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Biotechnology Advances 30 (2012) 14251431

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Production of shikimic acid


Saptarshi Ghosh a, Yusuf Chisti b, Uttam C. Banerjee a,
a
b

Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar-160 062, Punjab, India
School of Engineering, Massey University, Private Bag 11 222, Palmerston North, New Zealand

a r t i c l e

i n f o

Available online 14 March 2012


Keywords:
Shikimic acid
Oseltamivir
Quinic acid

a b s t r a c t
Shikimic acid is a key intermediate for the synthesis of the antiviral drug oseltamivir (Tamiu). Shikimic
acid can be produced via chemical synthesis, microbial fermentation and extraction from certain plants. An
alternative production route is via biotransformation of the more readily available quinic acid. Much of the
current supply of shikimic acid is sourced from the seeds of Chinese star anise (Illicium verum). Supply
from star anise seeds has experienced difculties and is susceptible to vagaries of weather. Star anise tree
takes around six-years from planting to bear fruit, but remains productive for long. Extraction and purication from seeds are expensive. Production via fermentation is increasing. Other production methods are
too expensive, or insufciently developed. In the future, production in recombinant microorganisms via fermentation may become established as the preferred route. Methods for producing shikimic acid are
reviewed.
2012 Elsevier Inc. All rights reserved.

Contents
1.
2.

Introduction . . . . . . . . . . . . . . . . . . .
Production methods . . . . . . . . . . . . . . .
2.1.
Extraction from plants . . . . . . . . . . .
2.2.
Fermentation processes . . . . . . . . . .
2.2.1.
The shikimic acid pathway . . . .
2.2.2.
Use of recombinant and engineered
2.3.
Chemical synthesis . . . . . . . . . . . .
2.4.
Microbial biotransformation . . . . . . . .
3.
Concluding remarks . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction
This review is focussed on the methods of producing shikimic acid
(3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid), a chemical
building block for the antiviral drug oseltamivir (Tamiu). Shikimic
acid is named after the Japanese shikimi (Illicium anisatum) ower
Abbreviations: DAHP, 3-Deoxy-D-arabino-heptulosonate-7-phosphate; DHQ,
3-Dehydroquinate or 3-dehydroquinic acid; DHS, 3-Dehydroshikimate or 3dehyroshikimic acid; DQD, 3-Dehydroquinate dehydrogenase; EPSP, 5-Enolpyruvylshikimate-3-phosphate; E4P, Erythrose-4-phosphate; GDH, Glucose dehydrogenase; NADP,
Nicotinamide adenine dinucleotide phosphate; NADPH, Reduced form of nicotinamide adenine dinucleotide phosphate; PEP, Phosphoenolpyruvate; QDH, Quinic acid dehydrogenase; SKDH, Shikimic acid dehydrogenase.
Corresponding author. Tel.: + 91 172 2214682 87; fax: + 91 172 2214692.
E-mail address: ucbanerjee@niper.ac.in (U.C. Banerjee).
0734-9750/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2012.03.001

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from which it was rst isolated. Shikimic acid is an intermediate of


the shikimic acid pathway (Herrmann and Weaver, 1999) that is involved in the synthesis of aromatic metabolites in plants and microorganisms (Ganem, 1978; Herrmann, 1995; Pittard, 1996; Wilson et
al., 1998). Metabolically essential products of the shikimic acid pathway include the three aromatic amino acids L-phenylalanine,
L-tryptophan and L-tyrosine. Shikimic acid pathway is not normally
associated with animals, but genes for coding some of the enzymes
of the pathway have been found in certain animals (Starcevic et al.,
2008). Properties and toxicology of shikimic acid have been discussed
elsewhere (Stavric and Stoltz, 1976). An insufciency of shikimic acid
has in the past affected the supply of oseltamivir (Farina and Brown,
2006).
As a highly functionalised, six-carbon ring with three chiral carbons and a carboxylic acid functional group, shikimic acid (Fig. 1) is

1426

S. Ghosh et al. / Biotechnology Advances 30 (2012) 14251431

extraction and purication processes are expensive. Use of recombinant bacteria for commercial production of shikimic acid is developing. Newer routes for producing oseltamivir without the use of
shikimic or quinic acids have appeared (Fukuta et al., 2006; Yeung
et al., 2006), but not been commercialized. Here we review the existing
and emerging methods for producing shikimic acid.
2. Production methods
2.1. Extraction from plants
Fig. 1. Structure of shikimic acid and oseltamivir.

a versatile enantiomerically pure precursor for making potentially


useful products. Interest in shikimic acid has been rekindled as it is
required for producing the avian u drug oseltamivir (Fig. 1). In
view of the potential impact of a u pandemic (Horimoto and
Kawaoka, 2001) and the limited usefulness of vaccines against rapidly
evolving u viruses, stockpiles of effective drugs are necessary for
managing a major outbreak. Oseltamivir is an effective treatment
for inuenza (Widmer et al., 2010), especially if administered early.
Oseltamivir is effective against both Type A and Type B inuenza
and it is used also in prophylaxis.
Oseltamivir is a viral neuraminidase inhibitor. Its synthesis from
quinic acid via shikimic acid has been described (Kim et al., 1997,
1998; Rohloff et al., 1998). Oseltamivir produced for early clinical trials
had been made from chemically synthesized shikimic acid (Federspiel
et al., 2001), but this method proved impractical for commercial production of the drug. Chemical and microbial methods for inexpensive
production of shikimic acid are being developed, but most of this
compound is currently extracted from the seeds of Chinese star
anise (Illicium verum) (Payne and Edmonds, 2005). The multistep

As an intermediate of the shikimic acid pathway, shikimic acid


is found widely in plants (Bohm, 1965; Dell and Frost, 1993;
Gurib-Fakim, 2006; Herrmann and Weaver, 1999), but generally occurs in low concentration. Seeds of Chinese star anise (Illicium verum)
are the main commercial source of shikimic acid. The botany and pharmacology of Chinese star anise have been reviewed (Wang et al., 2011).
Shikimic acid was rst isolated from owers of the highly toxic
Japanese star anise. Subsequently, it was reported in the leaves of
the sweetgum tree (Liquidambar styraciua) (Plouvier, 1961). The
seeds of the American sweetgum tree have been found to contain
up to 3.7% (w/w) shikimic acid (Enrich et al., 2008). Extraction of shikimic acid from bark and wood of the sweetgum has been reported
(Martin et al., 2010). A simple extraction method based on hot
water was used. Several varieties of pine, r and spruce are known
to produce shikimic acid. A hot water (4575 C) extraction of the
needles of Scots pine (Pinus sylvestris) yielded around 1.6% (w/w)
shikimic acid (Sui, 2008). Extraction from the needles of the pine
Pinus elliottii has been described (Xie et al., 2010). Attempts are
being made to identify other natural plant sources of shikimic acid
(Raghavendra et al., 2009).

Fig. 2. The shikimic acid or shikimate pathway.

S. Ghosh et al. / Biotechnology Advances 30 (2012) 14251431

Plants of the genus Illicium appear to the best source of shikimic


acid, but many Illicium species are poisonous (Lederer et al., 2006;
Yamada et al., 1968) and therefore not suitable for producing commercial shikimic acid. Japanese star anise (I. anisatum) is highly
toxic, but the Chinese star anise (I. verum) is edible. Nevertheless,
compounds found in Chinese star anise have been shown to be
toxic to infants (Ize-Ludlow et al., 2004).
Shikimic acid is highly soluble in water (180 g/L at 20 C), but not
in non-polar solvents. Therefore, a hot water extraction of the plant
tissue is used as the primary extraction step (Ohira et al., 2009; Sui,
2008; Xie et al., 2010; Ye et al., 2007). Shikimic acid content in the
plant tissue varies depending on the source of the tissue, the time of
harvest and other possible factors. Roughly 1 kg of shikimic acid can
be recovered from 30 kg of dry seed pods of Chinese star anise
(Ohira et al., 2009; Roche, 2006). Recovery is nearly complete (>95%)
within 10 min of water extraction at 70 C from seed pods ground to a
particle size of 355600 m (Ohira et al., 2009). The rate of recovery
can be further enhanced by using a higher extraction temperature.
Other extraction methods have been described, involving the use
of acids (Harring et al., 1998; Mueller, 2003), alcohols (Anderson et
al., 2001; Jaroszynska, 2003), complex formation (Miles et al., 1994;
Sadaka and Garcia, 1999) and microwave-assisted extraction
(Matallo et al., 2009). The crude water extract contains numerous
other water-soluble plant metabolites and requires extensive further
processing to yield pure shikimic acid. The recovery and purication
processes used in commercial production from Chinese star anise
are proprietary and involve multiple steps.
2.2. Fermentation processes
Microbial production of shikimic acid involves the shikimic acid (or
shikimate) pathway. Shikimic acid pathway occurs commonly in microorganisms and therefore they can be used to overproduce this compound from carbon sources such as glucose (Draths et al., 1999;
Simonart and Wiaux, 1960). Shikimic acid is a precursor for essential
amino acids and other metabolites in microorganisms. Its overproduction can be achieved for example by metabolic engineering to partly
block some of the biochemical pathways that consume shikimic acid
and by overexpression of the enzymes responsible for its synthesis. Production by fermentation using engineered microorganisms is already
supplementing the commercial supply of shikimic acid.
2.2.1. The shikimic acid pathway
The shikimic acid pathway is shown in Fig. 2. This metabolic pathway
is used to produce the aromatic amino acids that are essential to growth.
Shikimic acid is an intermediate in the pathway. This pathway has been
previously reviewed (Herrmann and Weaver, 1999).
The shikimic acid pathway begins with phosphoenolpyruvate
(PEP) and erythrose-4-phosphate (E4P) (Fig. 2). These two compounds must rst be generated from metabolism of glucose, or
other carbohydrate. PEP is produced through the glycolysis pathway
(Fig. 2) whereas the completely independent pentose phosphate

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Table 2
Modied E. coli strains with 3-dehydroshikimic acid yield and titer (Li et al., 1999).
Number Strain

Modication

DHS yield DHS titer


(mol/mol) (g/L)

aroFFBR expression controlled by


ParoF in absence of amplied tktA
aroFFBR expression controlled by
ParoF in absence of amplied tktA
aroFFBR expression controlled by
ParoF in the presence of amplied
tktA
KL3/
aroFFBR expression is controlled
pKD11.291A by ParoF in absence of amplied
tktA
KL3/
aroFFBR expression is controlled
pKL5.17A
by ParoF in presence of amplied
tktA
KL3/
aroFFBR expression under the
control of Ptac in presence of tktA
pKL4.124A

KL3/
pKL4.33B
KL3/
pKL4.66A
KL3/
pKL4.130B

2
3

0.17

20.3

0.16

38.5

0.30

69.0

0.18

41.2

0.24

58.1

0.28

66.0

pathway is used to generate E4P (Fig. 2). Thus, the shikimic acid pathway is dependent on the glycolytic pathway and the pentose phosphate pathway to provide the two starting materials it requires.
Therefore, metabolic engineering of the shikimic acid pathway alone
may be insufcient for increasing the yield of shikimic acid from the
carbon source used in a fermentation. Furthermore, because shikimic
acid is produced far downstream in the metabolic pathway relative to
the point where glucose rst enters the metabolism, channelling the
ow of carbon to production of shikimic acid can be difcult.
Once E4P and PEP have been generated through carbohydrate metabolism in other independent pathways, they are condensed in the
shikimic acid pathway through the action of the enzyme DAHP
synthase to produce DAHP. Action of three other enzymes then converts DAHP to shikimic acid. Chorismic acid is the nal product of
the SA pathway and this compound is the common precursor for
the biosynthesis of other aromatic products such as aromatic amino
acids, as it is shown in Fig. 2.
The shikimic acid pathway engineering has most commonly involved the bacterium Escherichia coli. This microorganism has three
isoforms of the DAHP synthase enzyme encoded by aroF, aroG, aroH
show feedback inhibition by the three aromatic amino acids, i.e.
L-tyrosine, L-phenylalanine and L-tryptophan respectively. The
DAHP synthase (aroG) of Bacillus subtilis is inhibited by the pathway
intermediate chorismate (Jensen and Nester, 1966a,b) (Fig. 2).
The enzyme DHQ synthase (aroB) in E. coli converts DAHP into
3-dehydroquinate (DHQ). The enzyme DHQ dehydratase (aroD) then
converts DHQ into 3-dehydroshikimate (DHS) by eliminating water.
Subsequently, NADPH-dependent shikimate dehydrogenase (aroE) reduces DHS to shikimic acid. The rate limiting enzymes of the shikimic
acid pathway of E. coli are DHQ synthase and shikimate kinase (Dell
and Frost, 1993) (Fig. 2). Shikimate kinase (aroL and aroK) is responsible
for converting shikimic acid to shikimate-3-phosphate (Fig. 2).
In E. coli, the enzymes DHQ synthase, DHQ dehydratase and
shikimate dehydrogenase (Fig. 2) are constitutively expressed
whereas the production of the DAHP synthases and one of the

Table 1
Modied E.coli strains with shikimic acid (SA) yield and titer.
Strain

Modication

SA yield
(mol/mol)

SA
titer (g/L)

SP1.1/pKD15.071B
(Chandran et al., 2003)
SP1.1pts/pSC6.090B
(Chandran et al., 2003)
SP1.1/pKD12.138
(Knop et al., 2001)
SP1.1/pKD12.112
(Draths et al., 1999)

Over expression of ppsA

0.23

66

PTS/glf+/tktA over
expression
Over expression of tktA

0.27

71

0.18

52

Insertion of aroB into the


serA locus of E.coli and
disruption of aroL and aroK

0.15

27.2

Table 3
Effect of carbon sources on the production of 3-dehydroshikimic acid by recombinant
E. coli strains (Li et al., 1999).
Strain

KL3/pKL4.124A
(tktA over expression)

KL3/pKL4.79B (tktA)

Carbon source

DHS titer
(g/L)
46.0
43.1
64.0

Carbon
source
Glucose
Xylose
Mixa

Glucose
Xylose
Mixa
a

DHS yield
(mol/mol)
0.28
0.33
0.41

Molar ratio of glucose/xylose/arabinose: 3:3:2.

DHS titer
(g/L)
36.4
41.7
53.0

DHS yield
(mol/mol)
0.22
0.32
0.36

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S. Ghosh et al. / Biotechnology Advances 30 (2012) 14251431

Fig. 3. Synthesis of shikimic acid via Diels-Alder reaction (McCrindle et al., 1960; Smissman et al., 1959). Based on Ambhaikar (2005).

shikimate kinases is transcriptionally regulated. Shikimic acid inhibits shikimate dehydrogenase (Dell and Frost, 1993).
An alternative microbial route to shikimic acid is through biotransformation of quinic acid. Certain microorganisms (Pseudomonas,
Achromobacter, Aspergillus and Neurospora crassa) can use quinic acid
(or its salt, quinate) as the sole carbon source (Case et al., 1978; Da
Silva et al., 1986; Rogoff, 1958) to produce aromatic amino acids via
the shikimic acid pathway. Quinate enters the pathway at the point
shown in Fig. 2. E. coli strains specically engineered for overproducing quinic acid from glucose have also been developed (Ran et al.,
2001).
2.2.2. Use of recombinant and engineered strains
Metabolically engineered bacteria provide an important emerging
route to production of shikimic acid via fermentation (Campbell et al.,
1993; Krmer et al., 2003). The bacterium E. coli has been the focus of
most metabolic engineering effort (Ahn et al., 2008; Escalante et al.,
2010; Johansson and Liden, 2006; Johansson et al., 2005, 2006;
Knop et al., 2001; Yi et al., 2002, 2003), but studies in other bacteria
have also been reported.
Several metabolic engineering approaches have been developed to
overproduce shikimic acid in E. coli (Ahn et al., 2008; Chandran et al.,
2003; Escalante et al., 2010; Gibson et al., 2001; Johansson et al.,
2005; Knop et al., 2001; Krmer et al., 2003). All these are based on
genetic modications to alter the central carbon metabolism and
the shikimic acid pathway (Table 1).
The shikimic acid pathway requires PEP and E4P (Fig. 2). The supply of PEP and E4P can be enhanced via metabolic engineering of the
glycolytic pathway and the pentose phosphate pathway, respectively.
Both these approaches have been demonstrated. Over expression of
transketolase (tktA) resulted in the increase of shikimic acid yield
from 0.12 to 0.18 mol/mol and titer from 38 to 52 g/L by enhancing
the concentration of E4P (Knop et al., 2001). In recombinant E. coli,

an increased availability of PEP has enhanced production of shikimic


acid (Chandran et al., 2003; Yi et al., 2002). The modication of the
glycolytic pathway involved over expression of PEP synthase (ppsA)
leading to increased shikimic acid titer to 66 g/L and yield on glucose
of 0.23 mol/mol (Chandran et al., 2003). Inactivation of the PTS operon
(PTS), expression of non-PTS glucose transporters like glucose facilitators (glf), glucokinase (glk) in combination with over expression of tktA
gene was reported to increase the shikimic acid titer to 71 g/L
(Chandran et al., 2003; Gibson et al., 2001). Using the engineered E.
coli strain, the shikimic acid concentration could be further raised to
84 g/L by supplementing the minimal medium with yeast extract
(Chandran et al., 2003). Shikimic acid could be produced during exponential growth on glucose to a nal concentration of 87 g/L (Chandran
et al., 2003). The nature of the carbon source affected the productivity
of shikimic acid (Ahn et al., 2008; Li et al., 1999).
In earlier studies, the shikimic acid titers from metabolically engineered E. coli strains were low partly due to simultaneous production
of quinic acid (Knop et al., 2001). Production of quinic acid during biosynthesis of shikimic acid is a consequence of the microbe-catalyzed
equilibration of the initially synthesized shikimic acid (Knop et al.,
2001). This problem appears to have been resolved in view of the aforementioned high titers.
Shikimic acid can be produced readily from DHS (Fig. 2). Therefore, some metabolic engineering studies have focussed on overproduction of DHS. Substantial overproduction of DHS in metabolically
engineered microorganisms has been achieved (Draths and Frost,
1990; Li and Frost, 1999; Li et al., 1999; Yi et al., 2002, 2003)
(Table 2). For example, E. coli constructs have produced as much as
60 g/L DHS from glucose in 60 h (Yi et al., 2003). A comparative analysis
of D-xylose, D-glucose and D-arabinose as carbon source for microbial
synthesis of DHS was also reported (Li and Frost, 1999) (Table 3). Different recombinant E. coli strains were used with various carbon sources
for DHS production.

Fig. 4. Shikimic acid synthesis of Koreeda and Ciufolini (1982) as summarized by Ambhaikar (2005).

S. Ghosh et al. / Biotechnology Advances 30 (2012) 14251431

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Fig. 5. Chemical conversion of ()-Quinic acid to ()-Shikimic acid (Dangschat and Fischer, 1938, 1950). Based on Ambhaikar (2005).

The activity of DAHP synthase (Fig. 2) controls the amount of cellular carbon directed into DHS synthesis. Transcriptional repression
and feedback inhibition of DAHP synthase by aromatic amino acids
are believed to control the activity of this enzyme. The amplied
expression of a mutant DAHP synthase, which is insensitive to
feedback inhibition by aromatic amino acids, has been used to enhance production of DHS. Metabolic engineering approaches have
been employed to produce shikimic acid in E. coli strains derived
from an evolved strain PB 12 lacking the PTS system but with ability
to grow on glucose. The double aroK -, aroL- mutant of strain PB
12.SA22 showed shikimic acid titer of 7 g/L and yield of 0.29 mol/mol
(Escalante et al., 2010).
In addition to recombinant E. coli, genetically modied or otherwise mutated Bacillus subtilis (Iomantas et al., 2002) and Citrobacter
freundii bacteria (Shirai et al., 2001) have been used to successfully
overproduce shikimic acid although the titers have not exceeded
about 20 g/L. It was reported that aroI (shikimate kinase) decient
strain of B. subtilis showed shikimic acid titer of 8.5 g/L with high
titer of DHS (9.5 g/L) as the by-product (Iomantas et al., 2002). Use
of glyphosate, an inhibitor of the enzyme EPSP synthase (Fig. 2), in
the fermentation medium to enhance accumulation of shikimic acid
by blocking its downstream consumption, has been described
(Bogosian, 2011). Metabolic engineering of microorganisms for producing shikimic acid has been reviewed by Krmer et al. (2003).
2.3. Chemical synthesis
Chemical synthesis of shikimic acid was rst achieved during the
1960s using the basic chemistry of Diels-Alder reaction (Fig. 3) to
form six membered rings (McCrindle et al., 1960; Smissman et al.,
1959), but the yield was low at 15%. Later attempts to increase
yield were not particularly successful (Grewe and Hinrichs, 1964).
In 1982, the efciency of Diels-Alder synthesis could be improved
(Fig. 4) to raise the yield to 29% (Koreeda and Ciufolini, 1982). A
more efcient synthesis further raised the yield to 55% (Koreeda et
al., 1990).
Synthesis of shikimic acid from benzene has been shown to be
possible (Birch et al., 1988). Synthesis via a palladium mediated elimination reaction was advanced by Yoshida and Ogasawara (2000).
Synthesis of ()-shikimic acid has been reported from sugars such
as D-mannose (Dangschat and Fischer, 1938, 1950; Fleet et al., 1984;
Jiang et al., 1994). Shikimic acid can be made also from ()-quinic
acid and its derivatives by chemical transformations (Box et al., 2002;
Cleophax et al., 1971, 1973; Federspiel et al., 2001; Kim et al., 1997,
1998; Rohloff et al., 1998) (Fig. 5). Other synthetic methods have

been described (Kancharla et al., 2009; Snchez-Abella et al., 2006).


Chemical synthesis of shikimic acid and its analogs is further reviewed
elsewhere (Campbell et al., 1993; Jiang and Singh, 1998).
Although several routes have been described for chemical synthesis,
production of shikimic acid by these methods is too expensive to be
of commercial use. Consequently, isolation from Chinese star anise
fruit remains the principal source of commercial shikimic acid
(Raghavendra et al., 2009).
The ten step commercial route of oseltamivir (Tamiu) synthesis
uses ()-shikimic acid as the starting material (Abrecht et al., 2004).
This precursor is converted into a diethyl ketal intermediate, which is
reductively opened to give 1,2-epoxide. This epoxide is then converted into oseltamivir via a ve step reaction involving 3 potentially
toxic and explosive azide intermediates (Fig. 6). This route gives 35%
yield of oseltamivir. Future methods for making oseltamivir may
completely circumvent the need for shikimic or quinic acids (Fukuta
et al., 2006; Yeung et al., 2006).

2.4. Microbial biotransformation


Many microorganisms are able to convert quinic acid to shikimic
acid (Adachi et al., 2003, 2006). Quinic acid (or quinate) is converted
to 3-dehydroquinate (DHQ) (Fig. 2) through the action of quinate dehydrogenase. DHQ is an intermediate of the shikimic acid pathway
and is transformed to shikimic acid in a two-step process (Fig. 2).
During exponential growth of Gluconobacter oxydans, nearly all quinate supplied in the medium could be converted to DHQ (Adachi et al.,
2003). This transformation could be achieved also with dried cells of
G. oxydans (Adachi et al., 2003) and with fresh cells immobilized in
alginate beads. The intermediate DHQ could be further transformed
to DHS in a reasonable yield by both growing and immobilized
cells. Lactobacillus pastorianus is capable of converting quinic acid to a
product that was claimed to be dihydroshikimic acid (Carr et al.,
1957), but was more likely DHS.
A two-step scheme for biotransformation of quinic acid to shikimic
acid has been published (Fig. 7) (Adachi et al., 2006). The rst step of
this scheme involves an oxidative fermentation with G. oxydans to essentially quantitatively convert quinic acid in the culture medium to
DHS. The latter is subsequently converted to shikimic acid in an
NADPH-dependent reaction using the enzyme shikimic acid dehydrogenase (SKDH) (Fig. 7) puried from cells of G. oxydans. The enzymatic biotransformation medium also contained glucose and the
enzyme glucose dehydrogenase to continuously regenerate NADPH
(Fig. 7).

Fig. 6. Synthesis of oseltamivir from shikimic acid.

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S. Ghosh et al. / Biotechnology Advances 30 (2012) 14251431

Fig. 7. Overall reaction for shikimic acid production (based on Adachi et al., 2006).

3. Concluding remarks
Shikimic acid is a precursor for the synthesis of the important antiviral drug oseltamivir. Extraction from Chinese star anise is the main
source for shikimic acid, but fermentation processes based on the recombinant bacterium E. coli have been shown to be a viable alternative
source. Chemical synthesis of shikimic acid is possible, but apparently
not commercially viable. Chemical methods of making oseltamivir
may entirely circumvent the need for shikimic acids, but are not commercially used. Bioconversion of quinic acid to shikimic acid is another
option for production, but appears not to have been developed to the
extent of the fermentation route from glucose.
References
Abrecht S, Harrington P, Iding H, Karpf M, Trussardi R, Wirz B, et al. The synthetic development of the anti-inuenza neuraminidase inhibitor oseltamivir phosphate
(Tamiu): a challenge for synthesis and process research. Chimia 2004;58:
6219.
Adachi O, Tanasupawat S, Yoshihara N, Toyama H, Matsushita M. 3-Dehydroquinate production by oxidative fermentation and further conversion of 3-dehydroquinate to the
intermediates in the shikimate pathway. Biosci Biotechnol Biochem 2003;67:
212431.
Adachi O, Ano Y, Toyama H, Matsushita K. High shikimate production from quinate
with two enzymatic systems of acetic acid bacteria. Biosci Biotechnol Biochem
2006;70:257982.
Ahn JO, Lee HW, Saha R, Park MS, Jung JK, Lee DY. Exploring the effects of carbon
sources on the metabolic capacity for shikimic acid production in Escherichia coli
using in silico metabolic predictions. J Microbiol Biotechnol 2008;18:177384.
Ambhaikar N. Shikimic acid. A seminar presentation dated 12 January 2005. La Jolla,
California, USA: The Scripps Research Institute; 2005http://www.scripps.edu/
chem/baran/images/grpmtgpdf/Ambhaikar_Jan_05.pdf.
Anderson KA, Cobb WT, Loper BR. Analytical method for determination of shikimic
acid: Shikimic acid proportional to glyphosate application rates. Commun Soil Sci
Plant Anal 2001;32:283140.
Birch AJ, Kelly LF, Weerasuria DV. Facile synthesis of (+)- and ()-shikimic acid with
asymmetric deuterium labelling, using tricarbonyliron as a lateral control group. J
Org Chem 1988;53:27881.
Bogosian, G., Frantz, J.P. Use of glyphosate to produce shikimic acid in microorganisms.
United States Patent Application Pub. No.: US 2011/0020885 Al; 2011.
Bohm BA. Shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid). Chem Rev
1965;65:43566.
Box JM, Harwood LM, Humphreys JL, Morris GA, Redon PM, Whitehead RC. Dehydration
of quinate derivatives: synthesis of a diuoromethylene homologue of shikimic acid.
Synlett 2002;2:35860.
Campbell MM, Sainsbury M, Searle PA. The biosynthesis and synthesis of shikimic acid,
chorismic acid, and related-compounds. Synth Stuttg 1993;2:17993.
Carr JG, Pollard A, Whiting GC, Williams AH. The reduction of quinic acid to dihydroshikimic acid by certain lactic acid bacteria. Biochem J 1957;66:2835.

Case ME, Pueyo C, Barea JL, Giles NH. Genetical and biochemical characterization of
QA-3 mutants and revertants in the QA gene cluster of Neurospora crassa. Genetics
1978;90:6984.
Chandran SS, Yi J, Draths KM, Von Daeniken R, Weber W, Frost JW. Phosphoenolpyruvate availability and the biosynthesis of shikimic acid. Biotechnol Prog 2003;19:
80814.
Cleophax J, Mercier D, Gro SD. A stereospecic conversion of ()-methyl
tri-O-benzoylquinate to the corresponding ()-methyl shikimate. Angew Chem
Int Ed 1971;10:6523.
Cleophax J, Leboul J, Mercier D, Gaudemer A, Gero SD. New and easy synthetic route to
shikimic and 4-epishikimic acid. Bull Soc Chim Fr Partie II 1973:29925.
Da Silva AJ, Whittington H, Clements J, Roberts C, Hawkins AR. Sequence analysis and
transformation by the catabolic 3-dehydroquinase (QUTE) gene from Aspergillus
nidulans. Biochem J 1986;240:4818.
Dangschat G, Fischer HOL. bergang der Chinasure in shikimisure. Naturwissenschaften
1938;26:5623.
Dangschat G, Fischer HOL. Congurational relationships between naturally occurring
cyclic plant acids and glucose transformation of quinic acid into shikimic acid. Biochem Biophys Acta 1950;4:199204.
Dell KA, Frost JW. Identication and removal of impediments to biocatalytic synthesis
of aromatics from D-glucose: rate limiting enzymes in the common pathway of
aromatic amino acid biosynthesis. J Am Chem Soc 1993;115:115819.
Draths KM, Frost JW. Genomic direction of synthesis during plasmid-based biocatalysis. J
Am Chem Soc 1990;112:96302.
Draths KM, Knop DR, Frost JW. Shikimic acid and quinic acid: replacing isolation from
plant sources with recombinant microbial biocatalysis. J Am Chem Soc 1999;121:
16034.
Enrich LB, Scheuermann ML, Mohadjer A, Matthias KR, Eller CF, Newman MS, et al.
Liquidambar styraciua: a renewable source of shikimic acid. Tetrahedron Lett
2008;49:25035.
Escalante A, Caldern R, Valdivia A, de Anda R, Hernndez G, Ramrez OT, et al. Metabolic engineering for the production of shikimic acid in an evolved Escherichia
coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase
system. Microb Cell Fact 2010;9:21.
Farina V, Brown JD. Tamiu: the supply problem. Angew Chem Int Ed 2006;45:73304.
Federspiel M, Fischer R, Hennig M, Mair HJ, Oberhauser T, Rimmler G, et al. Industrial synthesis of the key precursor in the synthesis of the anti-inuenza drug oseltamivir
phosphate (Ro 64-0796/002, GS-4104-02): Ethyl (3R, 4S, 5S)-4, 5-epoxy-3-(1ethyl-propoxy)-cyclohex-1-ene-1-carboxylate. Org Process Res Dev 2001;3:26674.
Fleet GWJ, Shing TKM, Warr SM. Enantiospecic synthesis of shikimic acid from
D-mannose: formation of a chiral cyclohexene by intramolecular olenation of a
carbohydrate-derived intermediate. J Chem Soc Perkin Trans 1 1984:9058.
Fukuta Y, Mita T, Fukuda N, Kanai M, Shibasaki M. De novo synthesis of Tamiu via a
catalytic asymmetric ring-opening of meso-aziridines with TMSN3. J Am Chem
Soc 2006;128:63123.
Ganem B. Shikimate-derived metabolites. 4. From glucose to aromatics recent developments in natural-products of shikimic acid pathway. Tetrahedron 1978;34:335383.
Gibson JM, Thomas PS, Thomas JD, Barker JL, Chandran SS, Harrup MK, et al. Benzenefree synthesis of phenol. Angew Chem Int Ed 2001;40:19458.
Grewe R, Hinrichs I. Eine neue synthese der shikimisaure. Chem Ber 1964;97:443.
Gurib-Fakim A. Medicinal plants: traditions of yesterday and drugs of tomorrow. Mol
Aspects Med 2006;27:1-93.
Harring T, Streibig JC, Husted S. Accumulation of shikimic acid: a technique for screening
glyphosate efcacy. J Agric Food Chem 1998;46:440612.

S. Ghosh et al. / Biotechnology Advances 30 (2012) 14251431


Herrmann KM. The shikimate pathway: early steps in the biosynthesis of aromatic
compounds. Plant Cell 1995;7:90719.
Herrmann KM, Weaver LM. The shikimate pathway. Annu Rev Plant Physiol Plant Mol
Biol 1999;50:473503.
Horimoto T, Kawaoka Y. Pandemic threat posed by avian inuenza A viruses. Clin
Microbiol Rev 2001;14:12949.
Iomantas, Y., Abalakina, E.G., Polanuer, B., Yampolskaya, T.A., Bachina, T.A., Kozlov, J.I.
Method for producing shikimic acid. US Patent 6436664; 2002.
Ize-Ludlow D, Ragone S, Bernstein JN, Bruck IS, Duchowny M, Pena BMG. Chemical
composition of Chinese Star anise (Illicium verum) and neurotoxicity in infants. J
Am Med Assoc 2004;291:5623.
Jaroszynska J. Isolation of free phenolic compounds from arboreal leaves by use of the
Florisil C18 system. Anal Bioanal Chem 2003;377:7028.
Jensen RA, Nester EW. Regulatory enzymes of aromatic amino acid biosynthesis in
Bacillus subtilis. J Biol Chem 1966a;241:336572.
Jensen RA, Nester EW. The enzymology of feedback inhibition of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase. J Biol Chem 1966b;241:337380.
Jiang SD, Singh G. Chemical synthesis of shikimic acid and its analogues. Tetrahedron
1998;54:4697753.
Jiang S, Mekki B, Singh G, Wightman H. Enantiospecic synthesis of ()-5-epi-shikimic
acid and a new route to ()-shikimic acid. Tetrahedron Lett 1994;35:55058.
Johansson L, Liden G. Transcriptome analysis of a shikimic acid producing strain of
Escherichia coli W3110 grown under carbon- and phosphate-limited conditions. J
Biotechnol 2006;126:52845.
Johansson L, Lindskog A, Silfversparre G, Cimander C, Nielsen KF, Lidn G. Shikimic acid
production by a modied strain of E. coli (W3110.shik1) under phosphate-limited
and carbon-limited conditions. Biotechnol Bioeng 2005;92:54152.
Johansson, L., Metabolic analysis of shikimic acid producing Escherichia coli. PhD thesis,
Lund University, Sweden, 2006.
Kancharla PK, Doddi VR, Kokatla H, Vankar YD. A concise route to ()-shikimic acid
and ()-5-epi-shikimic acid, and their enantiomers via Barbier reaction and
ring-closing metathesis. Tetrahedron Lett 2009;50:69514.
Kim CU, Lew W, Williams MA, Liu H, Zhang L, Swaminathan S, et al. Inuenza neuraminidase inhibitor possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid
analogues with potent anti-inuenza activity. J Am Chem Soc 1997;119:68190.
Kim CU, Lew W, Williams MA, Wu H, Zhang L, Chen X, et al. Structure activity relationship studies of novel carbocyclic inuenza neuraminidase inhibitors. J Med Chem
1998;41:245160.
Knop DR, Draths KM, Chandran SS, Barker JL, Frost JW. Hydroaromatic equilibrium during biosynthesis of shikimic acid. J Am Chem Soc 2001;123:1017382.
Koreeda M, Ciufolini MA. Natural product synthesis via allylsilanes. 1. Synthesis and reactions of (1E, 3E)-4-acetoxy-l-(trimethylsilyl)1,3-butadiene and its use in the
total synthesis of ()-shikimic acid. J Am Chem Soc 1982;104:230810.
Koreeda M, Teng K, Murata T. (1E,3E)-4-acetoxy-1-phenyldimethylsilyl-1,3-butadiene
as a surrogate for (1E,3E)-1,4-diacetoxy-1,3-butadiene: a highly efcient synthesis
of ()-shikimic acid. Tetrahedron Lett 1990;31:59976000.
Krmer M, Bongaerts J, Bovenberg R, Kremer S, Mller U, Orf S, et al. Metabolic engineering for microbial production of shikimic acid. Metab Eng 2003;5:27783.
Lederer I, Schulzki G, Gross J, Steffen JP. Combination of TLC and HPLC-MS/MS methods.
Approach to a rational quality control of Chinese star anise. J Agric Food Chem
2006;54:19704.
Li K, Frost JW. Microbial synthesis of 3-dehydroshikimic acid: a comparative analysis of
D-xylose, D-arabinose, and D-glucose carbon sources. Biotechnol Prog 1999;15:
87683.
Li K, Mikola MR, Draths KM, Worden RM, Frost JW. Fed-batch fermenter synthesis of
3-dehydroshikimic acid using recombinant Escherichia coli. Biotechnol Bioeng
1999;64:6173.
Martin E, Duke J, Pelkki M, Clausen EC, Carrier DJ. Sweetgum (Liquidambar styraciua
L.): extraction of shikimic acid coupled to dilute acid pretreatment. Appl Biochem
Biotechnol 2010;162:16608.
Matallo MB, Almeida SDB, Cerdeira AL, Franco DA, Blanco FMG, Menezes PTC, et al. Microwave-assisted solvent extraction and analysis of shikimic acid from plant tissues.
Planta Daninha 2009;27:98794.
McCrindle R, Overton KH, Raphael RA. A stereospecic total synthesis of D-()-shikimic
acid. J Am Chem Soc 1960:15606.
Miles D, Garcia AA, Sadaka M, Whited G. Recovery of shikimic acid using temperatureswing complexation extraction and displacement back extraction. Isolation Purif
1994;2:7582.
Mueller TC. Shikimate accumulates in both glyphosate-sensitive and glyphosateresistant horseweed (Conyza canadensis L. Cronq.). J Agric Food Chem 2003;51:
6804.

1431

Ohira H, Torii N, Aida TM, Watanabe M, Smith Jr RL. Rapid separation of shikimic acid
from Chinese star anise (Illicium verum Hook. f.) with hot water extraction. Sep
Purif Technol 2009;69:1028.
Payne R, Edmonds M. Isolation of shikimic acid from star anise seeds. J Chem Educ
2005;82:599600.
Pittard AJ. Biosynthesis of aromatic amino acids. In: Neidhardt FC, Curtiss III R,
Ingraham JL, Lin ECC, Low KB, Magasanic B, Reznikoff WS, Riley M, Schaechter M,
Umbarger HE, editors. Escherichia coli and Salmonella. Cellular and Molecular BiologyWashington, DC: American Society of Microbiology; 1996. p. 45884.
Plouvier V. Sur la recherche des acides quinique et shikimique, de la bergenine et des
heterosides chez quelques hamamelidacees. CR Hebd Seances Acad Sci 1961;252:
599.
Raghavendra TR, Vaidyanathan P, Swathi HK, Ramesha BT, Ravikanth G, Ganeshaiah
KN, et al. Prospecting for alternate sources of shikimic acid, a precursor of Tamiu,
a bird-u drug. Curr Sci 2009;96(6):7712.
Ran N, Knop DR, Draths KM, Frost JW. Benzene-free synthesis of hydroquinone. J Am
Chem Soc 2001;123:1092734.
Roche. Factsheet Tamiu. http://www.roche.com/med_mbtamiu05e.pdf2006.
Rogoff MH. An aromatic intermediate in the bacterial oxidation of quinic acid. J Gen
Microbiol 1958;19:3309.
Rohloff JC, Kenneth MK, Postich MJ, Becker MW, Chapman HH, Kelly DE, et al.
Practical total synthesis of the anti-inuenza drug GS-4104. J Org Chem
1998;63:454550.
Sadaka M, Garcia A. Extraction of shikimic and quinic acids. Chem Eng Commun
1999;173:91-102.
Snchez-Abella L, Fernndez S, Armesto N, Ferrero M, Gotor V. Novel and efcient synthesis of ()-methyl 4-epi-shikimate and 4,5-epoxy-quinic and ()-shikimic acid
derivatives as key precursors to prepare new analogues. J Org Chem 2006;71:
53969.
Shirai, M., Miyata, R., Sasaki, S., Sakamoto, K., Yahanda, S., Shibayama, K., Yonehara, T.,
Ogawa, K. Microorganism belonging to the genus Citrobacter and process for producing shikimic acid. European Patent 1092766; 2001.
Simonart P, Wiaux A. Production of shikimic acid by Penicillium griseofulvum Dierckx.
Nature 1960;186(4718):789.
Smissman EE, Suh JT, Oxman M, Daniels R. A stereospecic synthesis of DL-shikimic
acid. J Am Chem Soc 1959;81:290910.
Starcevic A, Akthar S, Dunlap WC, Shick JM, Hranueli D, Cullum J, et al. Enzymes of
the shikimic acid pathway encoded in the genome of a basal metazoan, Nematostella vectensis, have microbial origins. Proc Natl Acad Sci U S A 2008;105:
25337.
Stavric B, Stoltz DR. Shikimic acid. Food Cosmet Toxicol 1976;14:1415.
Sui RH. Separation of shikimic acid from Pine needles. Chem Eng Technol 2008;31:
46973.
Wang G-W, Hu W-T, Huang B-K, Qin L-P. Illicium verum: a review on its botany,
traditional use, chemistry and pharmacology. J Ethnopharmacol 2011;136:
1020.
Widmer N, Meylan P, Ivanyuk A, Aouri M, Decosterd LA, Buclin T. Oseltamivir in
seasonal, avian H5N1 and pandemic 2009 A/H1N1 inuenza pharmacokinetic
and pharmacodynamic characteristics. Clin Pharmacokinet 2010;49:74165.
Wilson DJ, Patton S, Florova G, Hale V, Reynolds KA. The shikimic acid pathway and
polyketide biosynthesis. J Ind Microbiol Biotechnol 1998;20:299303.
Xie JY, Chen XP, Chen F. Study on extraction of shikimic acid from pine needles of Pinus
elliottii by decompressing inner ebullition. Zhong Yao Cai 2010;33:14804. [In
Chinese].
Yamada K, Takada S, Nakamura S, Hirata Y. Structures of anisatin and neoanisatin toxic
sesquiterpenes from Illicium anisatum L. Tetrahedron 1968;24:199229.
Ye, Y., Junyi, S., Tang, C., Sugimoto, K., Ke, C., Li, X., Tan, M., Ge, F., Yang, X., Ding, J. Method for separating and purifying shikimic acid. China Patent 200510111303; 2007.
Yeung Y-Y, Hong S, Corey EJ. A short enantioselective pathway for the synthesis of the
anti-inuenza neuramidase inhibitor oseltamivir from 1, 3-butadiene and acrylic
acid. J Am Chem Soc 2006;128:63101.
Yi K, Li K, Draths KM, Frost JW. Modulation of phosphoenolpyruvate synthase expression increases shikimate pathway product yields in E. coli. Biotechnol Prog
2002;18:11418.
Yi J, Draths KM, Li K, Frost JW. Altered glucose transport and shikimate pathway product
yields in E. coli. Biotechnol Prog 2003;19:14509.
Yoshida N, Ogasawara K. An enantioconvergent route to ()-shikimic acid via a
palladium-mediated elimination reaction. Org Lett 2000;2:14613.