pAM322 in Gen 2.0 strains, which is
lacking in Gen 1.0 strains.
Initial development of a fermentation process for the produc-
tion of amorpha-4,11-diene used a derivative of the Gen 2.0 strain
Y227 known as Y337 in which galactose utilization had been
disabled by deletion of the GAL1 GAL10 GAL7 gene cluster.
A glucose-limited fed-batch fermentation process in which amor-
pha-4,11-diene production was induced by the addition of a low
concentration of galactose at the end of batch growth resulted in
production of 2.0 g∕L amorpha-4,11-diene. An initial attempt to
increase production by phosphate limitation, on the rationale
that restricting phosphate would limit growth and channel greater
carbon flux to product, resulted in more than doubling of amor-
pha-4,11-diene production. The biggest increase in production
was observed when ethanol was added to the feed, initially as
a glucose/ethanol mixed feed following the glucose batch phase
of growth. The rationale for adding ethanol to the feed was that
direct supply of ethanol may increase the supply of cytosolic acet-
yl-CoA to the mevalonate pathway (30). An alternative explana-
tion is that use of ethanol eliminated glucose repression, but this
scenario is considered unlikely as glucose was still present in the
feed, and the concentration of glucose in the feed stage of the
fed-batch fermentations was very low, and mostly undetectable.
However, transcript levels were not monitored during the differ-
ent fermentation procedures, and it is not possible to unequivo-
cally determine the reason for the increase in amorpha-4,11-
diene production in a glucose/ethanol mixed-feed fermentation.
The glucose/ethanol mixed-feed approach led to production of
16.5 g∕L of amorpha-4,11-diene with significantly increased
yield.
Chemical Conversion of Amorpha-4,11-diene to Dihydroartemisinic
Acid. Amorpha-4,11-diene, compound 1, was converted to dihy-
droartemisinic acid, compound 4, in three steps beginning with
selective hydroboration of the double bond at the 11-position
(Fig. 5). Using the hindered borane 9-borabicyclo[3.3.1]nonane
(9-BBN) a 79.3% yield of alcohol compound 2 was achieved as
an 85∶15 mixture (by 1H − NMR) of epimers with the desired
R-epimer predominating. Several methods reported to convert
primary alcohols to carboxylic acids in one step (24, 25) produced
complex mixtures from compound 2, presumably due to interac-
tion with the olefinic linkage at the 4-position. Thus a two-step
oxidation sequence was employed in which SO3 pyridine complex
(26) oxidized compound 2 to aldehyde compound 3 (76.4%) fol-
lowed by treatment of compound 3 with the oxidant NaOCl2 in
DMSO (27, 28) to form compound 4 (79.9%). The use of DMSO
as the solvent was crucial because the NaOCl formed in the re-
action preferentially reacts with it, thus protecting the double
bond at the 4-position. The overall yield of compound 4 from
compound 1 by this route was 48.4%.
Discussion
Initial work showed that deletion of GAL1, a gene required
for the utilization of galactose, allowed the use of galactose as a
gratuitous inducer while eliminating its use as a carbon source.
An attempt to increase production of artemisinic acid by codon
optimization of the heterologous genes from A. annua was unsuc-
cessful, perhaps reflecting the recent realization that expression
optimization by simple codon usage bias is of limited value (29).
However, the redesigned plasmid could not eliminate genes by
recombination and was used for subsequent work.
We chose to reengineer CEN.PK2 rather than S288C to pro-
duce Gen 2.0 strains in which every enzyme of the mevalonate
pathway leading to the production of farnesyl diphosphate was
transcribed from high-expression galactose-regulated promoters.
The enhanced sporulation efficiency of CEN.PK2 greatly simpli-
fied the construction of Gen 2.0 strains, and the better documen-
ted physiology of CEN.PK2 (20) suggested that this strain would
be suitable for fermentative production. The use of the galactose-
The final stage of fermentation development was conducted
with Y293, an amorph-4,11-diene-producing strain in which ex-
pression of Gal80p (the negative-regulator of galactose-regulon
gene expression) had been eliminated. The removal of Gal80p
expression eliminated the need to add galactose for induction of
amorpha-4,11-diene production. Y293 produced a similar con-
centration of amorph-4,11-diene to Y337 (which cannot metabo-
lize galactose), but Y293 did not require addition of galactose
to the fermentation. Two different strategies for feeding pure
ethanol as feed carbon source were evaluated. Whereas an un-
restricted ethanol feed (a pulse feed) produced the highest
concentration of amorpha-4,11-diene, its high OUR makes it
industrially infeasible. Conversely, the ethanol restricted-feed
process, capable of producing 37 g∕L of amorpha-4,11-diene, has
a lower OUR that may be amenable to adaptation to industrial-
scale fermentation. The use of S. cerevisiae for the production of
amorpha-4,11-diene has considerable advantages over the earlier
E. coli process (12), notably the absence of an expensive inducer
Fig. 5. Synthesis of dihydroartemisinic acid from amorpha-4,11-diene. Only
the R isomer is shown.
Westfall et al.
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such as IPTG, and the lower OUR required for the restricted-
feed ethanol process. In large-scale fermentations, high oxygen
uptake rates require high sparge rates, high agitation rates,
and/or supplementation with pure oxygen (31). In addition, the
heat generation resulting from rapid substrate and oxygen con-
sumption requires increased cooling capacity to maintain tem-
perature (32). Further process development is underway to
implement practical solutions at large scale.
artemisinin. A major challenge remains that the Gen 2.0 reengi-
neered strain of yeast did not produce significantly more artemi-
sinic acid than the original Gen 1.0 strains (Fig. 2B). Production
of artemisinic acid by S. cerevisiae at concentrations comparable
to that of amorpha-4,11-diene by Y293 would be advantageous,
as the chemical conversion of artemisinic acid to artemisinin
would be simpler than production from amorpha-4,11-diene. To
directly produce high levels of artemisinic acid from S. cerevisiae,
the underlying cause of the lower production of artemisinic acid
compared to amorpha-4,11-diene will need to be identified and
remedied.
The overall aim of the strain construction and fermentation
development was to create a process that could feasibly be devel-
oped for cost-effective production of artemisinin for the develop-
ing world. An earlier fermentation process for production of
artemisinic acid used galactose for both growth and induction
(14). A drawback with this process, other than the low titer of
artemisinic acid produced (2.3 g∕L), was the cost of galactose;
galactose is approximately 100-fold more expensive than glucose
(SI Materials and Methods). The advantage of eliminating catabo-
lism of galactose by deletion of the GAL1 GAL10 GAL7 gene
cluster is that galactose is required solely as an inducer, and the
quantities added to the fermentor are much lower (10 g∕L in the
induction feed medium; SI Materials and Methods). The final
strain used for fermentation development (Y293) contained a
deletion of GAL80 and did not require addition of galactose
for induction, thus providing a further cost saving. Ethanol is
approximately twice the cost of glucose on a weight basis (SI
Materials and Methods). However, addition of ethanol, initially
as a mixed-feed then as pure ethanol feed, resulted in approxi-
mately 20-fold increased production of amorpha-4,11-diene
[2.0 g∕L with restricted glucose feed, compared to 41 g∕L with
unrestricted ethanol feed (Fig. 4)]. The yield of amorpha-4,11-
diene production also increased significantly with ethanol feed,
rising from 1.11 Cmol % with restricted glucose feed to 18.67
Cmol % with restricted ethanol feed; this increased yield demon-
strates a dramatic increase in the conversion of substrate carbon
into amorpha-4,11-diene. Given that the market price of artemi-
sinin is over 200-fold higher than that of ethanol, and that the
production of amorpha-4,11-diene is a significant fraction of the
total production cost, it is economically reasonable to use ethanol
as a fermentation carbon source. Nonetheless, efforts are under-
way to increase the production and yield of amorpha-4,11-diene
from glucose.
Dihydroartemisinic acid (Fig. 5, compound 4) is a late-stage
intermediate in a number of total syntheses of artemisinin (33)
and its derivatives (34), and an efficient industrial-scale synthesis
from amorpha-4,11-diene would greatly aid in efforts to produce
large amounts of artemisinin-based antimalarial drugs. For small
scale (submolar) synthesis scale we used 9-borabicyclo[3,3,1]non-
ane for the hydroboration of amorpha-4,11-diene to form dihy-
droartemisinic alcohol (Fig. 5, compound 2) because it did not
affect the sensitive double bond at the 4-position. We elected to
use a two-step oxidation process because common reagents for
converting primary alcohols to carboxylic acids in one step are
either chromium based or contain components that were likely
to react with the sensitive double bond at the 4-position. The
readily available and scalable sulfur trioxide-pyridine complex
(35) smoothly converts compound 2 to dihydroartemisinic alde-
hyde (Fig. 5, compound 3), which is subsequently oxidized to
dihydroartemisinic acid (Fig. 5, compound 4) with inexpensive
inorganic reagents (26–28).
Materials and Methods
S. cerevisiae Strain Construction. Five separate DNA integration constructs
(Fig. S2) were made, all based on transcription of two mevalonate pathway
genes from the divergent GAL1,10 promoter, as follows: S. cerevisiae geno-
mic DNA covering several hundred nucleotides upstream of the desired
integration site was amplified by PCR with a flanking PmeI restriction enzyme
site. A selection marker was also PCR amplified from genomic DNA with 40 nt
of overlap to the downstream terminus of the upstream integration site
amplicon. The fragments were combined by overlap PCR (36). Several hun-
dred nucleotides of genomic DNA downstream of the desired integration
site were PCR amplified with a PmeI site at the downstream terminus. The
two amplicons were joined by overlap PCR to yield a fragment (no. 1) of
the following composition: (PmeI)-upstream integration fragment-selection
marker-(XmaI)-downstream integration fragment-(PmeI). The resulting am-
plicon was ligated into the TOPO pCR2.1 vector (Invitrogen). The genomic
locus encoding the open reading frame and transcriptional terminator of
the first gene to be expressed was PCR amplified with an XmaI site at its
upstream terminus. The PGAL1;10 promoter was PCR amplified with 40 nt
of overlap to the upstream gene, and the promoter and gene combined by
overlap PCR. The PGAL1;10 promoter also contained 40 nt of overlap to the
downstream gene to be expressed. The genomic locus of the second gene
to be expressed was PCR amplified, and the two amplicons combined by over-
lap extension to yield a fragment (no. 2) of the following composition:
(XmaI)-upstream gene-PGAL1;10 promoter-downstream gene-(XmaI). The re-
sulting amplicon was ligated into the TOPO ZERO Blunt II Cloning vector.
Fragments no. 1 and no. 2 were combined by digesting the TOPO pCR2.1 vec-
tor containing fragment no. 1 with XmaI, and subsequent ligation with frag-
ment no. 2 released from its TOPO ZERO Blunt II vector by XmaI digestion, to
yield a fragment of the following composition: (PmeI)-upstream integration
fragment-selection marker-(XmaI)-upstream gene-PGAL1;10 promoter-down-
stream gene-(XmaI)-downstream integration fragment-(PmeI). This com-
bined fragment was released from the TOPO ZERO Blunt II Cloning vector
by digestion with PmeI, purified, and used to transform S. cerevisiae CEN.PK2
derivatives. Full details of each construction are provided in SI Materials and
Methods; Table S2 lists all oligonucleotide primers..
Media and Growth Conditions. S. cerevisiae strains (Table 1) were grown on
synthetic complete medium agar plates at 30 °C with 2% glucose as the
carbon source unless otherwise indicated, lacking leucine where needed
for selection of plasmids (37). Flask and fermentor media were based on that
of van Hoek et al. (38) (SI Materials and Methods). Amorpha-4,11-diene pro-
duced in flask cultures was captured by addition of 20% vol∕vol isopropyl
myristate to the medium; amorpha-4,11-diene produced in fermentors
was captured by the addition of 10% vol∕vol methyl oleate. Fermentations
took place in 2 L Sartorius Biostat B plus twins with gas-flow ratio controllers.
Full details are provided in SI Materials and Methods.
Assay Methods. Amorpha-4,11-diene. Production of amorpha-4,11-diene was
monitored by gas chromatography with flame-ionization detection (GC/FID).
Fermentor samples were prepared by mixing 0.4 mL cell lysis reagent [two
parts Novagen YeastBuster protein reagent (EMD Biosciences, P/N 71186-4)
and one part 2 M HCl] with 0.1 mL whole broth and 1 mL ethyl acetate
containing 10 mg∕L transcaryophylene (surrogate; Sigma-Aldrich; ≥98.5%
purity; P/N 22075) in a 2 mL glass vial. For determination of amorpha-
4,11-diene from flask cultures the isopropyl myristate overlay was sampled,
and diluted directly into ethyl acetate/transcaryophylene. The sample was
mixed for 30 min on a vortex mixer. After phase separation 0.6 mL of the
ethyl acetate layer was transferred to a GC vial for analysis. The ethyl acet-
ate-extracted samples were analyzed using the GC/FID. Amorpha-4,11-diene
peak areas were converted to concentration values from external standard
calibrations using authentic compounds. A 1 μL sample was split 1∶20 and
separated using a DB-WAX column (50 m × 200 μm × 0.2 μm; P/N 128-7052;
This work links together each of the key steps of a complete
process for the production of semisynthetic artemisinin. The
construction of a complete mevalonate pathway under strong
induction, along with fermentation development, was crucial to
obtaining high production of amorpha-4,11-diene. Combining
high-level production of amorpha-4,11-diene with demonstration
of successful chemical conversion to dihydroartemisinic acid re-
presents a significant milestone toward the development of an
economically viable process for the production of semisynthetic
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Agilent) with hydrogen as the carrier gas at flow rate at 1.57 mL∕ min. The
temperature program for the analysis was as follows: the column
was initially held at 150 °C for 3.0 min, followed by a temperature gradient
of 5 °C∕ min to a temperature of 250 °C, where the column was held at 250 °C
for 5 min to elute all remaining components. Under these conditions trans-
caryophylene and amorpha-4,11-diene and elute at 4.95 and 5.77 min,
respectively.
TLC (20% EtOAC/hexane) revealed only a trace of compound 1 remaining.
A solution of NaOH (0.294 g, 0.734 mol) in 2.5 mL of H2O was added, the
mixture cooled to 0 °C with an ice bath and 0.75 mL of 30% H2O2 added.
The ice bath was removed and after 1 h the THF was evaporated under re-
duced pressure. The residue was subjected to extractive workup with ether
and H2O. The ether layer was dried (MgSO4) and evaporated under reduced
pressure. Flash chromatography of the crude product on silica gel (20%
EtOAc/hexane) provided 0.010 g of compound 1 and 0.119 g (79.3% based
on recovered stating material) of compound 2 as a colorless oil consisting of a
85∶15 mixture (1H − NMR) of 11- R : 11 − S isomers of compound 1 that ra-
pidly solidified. The 1H − NMR and MS spectra of the major isomer were iden-
tical to those previously published (39). For additional details, see Fig. 5.
Artemisinic acid. A 1 mL aliquot of well-mixed fermentation broth was diluted
in 9 mL of methanol þ 0.1% formic acid. The mixture is then mixed on a
vortex mixer for 30 min and centrifuged at 16;000 × g for 5 min. One hun-
dred microliters of the supernatant was diluted into 900 μL methanol þ 0.1%
formic acid. A 20 μL aliquot was injected on an Agilent 1200 HPLC with UV
detection at 212 nm. A Supelco Discovery C8 column (4.6 mm × 100 mm×
5.0 μm, Supelco, P/N 569423-U) equipped with the appropriate guard column
(4.0 mm × 20.0 mm, Supelco, P/N 59589-U) was used for separation, with
the following gradient at a flow rate of 1 mL∕ min (channel A, water þ
0.1% formic acid; channel B, methanol þ 0.1% formic acid): 0–0.5 min
70% B, gradually increasing to 97% B from 0.5 to 6.7 min, hold at 97% B
until 7 min, decrease to 70% B from 7–7.5 min, and reequilibrate to 70%
B from 7.5 to 9.5 min. The column was held at 25 °C during the separation.
Under these conditions, artemisinic acid was found to elute at 6.3 min. Ar-
temisinic acid peak areas were converted to concentrations from external
standard calibrations of authentic compounds.
Dihydroartemisinic aldehyde, compound 3. Dihydroartemisinic alcohol (com-
pound 2, 0.444 g, 2.00 mmol), triethylamine (1.12 mL, 0.808 g, 8.00 mmol),
and 10.4 mL of 5∶1 CH2Cl2∕DMSO were combined and cooled to −10 °C with
an ice/NaCl bath. The mixture was magnetically stirred and SO3 · py (0.796 g,
5.00 mmol) was added in three equal portions over 20 min. The ice bath was
removed and the reaction stirred for 15 h at ambient temperature at which
time GC/MS showed the reaction to be complete. The reaction mixture was
poured into 10 mL of 10% aqueous citric acid solution and stirred for 10 min.
The layers were separated and the organic phase washed with 10 mL of 10%
aqueous citric acid solution, 10 mL of saturated NaHCO3 solution, 10 mL of
NaCl solution, dried (MgSO4) and the CH2Cl2 removed under reduced pres-
sure. The residue was passed through a 1 × 2 cm plug of silica gel with 20%
EtOAc/hexane to afford 0.377 g (76.4%) of compound 3 with 1H − NMR and
MS spectra identical to those previously published (40). For additional details,
see Fig. 5.
Chemistry. General information. NMR spectra were recorded in deuterated
chloroform by Acorn NMR using a JEOL ECX-400 NMR spectrometer operat-
ing at 400 MHz for 1H and 100.5 MHz for 13C. Chemical shifts are reported
in δ units relative to SiMe4 as an internal standard (δ ¼ 0). GC/MS data were
obtained with an Agilent 5975 inert mass selective detector coupled to an
Agilent 6890 N network GC system using an Agilent 19091Z-005 50 m capil-
lary column. Helium was used as the carrier gas. Operating conditions: inlet
temperature 250 °C, initial temperature 50 °C for 0.5 min then ramp 5 °C∕ min
to 190 °C then ramp 60 °C∕ min to 300 °C and hold for 1 min. Total run time
11.33 min. All reagents and solvents were obtained from Sigma-Aldrich and
used as received. Thin layer chromatography (TLC) was performed on 2.5 ×
7.5 cm silica gel 60 F254 glass plates from EMD Chemicals, Inc. using ethyl
acetate/hexane mixtures as eluents. Column chromatography was conducted
with EtOAc/hexane mixtures as eluents and Merck grade 9385 silica gel
(230–400 mesh) obtained from Sigma-Aldrich.
Dihydroartemisinic acid, compound 4. Dihydroartemisinic aldehyde (0.250 g,
1.13 mmol) was dissolved in 20 mL of DMSO with mechanical stirring fol-
lowed by addition over 2 h of a solution of NaOCl2 (0.143 g, 1.58 mmol)
and NaH2PO4 (0.938 g, 6.92 mmol) in 10.0 mL of H2O. After stirring at am-
bient temperature for an additional 2 h, 15 mL of aqeous NaHCO3 solution
were added and the stirring continued for 15 h. The solution was acidified to
pH 2 (Colorphast Strip) by the dropwise addition of concentration H3PO4,
which resulted in the precipitation of compound 4 as fine white needles.
Yield, 0.214 g (79.9%). The 1H − NMR and MS spectra of compound 4 were
identical to those previously published (34). For additional details, see Fig. 5.
Dihydroartemisinic alcohol, compound 2. Amorpha-4,11-diene (compound 1,
0.150 g, 0.734 mmol) was dissolved in tetrahydrofuran (THF; 5.0 mL, Aldrich
Sure/Seal) and cooled to −78 °C under a blanket of N2. A 0.5 M solution of
9-BBN in THF (1.47 mL, 0.734 mol) was added via syringe drive over 19 min
with magnetic stirring. The dry-ice acetone bath was allowed to come to
room temperature and the reaction stirred for 16 h. The reaction was again
cooled to −78 °C and a second portion of 9-BBN (1.47 mL, 0.734 mol) added
via syringe drive over 4 min. The reaction was stirred for 16 h at which time
ACKNOWLEDGMENTS. We thank members of Jay Keasling’s laboratory for
EPY330 and for many productive conversations, and also Jasper Rine and
Hans van Dijken for advice and many fruitful discussions. We thank and
acknowledge our friends and colleagues at Sanofi–Aventis, especially Denis
Thibaut for valuable discussion around the use of ethanol as a carbon source,
Bruno Dumas, Corinne Masson-Brocard, Paul Baduel, and Henri Farret. This
research was conducted under the sponsorship of the Institute for OneWorld
Health through generous support of the Bill and Melinda Gates Foundation.
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