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polymer exhibits a broad range of interesting biological
properties, including anticancer,11,12) antitumor,14) and
anticoagulant15) activities. 6-Deoxy-L-mannose (L-rham-
nose) plays a major role in the gel formation processes
of bacterial polysaccharide (gellan) and deacetylated
rhamsan gum in aqueous solution.16,17) 6-Deoxy-L-
altrose is a component of Gram-negative pathogen
lipopolysaccharides, and most likely plays a role in
antigens. 6-Deoxy-D-altrose, which was isolated from
the fruiting bodies of the edible mushroom Lactarius
lividatus,18) has been used for a long time in Chinese
folk medicine for its antitumor and antiviral actvities.19)
In addition, 6-deoxy-D-glucose is used to study glucose
transport in human erythrocytes infected with the
malaria parasite, Plasmodium falciparum.20) 6-Deoxy-
6-iodo-D-glucose is used as a tool compound to assess
glucose transport, which is of major clinical importance
because changes in the glucose transport process occur
in a number of different pathologies.21) Based on their
interesting properties, deoxy monosaccharides have
become compounds of considerable interest. Unfortu-
nately, however, their characteristics and functions have
not been investigated to any great extent because of
their rarity. For this reason, the development of a meth-
odology allowing for the mass production of deoxy
monosaccharides is strongly desired to allow for their
properties to be thoroughly evaluated.
Traditionally, rare sugars have been synthesized by
chemical reactions, but these processes generally
require many steps that occur with low specificity and
poor purity profiles, resulting in low yields of the
desired products. Furthermore, they often result in the
formation of hazardous waste products.22,23) Based on
these limitations, a biochemical method using enzy-
matic reactions represents a potentially environmentally
friendly procedure for the biosynthesis of monosaccha-
rides, especially rare sugars. D-Xylose isomerase, which
catalyzes the conversion of D-glucose to D-fructose, is
widely used with amylases to produce a high-fructose
corn syrup sweetener from starch,24) and there are
many different enzymes that can catalyze isomerization
processes other than D-xylose isomerase. To establish
effective procedures for the production of rare sugars,
we constructed an Izumoring strategy involving a com-
bination of several microbial enzymes. An Izumoring
strategy is a structural framework containing 34 six-car-
bon monosaccharides and their alditols, 16 five-carbon
monosaccharides and their alditols, and 9 four-carbon
monosaccharides and their alditols, linked through a
series of enzymatic reactions.1,25) Rare sugar biosynthe-
sis is catalyzed by (i) alditol dehydrogenases, which
oxidize alditols to ketoses, (ii) aldose reductases, which
oxidize alditols to aldoses, (iii) D-tagatose 3-epimerase
(DTE), which epimerizes the C-3 positions of ketoses,
and (iv) aldose isomerases, which catalyze the isomeri-
zation reactions of ketoses and aldoses. It is thus possi-
ble to use this strategy for the mass production of rare
sugars from abundant and inexpensive sugars such as
D-glucose and D-fructose.
not a common monosaccharide in the Izumoring strat-
egy. One of these is L-rhamnose isomerase (L-RhI),
which shows the highest activity in the isomerization
between
L-rhamnose
(6-deoxy-L-mamnose)
and
L-rhamnulose (6-deoxy-L-fructose), and the other
enzyme is L-fucose isomerase, which shows the highest
activity in the isomerization between L-fucose (6-
deoxy-L-galactose) and L-fuculose (6-deoxy-L-tagatose).
These enzymes, however, are active towards common
monosaccharides, as in the conversion between D-allose
and D-psicose affected by L-rhamnose isomerase.26)
Hence, we thought that different sugar isomerases
showing high levels of activity towards common mono-
saccharides can also act on 6-deoxy monosaccharides,
and we constructed a deoxy-Izumoring strategy based
on the results of previous Izumoring strategies and our
own experiences. All 12 of the 5-deoxyhexoses and 10
of the 16 1- and 6-deoxyketohexoses were successfully
produced in our laboratory, by a combination of chemi-
cal and biotechnological methods.27,28) We are currently
investigating whether the D-arabinose isomerase (D-AI),
L-arabinose isomerase (L-AI), L-ribose isomerase (L-RI),
and DTE produced in our laboratory act on 6-deoxy
monosaccharides.
Here, we describe the development of a novel proce-
dure for the synthesis of three 6-deoxy-L-aldohexoses
by a combination of enzymatic reactions. To the best
of our knowledge, this study represents the first on the
production of 6-deoxy-L-allose and 6-deoxy-L-altrose
by microbial enzymes. Toluene-treated E. coli, harbor-
ing the L-RhI gene of Pseudomonas stutzeri LL172,29)
the DTE gene of Pseudomonas cichorii ST-24,30) the
D-AI gene of Bacillus pallidus Y25,31) the L-AI gene of
Enterobacter aerogenes IK7 (unpublished data), and
the L-RI gene of Acinetobacter calcoaceticus LR7C,32)
were used as biocatalysts in the production of 6-deoxy-
L-glucose, 6-deoxy-L-altrose, and 6-deoxy-L-allose. All
of the toluene-treated E. coli cells had recombinant pro-
teins as well as the original host proteins, and effec-
tively functioned in the same way as an immobilized
microbe. These 6-deoxy-L-aldohexoses were produced
from L-rhamnose via L-rhamnulose and 6-deoxy-L-psi-
cose. The overall procedure for the production of these
three deoxy-L-aldohexoses is summarized in Scheme 1.
Materials and methods
Chemicals. L-Rhamnose, D-arabinose, L-arabinose,
and L-ribose were purchased from Sigma (St. Louis,
MO). D-Tagatose was prepared in our laboratory by a
method described previously.33) Bacto tryptone and
Bacto yeast extract were purchased from Becton, Dick-
inson (Sparks, MD). All other chemicals and culture
media used were from Wako Pure Chemical Industry
(Osaka) and were of reagent grade.
Bacterial strains and culture conditions. The bac-
terial strains used in this study were recombinant
E. coli JM 109 harboring the L-RhI gene of P. stutzeri
LL172, the DTE gene of P. cichorii ST-24, the D-AI
gene of B. pallidus Y25, the L-AI gene of E. aerogenes
IK7, and the L-RI gene of A. calcoaceticus LR7C.
These bacteria were transferred from stock culture to
The microbial enzymes making up an Izumoring
strategy have broad substrate specificity, in that it is
possible for one of the enzymes to recognize and cata-
lyze the transformation of multiple monosaccharides.
There are two enzymes in which the real substrate is