J. Am. Chem. Soc. 1998, 120, 10545-10546
10545
Scheme 1a
Synthesis of Vanillin from Glucose
Kai Li and J. W. Frost*
Department of Chemistry
Michigan State UniVersity
East Lansing, Michigan 48824
ReceiVed May 21, 1998
Condensation of glyoxylic acid with benzene-derived guaiacol
(
Scheme 1) is currently the dominant route for vanillin manu-
1
facture. Natural vanillin is produced from glucovanillin (Scheme
1
) when the beans of the orchid Vanilla planifolia are submitted
1a
to a multistep curing process. Because of the extreme care that
must be exercised during vine cultivation, bean harvesting, and
hand pollination of flowers, natural vanillin can supply only 2 ×
4
7
1b
10 kg/yr of the world’s 1.2 × 10 kg/yr demand for vanillin.
This has resulted in the substitution of synthetic vanillin for natural
vanilla in most flavoring applications. Limited vanilla bean
supplies have also led to extensive research into the use of plant
tissue culture and microbes to convert ferulic acid (Scheme 1)
into vanillin suitable for labeling as a natural or nature-equivalent
flavoring.2 A synthesis of vanillin from glucose (Scheme 1) has
now been elaborated. Glucose is converted into vanillic acid by
a recombinant Escherichia coli biocatalyst under fed-batch
fermentor conditions. Reduction of vanillic acid to vanillin is
catalyzed by aryl aldehyde dehydrogenase isolated from Neuro-
spora crassa. This synthesis qualifies both as a route to natural
vanillin and as a first step toward large-scale, environmentally
benign manufacture of vanillin using biocatalysis.
a
Key: (a) KL7/pKL5.26A or KL7/pKL5.97A. (b) N. crassa aryl
aldehyde dehydrogenase. (c) Microbial catabolism. (d) HCO H, HCO H.
(e) Me SO . (f) (i) HCOCO H, (ii) O , (iii) H . (g) UDP-glucose:coniferyl
2 4 2 2
alcohol glucosyltransferase. (h) Unidentified enzymes. (i) â-glucosidase.
3
2
+
Scheme 2a
Vanillate-synthesizing E. coli KL7 biocatalysts carried a
mutated aroE locus and an aroBaroZ cassette inserted into the
serA locus. The lack of aroE-encoded shikimate dehydrogenase
resulted in the synthesis of 3-dehydroshikimic acid which was
converted into protocatechuic acid by genome-localized, aroZ-
encoded 3-dehydroshikimate dehydratase. Plasmid-localized
P
tacCOMT encoded catechol-O-methyltransferase for conversion
of protocatechuic acid into vanillic acid (Scheme 2). Plasmid
pKL5.97A carried two PtacCOMT loci, while only a single
P
tacCOMT locus was present in pKL5.26A. These plasmids also
FBR
FBR
carried an aroF and a serA insert. The aroF insert encodes
a 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase
isozyme insensitive to feedback inhibition which increased carbon
flow into the common pathway. Due to a mutation in the genomic
serA locus required for L-serine biosynthesis, growth in minimal
salts medium and plasmid maintenance followed from expression
of plasmid-localized serA. The two genomic copies of aroB
a
Key: (a) 3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase
FBR
(
aroF ). (b) 3-Dehydroquinate synthase (aroB). (c) 3-Dehydroquinate
dehydratase (aroD). (d) 3-Dehydroshikimate dehydratase (aroZ). (e)
Catechol-O-methyltransferase (COMT). (f) Aryl aldehyde dehydrogenase.
(g) D-glucose 6-phosphate dehydrogenase; D-glucose 6-phosphate; SAM,
S-adenosylmethionine; SAH, S-adenosylhomocysteine.
increased 3-dehydroquinate synthase (Scheme 2) activity to the
point where this enzyme no longer impeded carbon flow.3
KL7/pKL5.26A and KL7/pKL5.97A were cultured for 48 h
under fed-batch fermentor conditions at 37 °C, pH 7.0, and
dissolved oxygen at 20% of saturation. Extracellular accumula-
tion (Figure 1) of vanillic, isovanillic, protocatechuic, and
3-dehydroshikimic acids began in mid log phase of microbial
growth. 3-Dehydroshikimic acid usually constituted 5-10 mol
*
Author to whom correspondence should be addressed. Phone: 517-355-
715, ext 115. FAX: 517-432-3873. E-mail: frostjw@argus.cem.msu.edu.
1) (a) Ranadive, A. S. In Spices, Herbs, and Edible Fungi; Charalambous,
G., Ed.; Elsevier: Amsterdam, 1994; p 517. (b) Clark, G. S. Perfum. FlaVor.
990, 15, 45. (c) Esposito, L.; Formanek, K.; Kientz, G.; Mauger, F.;
9
(
%
of the total product mixture, indicating that the rates for its
1
Maureaux, V.; Robert, G.; Truchet, F. In Kirk-Othmer Encyclopedia of
Chemical Technology, 4th ed.; Kroschwitz, J. I., Howe-Grant, M., Eds.;
Wiley: New York, 1997; Vol. 24, p 812.
biosynthesis and dehydration were nearly equal. However, the
molar dominance of protocatechuic acid (Figure 1, Table 1)
relative to vanillic acid pointed to inadequate catechol-O-
methyltransferase activity. Although increasing the specific
activity (Table 1) of catechol-O-methyltransferase in KL7/
pKL5.97A relative to KL7/pKL5.26A had little impact on the
concentrations (Table 1) of synthesized vanillic acid, supplemen-
tation with L-methionine nearly doubled the amount of vanillic
acid synthesized by both biocatalysts (Table 1). The 4-fold to
6-fold molar excess of vanillic acid synthesized relative to
(
2) (a) Falconnier, B.; Lapierre, C.; Lesage-Meessen, L.; Yonnet, G.;
Brunerie, P.; Colonna-Ceccaldi, B.; Corrieu, G.; Asther, M. J. Biotechnol.
994, 37, 123. (b) Lesage-Meessen, L.; Delattre, M.; Haon, M.; Thibault,
J.-F.; Ceccaldi, B. C.; Brunerie, P.; Asther, M. J. Biotechnol. 1996, 50, 107.
c) Lesage-Meessen, L.; Haon, M.; Delattre, M.; Thibault, J.-F.; Ceccaldi, B.
1
(
C.; Asther, M. Appl. Microbiol. Biotechnol. 1997, 47, 393. (d) Labuda, I. M.;
Goers, S. K.; Keon, K. A. U.S. Patent 5,279,950, 1994. (e) Westcott, R. J.;
Cheetham, P. S. J.; Barraclough, A. J. Phytochemistry 1994, 35, 135.
(
3) Snell, K. D.; Draths, K. M.; Frost, J. W. J. Am. Chem. Soc. 1996, 118,
5
605.
S0002-7863(98)01774-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/25/1998