Synthesis of gallic acid and pyrogallol from glucose: Replacing natural product isolation with microbial catalysis [9]

Full text of DOI:10.1021/ja000853r

9042
J. Am. Chem. Soc. 2000, 122, 9042-9043
Scheme 1a
Synthesis of Gallic Acid and Pyrogallol from
Glucose: Replacing Natural Product Isolation with
Microbial Catalysis
†
†
,†,‡
Spiros Kambourakis, K. M. Draths, and J. W. Frost*
Departments of Chemistry and Chemical Engineering
Michigan State UniVersity, East Lansing, Michigan 48824
ReceiVed March 9, 2000
1
Among a spectrum of uses, gallic acid and pyrogallol are often
incorporated into chemical syntheses to provide the trihydrox-
ylated aromatic ring of biologically active molecules such as
the antibiotic trimethoprim 1, the muscle relaxant gallamine tri-
ethiodide 2, and the insecticide bendiocarb 3. The availability of
a
Enzymes: (a) DAHP synthase (aroF); (b) DHQ synthase (aroB);
(c) DHQ dehydratase (aroD); (d) DHS dehydratase (aroZ); (e) p-hydroxy-
benzoate hydroxylase (pobA*); (f) PCA decarboxylase (aroY); (g) DHS
dehydrogenase. Abbreviations: E4P, D-erythrose 4-phosphate; PEP,
phosphoenolpyruvic acid; DAH(P), 3-deoxy-D-arabino-heptulosonic acid
(7-phosphate); DHQ, 3-dehydroquinic acid; DHS, 3-dehydroshikimic acid;
gallic acid is restricted by its current isolation from insect
carapices (gall nuts) harvested in China, and an isolate (tara
powder) derived from the ground seed pod of a tree found in
GA, gallic acid; PCA, protocatechuic acid.
1
pneumoniae and the encoding aroZ gene has been isolated.8
Mutagenesis of the pobA gene, which encodes p-hydroxybenzoate
hydroxylase in Pseudomonas aeruginosa, produced pobA*, which
encodes a mutant enzyme capable of hydroxylating PCA to form
Peru. Thermal decarboxylation of gallic acid in copper autoclaves
1
2
affords pyrogallol. As part of a larger effort to supplant isolation
of natural products from scarce natural sources, gallic acid and
pyrogallol were targeted for microbe-catalyzed synthesis from
abundant glucose. In lieu of an elaborated biosynthetic pathway
leading to gallic acid, a pathway had to be created. The result is
Escherichia coli KL7/pSK6.161, which synthesizes 20 g/L of
gallic acid in 12% yield from glucose. E. coli RB791serA::aroB/
pSK6.234 then converts gallic acid into pyrogallol in yields of
9
gallic acid. Expression of aroZ and pobA* in a DHS-synthesizing
microbe thus provided the basis for creating a gallic acid
biosynthetic pathway in E. coli KL7/pSK6.161.
E. coli KL7 carried a mutation in its aroE gene and an
aroBaroZ insert. Plasmid pSK6.161 contained pobA* and aroF
FBR
9
3-97%.
inserts. DHS was synthesized by KL7 due to the absence of aroE-
encoded shikimate dehydrogenase. This DHS was dehydrated
by the K. pneumoniae aroZ dehydratase to PCA (Scheme 1).
Hydroxylation of the PCA by the P. aeruginosa pobA* hydrox-
ylase then afforded the desired gallic acid (Scheme 1). Since
the amount of DHS synthesized is significantly reduced by feed-
3
Biosynthesis of gallic acid has been narrowed to two possible
routes. Oxidation (Scheme 1) of 3-dehydroshikimic acid (DHS)
to a diketo intermediate followed by spontaneous aromatization
might lead to gallic acid. Alternatively, gallic acid may result
from the dehydration of DHS followed by hydroxylation (Scheme
10
1) of the intermediate protocatechuic acid (PCA). Gallic acid has
back inhibition of DAHP synthase, a feedback-insensitive mutant
4
11
FBR
been detected in cultures of Phycomyces blakesleeanus, Pseudo-
monas fluorescens, Entorobacter cloacae, Aspergillus terreus,
and recombinant E. coli. However, neither DHS dehydrogenase-
catalyzed oxidation of DHS (Scheme 1) nor PCA hydroxylase-
catalyzed hydroxylation of PCA (Scheme 1) has been detected
in these or any other microbes.
enzyme encoded by aroF
was used. With the aroBaroZ
5
5
6
insert, E. coli KL7 was equipped with two copies of aroB to
increase DHQ synthase activity and eliminate DAH accumulation
(Scheme 1).12
Cultivation of E. coli KL7/pSK6.161 for 48 h in a fermentor
under glucose-rich, nitrogen-rich culture conditions led (Figure
7
We therefore explored recruitment of enzyme activities that,
although not associated with gallic acid biosynthesis in their native
microbial hosts, are capable of catalyzing Scheme 1 reactions.
DHS dehydratase (Scheme 1) has been detected in Klebsiella
1
) to the formation of gallic acid (20 g/L), DAH (8.9 g/L), DHS
(11 g/L), PCA (0.90 g/L), and glutamic acid (14 g/L). Gallic acid
and PCA were separated from the DAH and glutamic acid upon
extraction from cell-free culture supernatants using EtOAc. After
concentration of the charcoal-decolorized organic layer, addition
†
Department of Chemistry.
‡
Department of Chemical Engineering.
(
1) Leston, G. In Kirk-Othmer Encyclopedia of Chemical Technology;
(8) (a) Draths, K. M.; Frost, J. W. J. Am. Chem. Soc. 1994, 116, 399. (b)
Draths, K. M.; Frost, J. W. In Benign by Design; Anastas, P. T., Farris, C. S.,
Eds.; ACS Symp. Ser. No. 577; American Chemical Society: Washington,
DC, 1994; Chapter 3, p 32. (c) Draths, K. M.; Frost, J. W. J. Am. Chem. Soc.
1995, 117, 2395.
(9) (a) Entsch, B.; Palfey, B. A.; Ballou, D. P.; Massey, V. J. Biol. Chem.
1991, 266, 17341. (b) Eschrich, K.; van der Bolt, F. J. T.; de Kok, A.; van
Berkel, W. J. H. Eur. J. Biochem. 1993, 216, 137.
(10) Ogino, T.; Garner, C.; Markley, J. L.; Herrmann, K. M. Proc. Natl.
Acad. Sci. U.S.A. 1982, 79, 5828.
Kroschwitz, J. I., Howe-Grant, M., Eds.; Wiley: New York, 1996; Vol. 19,
p 778.
(
2) (a) Draths, K. M.; Knop, D. R.; Frost, J. W. J. Am. Chem. Soc. 1999,
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21, 1603. (b) Li, K.; Frost, J. W. J. Am. Chem. Soc. 1998, 120, 10545.
(
3) Werner, I.; Bacher, A.; Eisenreich, W. J. Biol. Chem. 1997, 272, 25474.
4) (a) Haslam, E.; Haworth, R. D.; Knowles, P. F. J. Chem. Soc. 1961,
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5) Korth, H. Arch. Mikrobiol. 1973, 89, 67.
6) Kawakubo, J.; Nishira, H.; Aoki, K.; Shinke, R. Biosci. Biotech.
Biochem. 1993, 57, 1360.
(11) Weaver, L. M.; Herrmann, K. M. J. Bacteriol. 1990, 172, 6581.
(12) Snell, K. D.; Draths, K. M.; Frost, J. W. J. Am. Chem. Soc. 1996,
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7) Li, K.; Mikola, M. R.; Draths, K. M.; Worden, R. M.; Frost, J. W.
Biotechnol. Bioeng. 1999, 64, 61.
1
0.1021/ja000853r CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/01/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 37, 2000 9043
Figure 2. Anaerobic decarboxylations catalyzed by E. coli RB791serA::
aroB/pSK6.234: circle, gallic acid (GA); square, pyrogallol (PGL);
triangle, PCA; and tilted square, catechol.
Figure 1. Fed-batch, aerobic cultivation of E. coli KL7/pSK6.161: 2,
cell growth; open bar, gallic acid (GA); cross-hatched bar, PCA; and
horizontally ruled bar, DHS.
After RB791serA::aroB/pSK6.234 completed its growth on
glucose in a fermentor, gallic acid and PCA were added to give
concentrations of 115 and 4.5 mM, respectively. Glucose addition
and aeration were then terminated, and the fermentor was
of petroleum ether led to precipitation of gallic acid as a white
powder free of PCA contamination.
DAH accumulation during gallic acid synthesis was unexpected
given that this metabolite has not been observed in E. coli
strains such as KL7, which possess two copies of aroB.12 We
subsequently discovered that PCA competitively inhibits aroB-
encoded 3-dehydroquinate synthase.13 The absence of acetic acid
accumulation and the formation of substantial amounts of glutamic
acid during gallic acid synthesis were also unexpected. Typically,
acetic acid is formed during the later stages of E. coli cultures
when glucose is used as the carbon source.14 The initial increase
and subsequent decrease in PCA concentrations (Figure 1) may
indicate that KL7/pSK6.161 can recapture PCA initially exported
into the culture medium.
2
sparged with a continuous flow of N . Within 14 h (Figure 2),
gallic acid and PCA were converted into a solution containing
112 mM pyrogallol (97%) and 4 mM catechol. Repetition of the
decarboxylation with culture supernatant produced by gallate-
synthesizing KL7/pSK6.161 resulted in a 93% yield of pyrogallol.
The concentrations of DHS, DAH, and glutamic acid remained
unchanged. Pyrogallol was isolated from cell-free culture super-
natants by extraction with EtOAc. Removal of solvent from the
charcoal-decolorized organic layer followed by heating under
vacuum to remove catechol contamination afforded pyrogallol
as a white solid.
As with the synthesis of gallic acid, we attempted to synthesize
pyrogallol directly from glucose. Pyrogallol is typically generated
as a catabolic intermediate by microbes using either gallic acid
or tannic acid as a sole source of carbon during growth.15 The
most extensively characterized nonoxidative decarboxylase ca-
pable of converting gallic acid into pyrogallol has been isolated
from Pantoea agglomerans T71.16 Our approach to conversion
of gallic acid into pyrogallol was based on the fortuitous discovery
Significant improvements in KL7/pSK6.161 remain to be
elaborated before the current 12% yield for synthesis of gallic
acid from glucose reaches the maximum theoretical8 43% yield.
Increasing the in vivo activities of AroB, AroZ, and PobA* along
with suppression of glutamic acid synthesis are obvious (albeit
nontrivial) avenues that warrant exploration in the future. While
the synthesis of gallic acid from glucose requires controlled
expression of multiple genes, microbe-catalyzed decarboxylation
of gallic acid benefits from being a much simpler biocatalytic
conversion. The high-yielding decarboxylation of gallic acid
catalyzed by RB791serA::aroB/pSK6.234 and the ease of purify-
ing product pyrogallol are already at the stage where this
c,17
8
that aroY-encoded PCA decarboxylase isolated from K. pneu-
moniae also catalyzes the decarboxylation of gallic acid to
pyrogallol. PCA decarboxylase has previously been used in
microbial syntheses of catechol and adipic acid from glucose.8
Synthesis of pyrogallol from glucose was attempted using E.
coli KL7/pSK6.232. Plasmid pSK6.232 carried an aroY insert in
1
biocatalytic route is an attractive alternative to currently employed
chemical decarboxylation of gallic acid.
FBR
addition to pobA* and aroF . KL7/pSK6.232 cultured under
Microbe-catalyzed syntheses of natural products are typically
based on pathways where the intermediates, enzymes, and en-
fermentor conditions failed to grow beyond 18 h of cultivation.
Neither gallic acid nor pyrogallol formation could be detected.
Only formation of catechol was observed suggesting that the rate
of in vivo decarboxylation of PCA was significantly more rapid
than PobA*-catalyzed hydroxylation of PCA. Catechol formation
and its associated toxicity8 toward microbes likely explain the
early cessation of growth.
2
a
coding genes have been identified and characterized. Microbe-
catalyzed synthesis of gallic acid, by contrast, is based on a hypo-
thetical biosynthetic pathway. By exploiting modified enzyme
function (PobA*) and the diversity of enzyme function (AroZ
and AroY), gallic acid and pyrogallol have been successfully
c
2
b
synthesized from glucose. In the process, another example is
provided for how chemistry can reach beyond known biosynthetic
pathways to create new catalytic function appropriate for natural
product synthesis.
Synthesis of pyrogallol subsequently switched to a strategy
where gallic acid solutions containing PCA were added to the
culture medium after growth of the E. coli catalyst was complete.
The microbe, E. coliRB791serA::aroB/pSK6.234, overexpressed
only plasmid-localized, aroY-encoded decarboxylase activity. This
Acknowledgment. Research was supported by the National Science
Foundation and the Environmental Protection Agency. Professor David
P. Ballou (University of Michigan) kindly provided pobA*.
strategy benefited from catechol’s attenuated toxicity toward
pregrown as opposed to actively growing E. coli.8c However, it
was unclear whether AroY decarboxylase in RB791serA::aroB/
pSK6.234 would have access to the added gallic acid.
Supporting Information Available: Genetic manipulations, fermen-
tation, and purification methods (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
(
13) Chandran, S.; Frost, J. W. Unpublished results.
(
14) Konstantinov, K. B.; Nishio, N.; Seki, T.; Yoshida, T. J. Ferm. Bioeng.
1
991, 71, 350.
(
15) Armstrong, S. M.; Patel, T. R. J. Basic Microbiol. 1994, 34, 123.
16) Zeida, M.; Wieser, M.; Yoshida, T.; Sugio, T.; Nagasawa, T. Appl.
JA000853R
(
EnViron. Micriobiol. 1998, 64, 4743.
(17) F o¨ rberg, C.; Eliaeson, T.; H a¨ ggstr o¨ m, L. J. Biotechnol. 1988, 7, 319.