establishes RifI as an in vivo aminoshikimate dehydrogenase.
The synthesis of aminoshikimic acid when expression of RifI
is increased also adds to questions relating to why A.
mediterranei possesses the ability to synthesize this mol-
ecule.7
As an alternative route to aminoshikimic acid, we elabo-
rated a synthesis that did not employ A. mediterranei
constructs and that minimized the number of A. mediterranei
genes requiring heterologous expression. This was ac-
complished using Bacillus pumilus ATCC 21143 to synthe-
size kanosamine from glucose.9 Derivatives of E. coli SP1.1
were then employed to synthesize aminoshikimic acid from
kanosamine.
Synthesis of kanosamine required careful attention to the
purity of the B. pumilus strain employed as well as culture
conditions. A multistep strain purification procedure was
utilized9,10 which included evaluation for catalase activity,
starch hydrolysis, catabolism of citric acid and various
carbohydrates, growth under anaerobic conditions, growth
in the presence of elevated NaCl concentrations, and a
Voges-Proskauer test for acetoin formation. The purified
B. pumilus ATCC 21143 synthesized 1-2 g/L of kanosamine
using culture conditions reported in the literature.9 Culture
conditions were subsequently modified to improve the
concentrations of kanosamine synthesized by B. pumilus
ATCC 21143. Employing glucose as the carbon source,
soybean meal or peanut meal as the nitrogen source, and
fermentor-controlled conditions, B. pumilus ATCC 21143
synthesized 25 g/L of kanosamine in 28% yield from glucose
(Figure 1).
in E. coli SP1.1, which prevented phosphorylation of
aminoshikimic acid. The corresponding absence of shikimic
acid phosphorylation and de novo biosynthesis of aromatic
amino acids required the addition of L-phenylalanine, L-
tyrosine, and L-tryptophan to all cultures of E. coli SP1.1.
These aromatic amino acid supplements also feedback
inhibited the native isozymes of DAHP synthase.11b This
inhibition minimized unwanted biosynthesis of shikimic acid
from glucose.
Plasmid pJG5.166A carried aroE and tktA loci encoding
shikimate dehydrogenase and transketolase, respectively,
from E. coli along with the rifH locus from A. mediterranei.
The resulting amplified expression of aroE-encoded shiki-
mate dehydrogenase was intended to prevent unwanted
synthesis of aminoDHS (Scheme 1) as a byproduct. Ampli-
fied expression of tktA was designed to increase the rate of
transketolase-catalyzed ketol transfer from 3-amino-3-deoxy-
D-fructose 6-phosphate (aminoF6P, Scheme 1) to generate
iminoE4P. The A. mediterranei rifH insert encoding
aminoDAHP synthase was provided with an E. coli promoter
and ribosomal binding site.
For the conversion of kanosamine into aminoshikimic acid,
E. coli enzymes catalyzed the transport and phosphorylation
of kanosamine, isomerization of kanosamine 6-phosphate,
and fragmentation of aminoF6P to form iminoE4P. Con-
densation of phosphoenolpyruvate with iminoE4P catalyzed
by aminoDAHP synthase was the only A. mediterranei
enzyme activity expressed in E. coli. AminoDAHP was
converted into aminoshikimic acid via the sequential action
of the E. coli shikimate pathway enzymes 3-dehydroquinate
synthase, 3-dehydroquinate dehydratase, and shikimate de-
hydrogenase.
As a control, E. coli SP1.1/pJG5.166A was cultured in
the absence of added kanosamine under fermentor-controlled
conditions. The 2.1 g/L of shikimic acid (entry 2, Table 1)
synthesized by E. coli SP1.1/pJG5.166A over 48 h reflects
the small amount of native DAHP synthase activity that
escaped feedback inhibition by the aromatic amino acid
supplements added to the culture medium. Repetition of these
culture conditions, but with a total of 7.5 g/L of kanosamine
added in equal (2.5 g) increments at 18, 24, and 30 h, resulted
in the synthesis of 0.81 g/L of aminoshikimic acid in 16%
yield from kanosamine and in an overall 4% yield from
glucose (entry 3, Table 1) at 33 °C.
Phosphorylation of kanosamine could not be detected by
enzyme assay in cell-free lysate prepared from E. coli SP1.1/
pJG5.166A. As a consequence, plasmid pJG6.181B was
constructed with a Zymomonas mobilis glk insert12 encoding
glucokinase. This modification was introduced to explore
whether increased kanosamine kinase activity and an at-
tendant increase in the rate of kanosamine phosphorylation
might result in an increase in the concentration and yield of
synthesized aminoshikimic acid. The assayed specific activity
for phosphorylation of kanosamine to kanosamine 6-phos-
phate in E. coli SP1.1/pJG6.181B was 0.02 units/mg.
Cultivation of E. coli SP1.1/pJG6.181B for 48 h (Figure 2)
Figure 1. Synthesis of kanosamine from glucose by B. pumilus
ATCC 21143 under fermentor-controlled conditions.
E. coli SP1.1, which had been previously prepared in our
laboratory for synthesis of shikimic acid,11 was chosen as
the host strain for the synthesis of aminoshikimic acid from
kanosamine. Both isozymes of shikimate kinase are inactive
(11) (a) Draths, K. M.; Knop, D. R.; Frost, J. W. J. Am. Chem. Soc.
1999, 121, 1603. (b) Knop, D. R.; Draths, K. M.; Chandran, S. S.; Barker,
J. L.; von Daeniken, R.; Weber, W.; Frost, J. W. J. Am. Chem. Soc. 2001,
123, 10173. (c) Chandran, S. S.; Yi, J.; Draths, K. M.; von Daeniken, R.;
Weber, W.; Frost, J. W. Biotechnol. Prog. 2003, 19, 808.
(12) Parker, C.; Barnell, W. O.; Snoep, J. L.; Ingram, L.; Conway, T.
O. Mol. Microbiol. 1995, 15, 795.
Org. Lett., Vol. 6, No. 10, 2004
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Guo, Jiantao
