JBA-06566; No of Pages 7

Biotechnology Advances xxx (2012) xxx–xxx

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

a r t i c l e i n f o a b s t r a c t

Article history: Shikimic acid is a key intermediate for the synthesis of the antiviral drug oseltamivir (Tamiflu®). Shikimic
Received 23 November 2011 acid can be produced via chemical synthesis, microbial fermentation and extraction from certain plants. An
Received in revised form 1 March 2012 alternative production route is via biotransformation of the more readily available quinic acid. Much of the
Accepted 5 March 2012
current supply of shikimic acid is sourced from the seeds of Chinese star anise (Illicium verum). Supply
Available online xxxx
from star anise seeds has experienced difficulties and is susceptible to vagaries of weather. Star anise tree
Keywords:
takes around six-years from planting to bear fruit, but remains productive for long. Extraction and purifica-
Shikimic acid tion from seeds are expensive. Production via fermentation is increasing. Other production methods are
Oseltamivir too expensive, or insufficiently developed. In the future, production in recombinant microorganisms via fer-
Quinic acid mentation may become established as the preferred route. Methods for producing shikimic acid are
reviewed.
© 2012 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2. Production methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.1. Extraction from plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2. Fermentation processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2.1. The shikimic acid pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2.2. Use of recombinant and engineered strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.3. Chemical synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.4. Microbial biotransformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction from which it was first isolated. Shikimic acid is an intermediate of

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 (Tamiflu®). Shikimic
acid is named after the Japanese shikimi (Illicium anisatum) flower
Abbreviations: DAHP, 3-Deoxy-D-arabino-heptulosonate-7-phosphate; DHQ,
3-Dehydroquinate or 3-dehydroquinic acid; DHS, 3-Dehydroshikimate or 3-
dehyroshikimic acid; DQD, 3-Dehydroquinate dehydrogenase; EPSP, 5-Enolpyruvylshiki-
mate-3-phosphate; E4P, Erythrose-4-phosphate; GDH, Glucose dehydrogenase; NADP,
Nicotinamide adenine dinucleotide phosphate; NADPH, Reduced form of nicotinamide ad-
enine dinucleotide phosphate; PEP, Phosphoenolpyruvate; QDH, Quinic acid dehydroge-
nase; 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).
the shikimic acid pathway (Herrmann and Weaver, 1999) that is in-
volved in the synthesis of aromatic metabolites in plants and micro-
organisms (Ganem, 1978; Herrmann, 1995; Pittard, 1996; Wilson et
al., 1998). Metabolically essential products of the shikimic acid path-
way 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 insufficiency 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 car-
bons and a carboxylic acid functional group, shikimic acid (Fig. 1) is

0734-9750/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2012.03.001

Please cite this article as: Ghosh S, et al, Production of shikimic acid, Biotechnol Adv (2012), doi:10.1016/j.biotechadv.2012.03.001
2 S. Ghosh et al. / Biotechnology Advances xxx (2012) xxx–xxx
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 flu drug oseltamivir (Fig. 1). In
view of the potential impact of a flu pandemic (Horimoto and
Kawaoka, 2001) and the limited usefulness of vaccines against rapidly
evolving flu viruses, stockpiles of effective drugs are necessary for
managing a major outbreak. Oseltamivir is an effective treatment
for influenza (Widmer et al., 2010), especially if administered early.
Oseltamivir is effective against both Type A and Type B influenza
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 pro-
duction 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
Fig. 2. The shikimic acid or shikimate pathway.
Please cite this article as: Ghosh S, et al, Production of shikimic acid, Biotechnol Adv (2012), doi:10.1016/j.biotechadv.2012.03.001
extraction and purification processes are expensive. Use of recombi-
nant bacteria for commercial production of shikimic acid is develop-
ing. 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
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 oc-
curs in low concentration. Seeds of Chinese star anise (Illicium verum)
are the main commercial source of shikimic acid. The botany and phar-
macology of Chinese star anise have been reviewed (Wang et al., 2011).
Shikimic acid was first isolated from flowers of the highly toxic
Japanese star anise. Subsequently, it was reported in the leaves of
the sweetgum tree (Liquidambar styraciflua) (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 shi-
kimic 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, fir and spruce are known
to produce shikimic acid. A hot water (45–75 °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).
 S. Ghosh et al. / Biotechnology Advances xxx (2012) xxx–xxx 3

Plants of the genus Illicium appear to the best source of shikimic Table 2
acid, but many Illicium species are poisonous (Lederer et al., 2006; Modified E. coli strains with 3-dehydroshikimic acid yield and titer (Li et al., 1999).

Yamada et al., 1968) and therefore not suitable for producing com- Number Strain Modification DHS yield DHS titer
mercial shikimic acid. Japanese star anise (I. anisatum) is highly (mol/mol) (g/L)
toxic, but the Chinese star anise (I. verum) is edible. Nevertheless, 1 KL3/ aroFFBR expression controlled by 0.17 20.3
compounds found in Chinese star anise have been shown to be pKL4.33B ParoF in absence of amplified tktA
toxic to infants (Ize-Ludlow et al., 2004). 2 KL3/ aroFFBR expression controlled by 0.16 38.5
pKL4.66A ParoF in absence of amplified tktA
Shikimic acid is highly soluble in water (180 g/L at 20 °C), but not
3 KL3/ aroFFBR expression controlled by 0.30 69.0
in non-polar solvents. Therefore, a hot water extraction of the plant pKL4.130B ParoF in the presence of amplified
tissue is used as the primary extraction step (Ohira et al., 2009; Sui, tktA
2008; Xie et al., 2010; Ye et al., 2007). Shikimic acid content in the 4 KL3/ aroFFBR expression is controlled 0.18 41.2
plant tissue varies depending on the source of the tissue, the time of pKD11.291A by ParoF in absence of amplified
tktA
harvest and other possible factors. Roughly 1 kg of shikimic acid can
5 KL3/ aroFFBR expression is controlled 0.24 58.1
be recovered from 30 kg of dry seed pods of Chinese star anise pKL5.17A by ParoF in presence of amplified
(Ohira et al., 2009; Roche, 2006). Recovery is nearly complete (>95%) tktA
within 10 min of water extraction at 70 °C from seed pods ground to a 6 KL3/ aroFFBR expression under the 0.28 66.0
pKL4.124A control of Ptac in presence of tktA
particle size of 355–600 μm (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 pathway is used to generate E4P (Fig. 2). Thus, the shikimic acid path-
al., 2001; Jaroszynska, 2003), complex formation (Miles et al., 1994; way is dependent on the glycolytic pathway and the pentose phos-
Sadaka and Garcia, 1999) and microwave-assisted extraction phate pathway to provide the two starting materials it requires.
(Matallo et al., 2009). The crude water extract contains numerous Therefore, metabolic engineering of the shikimic acid pathway alone
other water-soluble plant metabolites and requires extensive further may be insufficient for increasing the yield of shikimic acid from the
processing to yield pure shikimic acid. The recovery and purification carbon source used in a fermentation. Furthermore, because shikimic
processes used in commercial production from Chinese star anise acid is produced far downstream in the metabolic pathway relative to
are proprietary and involve multiple steps. the point where glucose first enters the metabolism, channelling the
flow of carbon to production of shikimic acid can be difficult.
2.2. Fermentation processes Once E4P and PEP have been generated through carbohydrate me-
tabolism in other independent pathways, they are condensed in the
Microbial production of shikimic acid involves the shikimic acid (or shikimic acid pathway through the action of the enzyme DAHP
shikimate) pathway. Shikimic acid pathway occurs commonly in micro- synthase to produce DAHP. Action of three other enzymes then con-
organisms and therefore they can be used to overproduce this com- verts DAHP to shikimic acid. Chorismic acid is the final product of
pound from carbon sources such as glucose (Draths et al., 1999; the SA pathway and this compound is the common precursor for
Simonart and Wiaux, 1960). Shikimic acid is a precursor for essential the biosynthesis of other aromatic products such as aromatic amino
amino acids and other metabolites in microorganisms. Its overproduc- acids, as it is shown in Fig. 2.
tion can be achieved for example by metabolic engineering to partly The shikimic acid pathway engineering has most commonly in-
block some of the biochemical pathways that consume shikimic acid volved the bacterium Escherichia coli. This microorganism has three
and by overexpression of the enzymes responsible for its synthesis. Pro- isoforms of the DAHP synthase enzyme encoded by aroF, aroG, aroH
duction by fermentation using engineered microorganisms is already show feedback inhibition by the three aromatic amino acids, i.e.
supplementing the commercial supply of shikimic acid. L-tyrosine, L-phenylalanine and L-tryptophan respectively. The
DAHP synthase (aroG) of Bacillus subtilis is inhibited by the pathway
2.2.1. The shikimic acid pathway intermediate chorismate (Jensen and Nester, 1966a,b) (Fig. 2).
The shikimic acid pathway is shown in Fig. 2. This metabolic pathway The enzyme DHQ synthase (aroB) in E. coli converts DAHP into
is used to produce the aromatic amino acids that are essential to growth. 3-dehydroquinate (DHQ). The enzyme DHQ dehydratase (aroD) then
Shikimic acid is an intermediate in the pathway. This pathway has been converts DHQ into 3-dehydroshikimate (DHS) by eliminating water.
previously reviewed (Herrmann and Weaver, 1999). Subsequently, NADPH-dependent shikimate dehydrogenase (aroE) re-
The shikimic acid pathway begins with phosphoenolpyruvate duces DHS to shikimic acid. The rate limiting enzymes of the shikimic
(PEP) and erythrose-4-phosphate (E4P) (Fig. 2). These two com- acid pathway of E. coli are DHQ synthase and shikimate kinase (Dell
pounds must first be generated from metabolism of glucose, or and Frost, 1993) (Fig. 2). Shikimate kinase (aroL and aroK) is responsible
other carbohydrate. PEP is produced through the glycolysis pathway for converting shikimic acid to shikimate-3-phosphate (Fig. 2).
(Fig. 2) whereas the completely independent pentose phosphate 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
Modified E.coli strains with shikimic acid (SA) yield and titer.
Table 3
Strain Modification SA yield SA Effect of carbon sources on the production of 3-dehydroshikimic acid by recombinant
(mol/mol) titer (g/L) E. coli strains (Li et al., 1999).
SP1.1/pKD15.071B Over expression of ppsA 0.23 66
Strain KL3/pKL4.124A KL3/pKL4.79B (tktA−)
(Chandran et al., 2003)
(tktA over expression)
SP1.1pts/pSC6.090B PTS−/glf+/tktA over 0.27 71
(Chandran et al., 2003) expression Carbon source DHS titer DHS yield Carbon DHS titer DHS yield
SP1.1/pKD12.138 Over expression of tktA 0.18 52 (g/L) (mol/mol) source (g/L) (mol/mol)
(Knop et al., 2001) Glucose 46.0 0.28 Glucose 36.4 0.22
SP1.1/pKD12.112 Insertion of aroB into the 0.15 27.2 Xylose 43.1 0.33 Xylose 41.7 0.32
(Draths et al., 1999) serA locus of E.coli and Mixa 64.0 0.41 Mixa 53.0 0.36
disruption of aroL and aroK a
Molar ratio of glucose/xylose/arabinose: 3:3:2.

Please cite this article as: Ghosh S, et al, Production of shikimic acid, Biotechnol Adv (2012), doi:10.1016/j.biotechadv.2012.03.001
4 S. Ghosh et al. / Biotechnology Advances xxx (2012) xxx–xxx

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 in-
hibits shikimate dehydrogenase (Dell and Frost, 1993).
An alternative microbial route to shikimic acid is through bio-
transformation 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 specifically engineered for overprodu-
cing 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; Krämer 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; Krämer et al., 2003). All these are based on
genetic modifications to alter the central carbon metabolism and
the shikimic acid pathway (Table 1).
The shikimic acid pathway requires PEP and E4P (Fig. 2). The sup-
ply 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 modification 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 facilita-
tors (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 expo-
nential growth on glucose to a final 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 engi-
neered E. coli strains were low partly due to simultaneous production
of quinic acid (Knop et al., 2001). Production of quinic acid during bio-
synthesis 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 afore-
mentioned high titers.
Shikimic acid can be produced readily from DHS (Fig. 2). There-
fore, some metabolic engineering studies have focussed on overpro-
duction 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). Differ-
ent 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).

Please cite this article as: Ghosh S, et al, Production of shikimic acid, Biotechnol Adv (2012), doi:10.1016/j.biotechadv.2012.03.001
 S. Ghosh et al. / Biotechnology Advances xxx (2012) xxx–xxx
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 cel-
lular 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 amplified
expression of a mutant DAHP synthase, which is insensitive to
feedback inhibition by aromatic amino acids, has been used to en-
hance 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 modified or other-
wise 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) deficient
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 pro-
ducing shikimic acid has been reviewed by Krämer et al. (2003).
2.3. Chemical synthesis
Chemical synthesis of shikimic acid was first 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 efficiency of Diels-Alder synthesis could be improved
(Fig. 4) to raise the yield to 29% (Koreeda and Ciufolini, 1982). A
more efficient 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 elim-
ination 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
Fig. 6. Synthesis of oseltamivir from shikimic acid.
Please cite this article as: Ghosh S, et al, Production of shikimic acid, Biotechnol Adv (2012), doi:10.1016/j.biotechadv.2012.03.001
5
have been described (Kancharla et al., 2009; Sánchez-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 (Tamiflu®) 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 con-
verted into oseltamivir via a five 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 de-
hydrogenase. 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 qui-
nate 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 algi-
nate 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 first step of
this scheme involves an oxidative fermentation with G. oxydans to es-
sentially 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 dehy-
drogenase (SKDH) (Fig. 7) purified from cells of G. oxydans. The en-
zymatic biotransformation medium also contained glucose and the
enzyme glucose dehydrogenase to continuously regenerate NADPH
(Fig. 7).
6 S. Ghosh et al. / Biotechnology Advances xxx (2012) xxx–xxx
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 anti-
viral drug oseltamivir. Extraction from Chinese star anise is the main
source for shikimic acid, but fermentation processes based on the re-
combinant 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 com-
mercially 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.
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