Chapter 11
3-Dehydroshikimic Acid: A Building Block for Chemical Synthesis from Renewable Feedstocks 1
1
1
1,2
Κ. M . Draths , Spiros Kambourakis , Kai Li , and J. W. Frost * 1
2
Departments of C h e m i s t r y and Chemical Engineering, Michigan State University, East Lansing, MI 48824
3-Dehydroshikimic acid is an ideal building block for chemical synthesis from feedstocks derived from renewable resources. Microbial catalysts that synthesize 3dehydroshikimic acid from starch-derived glucose or glucose:xylose:arabinose mixtures that mimic corn fiber hydrolysate achieve high titers of product in high yield. Catalytic conditions ranging from microbial catalysis to traditional chemical catalysis have been developed that transform 3-dehydroshikimic acid into protocatechuic acid, vanillin, catechol, adipic acid, gallic acid, and pyrogallol.
Chemical synthesis has traditionally relied on a building block strategy where relatively few chemicals are identified as primary building blocks that are subsequently transformed into secondary building blocks and other materials of industrial interest. Of the top 50 large volume chemicals produced annually in the United States, the 28 organic compounds on the list are derived from only 8
© 2001 American Chemical Society
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primary building blocks (/). One consequence of the building block approach is that the vast majority of industrial chemicals are synthesized from a limited pool of feedstocks. Of the approximately 170 compounds produced annually in the U.S. in volumes exceeding 4.5 million kilograms, 98% are derived from petroleum and natural gas (/). Given that both these feedstocks are nonrenewable resources, the ability to synthesize the bulk of industrial chemicals is not limitless. Successful transfer of some chemical synthesis from nonrenewable feedstocks to those derived from renewable resources requires identification of new building blocks to emulate this strategy. Ideal building blocks must be easily synthesized in high yield from inexpensive, readily available renewable feedstocks and must be converted through various catalytic transformations into a variety of materials of industrial interest. 3-Dehydroshikimic acid (DHS) is presented as a useful chemical building block. Microbial catalysts have been created that convert glucose or mixtures of glucose, xylose, and arabinose into DHS in high yield. Glucose is currently obtained from starch, although cellulose may ultimately be a less expensive source, whereas glucose, xylose, and arabinose mixtures model the feedstock that may eventually be derived from inexpensive corn fiber. Optimization of microbial catalysts that utilize inexpensive feedstocks that might become available in the future is also possible. Using combinations of microbial catalysis and traditional chemical catalysis, DHS is transformed into a spectrum of chemicals of industrial interest (Figure 1).
Microbe-Catalyzed Synthesis of DHS 3-Dehydroshikimic acid is a hydroaromatic intermediate of the aromatic amino acid biosynthetic pathway (Figure 2) (2). Existing in microorganisms and plants, this pathway gives rise to the aromatic amino acids as well as several aromatic vitamins. As an intermediate of biosynthesis, quantities of DHS found in natural sources are extremely limited. Development of a process for largescale DHS synthesis may lead to interest in DHS in its own right. For example, DHS was recently found to be a potent antioxidant (5). Used in commercial preparation of foods, materials, medicinals, and cosmetics, antioxidants are designed to interfere with oxidative decomposition processes. When DHS was tested for its ability to inhibit peroxide and hydroperoxide formation in lard, DHS provided complete protection against oxidation for a period of 28 days and was equal to or superior to the antioxidant activities of propyl gallate, gallic acid, and TBHQ (5), all of which are used as commercial antioxidants. Furthermore, as a small chiral molecule possessing several stereocenters and useful functionality, DHS might be an attractive synthon in the pharmaceutical arena if it were available in sufficient quantities.
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C0 H 1
CO,H
2
a or b
Π
e
OH
OH
DHS
PCA
OH catechol
e
Ι
CIS, CIS-
muconic acid
fl
OH pyrogallol
Figure 1. DHS as a chemical building block. Boxed structures are of current industrial value. Catalysts: (a) DHS dehydratase (aroZ); (b) reflux; (c) catechol-O-methyltransferase (comt); (d) aryl-aldehyde dehydrogenase; (e) PCA decarboxylase (aroY); (f) catechol 1,2-dioxygenase (catA); (g) Pt/C, Η2; (h) p-hydroxybenzoate hydroxylase (pobA*); (i)Cu(OAc) 2, HO Ac. tktA t k t B
HO
»
Η °3 2
transketolase
Ρ Ο
Ο
νΛ^Η QU
E4P
CO H
p aroG aro
aroH^
HO, C0 H
2
2
O"^
aroB
DAHP / V l O H synthase RO OH
H 0 PO^ PEP 2
HO_C0 H
DAHP DAH
3
C0 H
P0 H H 3
DHÇf synthase
O^V\)H OH DHQ
2
C0 H
2
2
aroD^ DHQ v. . ^ Q^\m dehydratase OH DHS
>
.shikimate dehydrogenase
—O H
V. ^ O — H OH shikimic acid , , k
pl
v r
L _ T r
P aromatic vitamins
Figure 2. The aromatic amino acid biosyntheticpathway.
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Synthesis of DHS from Glucose Creation of microbial catalysts for high-yielding synthesis of DHS from glucose began with Escherichia coli KL3, which possesses a mutation in the shikimate dehydrogenase-encoding aroE gene (4). KL3 is unable to convert DHS into shikimic acid and exports DHS into the growth medium. KL3 has a second copy of the 3-dehydroquinate (DHQ) synthase-encoding aroB gene inserted into its 3-phosphoglycerate dehydrogenase-encoding serA gene. Insertion of a second copy of aroB was needed for compete conversion of DAHP into DHQ (5). By inserting aroB into serA, KL3 was rendered incapable of synthesizing the serine it would require for growth in minimal medium. KL3 was transformed with pKL4.79B, a plasmid designed to study the impact of DAHP synthase specific activity on DHS synthesis (4). As the first committed enzyme of aromatic biosynthesis, DAHP synthase regulates carbon flow into the pathway. Since feedback inhibition of DAHP synthase by the aromatic amino acids is known to be the most significant mode of regulation (f5), an aroF locus encoding a feedback insensitive isozyme of DAHP synthase (7) was inserted into pKL4.79A. Expression of a r o F behind the regulatable P promoter and inclusion of the gene encoding the regulatory L a d protein (lacN) on pKL4.79B allowed expression of DAHP synthase to be titrated by addition of IPTG to the culture medium. Maintenance of pKL4.79B in KL3 was ensured by including a functional copy of serA in the plasmid. Growth of KL3/pKL4.79B in culture medium lacking serine forces the host strain to maintain the plasmid. Fed-batch fermentations of KL3/pKL4.79B were performed in a 2-L benchtop fermentor (4). Various concentrations of IPTG were added at 6 h intervals. As seen in Table I, DAHP synthase specific activity increased as a function of IPTG addition. Incremental increases in IPTG concentrations led to corresponding improvements in DHS titers (entries 1-4, Table I), reaching a maximum of 52 g/L of DHS (entry 4, Table I) and a 20 % conversion (mol DHS/mol glucose). However, when the IPTG concentration was increased further, a pronounced decrease in DHS titer was observed (entry 5, Table I), implying that there is a level of DAHP synthase activity above which further increases have a negative impact on DHS-synthesizing ability. Further improvement in DHS titers were realized by addition of a copy of the gene encoding transketolase (tktA) to pKL4.79B to form pKL4.124A (8, 9). Transketolase is one of two cellular enzymes responsible for formation of Derythrose 4-phosphate (E4P). An unstable molecule which has been shown to dimerize and polymerize, E4P has never been detected in a living cell (10). Fermentation of KL3/pKL4.124A at the IPTG concentration which had previously proven optimal yielded 66.3 g/L of DHS (entry 6, Table I), representing a 28% conversion (mol DHS/mol glucose) (4). ¥BR
F B R
tac
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Table I. Product titers, yields, and DAHP synthase activities for KL3/pKL4.79B and KL3/pKL4.124A. rf
Entry \
a
2
