J. Am. Chem. Soc. 1999, 121, 9461-9462
9461
Scheme 1
Utilizing Succinic Acid as a Glucose Adjunct in
Fed-Batch Fermentation: Is Butane a Feedstock
Option in Microbe-Catalyzed Synthesis?
Kai Li and J. W. Frost*
Department of Chemistry, Michigan State UniVersity
East Lansing, Michigan 48824
ReceiVed July 14, 1999
Although the carbohydrate phosphotransferase system1 supports
rapid bacterial growth, this transport mechanism imposes an upper
limit on the yields and concentrations of chemicals which can be
synthesized when microbes such as Escherichia coli employ
glucose as a carbon source. Phosphotransferase-catalyzed phos-
phoryl group transfer from phosphoenolpyruvic acid (PEP)
simultaneously drives glucose transport and glucose phosphory-
lation. Because byproduct pyruvic acid is not significantly
recycled to PEP in phosphotransferase-utilizing microbes, three
carbon atoms are lost from chemical synthesis for every six carbon
atoms transported into the cytoplasm.2 A possible means for
circumventing this limitation has been evaluated which exploits
butane-derived3 succinic acid (Scheme 1) as an adjunct to starch-
derived glucose. By overexpressing selected enzymes and evading
catabolite repression, succinic acid is apparently exploited as a
transportable precursor of PEP, resulting in the synthesis of
increased concentrations of 3-dehydroshikimic acid (DHS) by E.
coli. DHS is a potent antioxidant4 and the most advanced common
biosynthetic intermediate in the biocatalytic synthesis of adipic
acid,5 catechol,6 vanillin,7 shikimic acid,8 phenylalanine,9 tryp-
tophan,10 and indigo.11
The native tricarboxylic acid cycle was used to convert
transported glucose adjuncts into oxaloacetic acid (Scheme 1).
Plasmid-localized, pck-encoded12 PEP carboxykinase then con-
verted oxaloacetic acid into PEP. This increased level of PEP
generation was channeled into the common pathway of aro-
matic amino acid biosynthesis (Scheme 1) by plasmid-localized,
PtacaroFFBR-encoded13 3-deoxy-D-arabino-heptulosonic acid 7-phos-
phate (DAHP) synthase. Because D-erythrose 4-phosphate (E4P)
availability can limit DAHP synthase activity, the impact of
plasmid-localized tktA-encoded14 transketolase was examined.
Fed-batch fermentor cultivation of DHS-synthesizing constructs
relied on a dissolved O2 sensor to control addition of the adjunct/
glucose mixtures.15 This addition strategy prevents glucose
concentrations from reaching a level16 where catabolic repression
of adjunct transport17 occurs.
* Corresponding author. Phone: 517-355-9715 ext. 115. E-mail: frostjw@
argus.cem.msu.edu. Fax: 517-432-3873.
DHS (57 g/L) concentrations synthesized (Table 1) from
succinate/glucose (1:1, mol/mol) by KL3/pKL6.218A, which
overexpressed DAHP synthase, transketolase, and PEP carboxy-
kinase, were significantly higher than DHS (44 g/L) concentrations
synthesized from glucose. Another measure of the increased
carbon flow directed into aromatic amino acid biosynthesis is the
accumulation (Figure 1, Table 2) of 3-deoxy-D-arabino-heptu-
losonic acid (DAH), 3-dehydroquinic acid (DHQ), and gallic acid.
DAH results from enzymatic hydrolysis of DAHP, while gallic
acid is derived from DHS.18 Significantly higher concentrations
of DHQ were synthesized from succinate/glucose versus glucose
by KL3/pKL6.218A (Table 2). While no DAH accumulation was
observed (Table 2) during cultivation of KL3/pKL6.218A on
glucose, substantial DAH was produced by this construct from
the succinate/glucose mixture.
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C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M.,
Umbarger, H. E., Eds.; ASM Press: Washington, DC, 1996; pp 1149-1174.
(2) (a) Patnaik, R.; Liao, J. C. Appl. EnViron. Microbiol. 1994, 60, 3903.
(b) Patnaik, R.; Spitzer, R. G.; Liao, J. C. Biotechnol. Bioeng. 1995, 46, 361.
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The importance of PEP carboxykinase is evident (Table 1) from
the reduced concentrations of DHS (46 g/L) synthesized from
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10.1021/ja992477k CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/24/1999