Synthesis of vanillin from glucose [2]

Full text of DOI:10.1021/ja9817747

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

10546 J. Am. Chem. Soc., Vol. 120, No. 40, 1998
Communications to the Editor
Use of an intact microbe to reduce vanillic acid will be essential
for future large-scale vanillin synthesis. Vanillic acid synthesized
by one microbe from glucose could be reduced to vanillin by a
second, different microbe. Conversion of glucose into vanillin
using a single vanillate-synthesizing microbe expressing aryl
aldehyde dehydrogenase may also be possible. Irrespective of
the strategy employed, improved protocatechuic acid methylation
will be essential. The lack of significantly improved protocat-
echuic acid methylation with increased catechol-O-methyltrans-
ferase activity and the improvement in methylation observed with
L-methionine supplementation suggest that cosubstrate S-adeno-
6
sylmethionine availability and/or feedback inhibition may be
limiting in vivo methyltransferase activity. Improving regiose-
lectivity for protocatechuic acid meta-oxygen methylation using
4
a different isozyme of widely distributed catechol-O-methyl-
transferase would also be advantageous.
Biocatalytic synthesis of vanillin from glucose has a number
of advantages relative to other biocatalytic vanillin syntheses
(Scheme 1). Coniferol, formed during phenylpropanoid biosyn-
thesis, is converted into coniferin by a glucosyltransferase in V.
Figure 1. Fermentor culture of KL7/pKL5.97A supplemented with
L-methionine.
1a
planifolia. Coniferin is then transformed into glucovanillin
1a
which is finally hydrolyzed by a â-glucosidase. Synthesis of
vanillin via 3-dehydroshikimic, protocatechuic, and vanillic acids
circumvents phenylpropanoid biosynthesis and glucosylation/
deglucosylation reactions. This substantially reduces the number
of enzymes required to synthesize vanillin. Ferulic acid (Scheme
Table 1. Products Formed after 48 h under Fed-Batch Fermentor
Conditions as a Function of Catechol-O-methyltransferase Activity
and L-Methionine Supplementation
KL7/pKL5.26Aa
KL7/pKL5.97Ab
1
) can be microbially converted into vanillin. However, these
c
L-methionine
-
+
-
+
2
routes produce vanillin titers below 1 g/L. Cultured plant tissue
in medium supplemented with ferulic acid has also been used to
d
COMT
vanillic acid
0.0060
2.5
0.0055
0.012
0.010
e
4.9
1.3
7.1
1.0
3.0
0.6
12.9
1.0
5.0
1.2
10.5
1.8
e
produce vanillin although this conversion is slow and scale-up is
isovanillic acid
protocatechuic acid
-dehydroshikimic acid
0.4
9.7
0.9
e
2d,e
relatively difficult.
A problem with all vanillin syntheses from
e
3
ferulic acid is the absence of low-cost, commercial production
of this phenylpropanoid.
a
aroFFBR
tacCOMTserA. b aroFFBR tacCOMTPtacCOMTserA. c 0.4 g/L
P
d
P
added every 6 h beginning at 12 h. Specific activity, µmol/min/mg.
g/L.
Biocatalytic synthesis of vanillin from glucose, although a
longer-term commercial prospect, also has advantages relative
to synthetic vanillin manufacture1 (Scheme 1). Phenol and
e
c
7
guaiacol are toxic and are derived from carcinogenic benzene.
isovanillic acid (Table 1) conforms to the reported selectivity of
catechol-O-methyltransferase toward meta-hydroxyl group meth-
ylation.4
Nontoxic 3-dehydroshikimic, protocatechuic, and vanillic acids
are derived from innocuous glucose. Corrosive H O used for
2 2
5
the oxidation of phenol into catechol requires special handling
Aryl aldehyde dehydrogenase in N. crassa mycelial extract
7
precautions while biocatalytically synthesized vanillin derives
was purified from an unwanted dehydrogenase which reduced
vanillin to vanillyl alcohol. Vanillic, protocatechuic, and iso-
vanillic acids were extracted into EtOAc after acidification of
the fermentor broth. A subsequent reprecipitation step increased
the vanillic acid/protocatechuic acid ratio from 1:2 to 2.5:1 (mol/
its oxygen atoms from the oxygen atoms of glucose. Dimethyl
7
sulfate, a carcinogen, has historically been used to methylate
catechol. Protocatechuic acid methylation employs S-adenosyl-
methionine generated and consumed intracellularly. Finally,
synthetic vanillin manufacture is based on the use of nonrenewable
petroleum, whereas glucose is derived from abundant, renewable
starch. This difference in feedstock utilization is important, given
projected fierce international competition as global petroleum
production diminishes.8
mol). The resulting aromatic mixture was incubated with glucose
+
6
-phosphate dehydrogenase (to recycle NADP ) and aryl aldehyde
+
dehydrogenase at 30 °C and pH 8.0 using 0.07 equiv of NADP
and 2 equiv of ATP relative to vanillic acid. Reduction of vanillic
acid to vanillin (Scheme 2) proceeded in 92% yield in 7 h.
Reduction of protocatechuic acid was slower with a 33% yield
of protocatechualdehyde obtained after 7 h. Vanillin was
Acknowledgment. Research was supported by the United States
Department of Agriculture and the National Science Foundation. Orion
Corporation provided the COMT gene.
2 2
extracted from the enzymatic reduction with CH Cl , leaving
protocatechualdehyde and protocatechuic acid in the aqueous
phase. Isovanillin at 10 mol % remained as the only contaminant.
Extraction of the fermentor broth, selective precipitation to remove
excess protocatechuic acid, aryl aldehyde dehydrogenase reduc-
Supporting Information Available: Enzyme isolation, fermentor
synthesis, and enzymatic reduction (7 pages, print/PDF). See any current
masthead page for ordering information and Web access instructions.
tion, and the final CH
2 2
Cl extraction led to a 66% overall yield
(mol/mol) for the conversion of vanillic acid into vanillin.
JA9817747
(
4) Guldberg, H. C.; Marsden, C. A. Pharmacol. ReV. 1975, 27, 135.
5) (a) Gross, G. G.; Bolkart, K. H.; Zenk, M. H. Biochem. Biophys. Res.
(
(6) Coward, J. K.; Slisz, E. P.; Wu, F. Y.-H. Biochemistry 1973, 12, 2291.
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