63NISR Research GRANT
2005 Research Grant

Microbial Biochemistry for Stable 5-keto-gluconate Fermentation
Hirohide TOYAMA
Department of Biological Chemistry, Faculty of Agriculture, and Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University

Research aims
5-Keto-D-gluconic acid (5KGA) has been reported as aprecursor for synthesis of many kinds of valuable substances1) including Vitamin C, tartaric acid, xylaric acid,and 4-hydroxy-5-methyl-dehydrofuranone-3, a valuable flavor compound. It has been reported that Gluconobacter sp.
can produce 5KGA and 2-keto-D-gluconic acid (2KGA)
from D-gluconic acid, and some strains further oxidize
2KGA to 2,5-diketo-D-gluconic acid (25DKGA). Among
several Gluconobacter stains, G. suboxydans IFO 12528
has been reported to produce 5KGA at high amount withlower amount of 2KGA. However, this strain is amesophilic strain which has optimum temperature for
5KGA production at 20°C2). Therefore, thermotolerant
5KGA-producing strains are in demand for industrial production.
The aim of this study is to isolate the thermotolerantstrains which are able to produce 5KGA at higher temperatures and improve their production of 5KGA. On the otherhand, for easy quantifications of 5KGA and 2KGA, 5-ketogluconate reductase (5KGR) and 2-ketogluconate reductase (2KGR) are useful, but preparation of these enzymes
from Gluconobacter is quite laborious. Therefore, overexpression of both enzymes in Escherichia coli was also carried out in this study.
Methods(1) Screening 5KGA-producing thermotolerant Gluconobacter strains
All thermotolerant Gluconobacter strains used in screening experiment were isolated from various sources in Thailand3). For rough screening, a single colony of all isolates
were inoculated to the medium containing 2% D-glucose,
2% sodium D-gluconate, 0.3% Polypeptone and 0.3% yeast
extract (G-GA medium), and cultured for 48 hours at 30°C
and 60 hours at 37°C. The strains which produced browncompound in the culture medium were screened out as
25DKGA-producing strains. Then, 200 m l of the culture
medium of the remaining isolates were reacted with 1 ml ofResorcinol reagent (0.5% (w/v) Resorcinol 49 ml, conc.
HCl 168 ml and distilled water 273 ml) at 80°C for 20 minutes. The reaction product of D-glucose and 5KGA with
Resorcinol reagent appeared as red and dark brownish
green precipitates, respectively, whereas D-gluconate and2KGA did not generate any color products. The strains producing 5KGA were selected, pre-cultured in Potato medium

for 1 day, and then 10 m l of the pre-cultures was seeded to 1
ml of the G-GA medium and incubated at 30°C for 36hours and 37°C for 48 hours. The culture mediums werethen collected and reacted with Resorcinol reagent, and finally the four strains which showed high productivity at37°C were selected. 5KGA and 2KGA production was also
examined by thin layer chromatography and enzyme assay
by using 5KGR4) and 2KGR5), respectively.
(2) Overexpression of 5KGRThe gno (GOX2187) was obtained by PCR with genomic
DNA of G. suboxydans IFO12528 (identical to G. oxydans
ATCC621) as a template. A pair of PCR primers was designed to cover the locus GOX2187 including the probable
Shine-Dalgarno sequence (SD) of this gene. The PCR product was then cloned into pGEM-T Easy vector and sequenced. The PCR product was then cloned into pUC119 inSphI and PstI sites. The resulting plasmid, pUC-gno, has
the gno gene inserted under lac promoter. 5KGR activity
was not observed in E. coli DH5a carrying pUC-gno
whereas a very low activity was observed at 37°C and noactivity at lower temperatures (30 and 25°C) in the strains
JM109 and TG1.
Next, the gno gene inserted in pUC-gno was then subcloned into pET-28a() at the NcoI and BamHI sites. The
resulting plasmid was designated as pET-gno. 5KGR activity was detected in cell-free extracts of E. coli BL21(DE3)carrying pET-gno, however, the activity observed was stilllow.
Finally, a pair of PCR primers was designed to cover gno
gene from start to stop codon with additional NcoI site tothe 5 end and BamHI site to the 3 end of the gene. To create a new NcoI site at the start codon of gno, a base change
is required, which resulted in a change of the second aminoacid residue from Ser to Ala. The PCR product was cloned
into pGEM-T Easy vector and sequenced, designated aspGEM-gno-NB. The PCR product was then subcloned intopET-28a() at NcoI and BamHI site and the resulted plasmid was named as pET-gno-NB. This plasmid was introduced into E. coli BL21(DE3) and used for 5KGR production.
(3) Overexpression of 2KGR
2KGR was partially purified from the soluble fraction ofG. oxydans IFO12528. N-terminal amino acid sequenceswere determined by protein sequencer PPSQ21 (Shimadzu), after blotted onto a sheet of PVDF membrane.
A pair of PCR primers was designed to cover the locus

64GOX0417 from start to stop codon including the 5 upstream region of 103 base pairs of this locus which contains
SD sequence of this gene. The PCR product was cloned
into pGEM-T Easy vector. The resulting plasmid, pGEMGOX0417, was isolated and sequenced. 2KGR activity wasobserved from E. coli DH5a carrying pGEM-GOX0417 at
about 20 times greater than in soluble fraction of Gluconobacter strains, but the expression level was still verylow.
Next, the inserted fragment on pGEM-GOX0417 wassubcloned into pET-28a() at XbaI and SalI site, resulted
in the pET-GOX0417 plasmid. This plasmid was transformed to E. coli BL21 (DE3) and checked for expression.
It was found that 2KGR activity was slightly increased
comparing with E. coli DH5a carrying pGEM-GOX0417.
And finally, the 5 upstream sequence of GOX0417 with aspace of 7 bases from start codon was changed fromAATGGA to GAAGGA to improve translation efficiency
by DpnI-mediated site-directed mutagenesis, resulting theplasmid pET-GOX0417 M6. This plasmid was introduced
into E. coli BL21(DE3) and used for 2KGR production.
Results(1) Screening 5KGA-producing thermotolerant Gluconobacter strains
Total 84 thermotolerant Gluconobacter strains isolated

from Thailand were tested for 5KGA production in themedium containing both D-glucose and D-gluconate.
Among them, 29 isolates showed the ability to produce
5KGA without producing brown pigment derived from25DKGA at 37°C, but only four strains showed high ability
to accumulate 5KGA at 37°C. Among four strains, thethree isolates produced 2KGA as the major product withlower amount of 5KGA at 30°C and 37°C (Figure 1). Interestingly, the other strain was found to be defective in 2KGA
production at 37°C whereas 5KGA was produced in nearly
the same amount as that at 30°C. All four strains showedbiphasic growth at 30°C. Production of both ketogluconates
occurred simultaneously at the first growing phase and continued after the growth slowed down. The second growthphase was observed when consumption of both 2KGA and5KGA occurred. On the other hand, such assimilation ofboth 2KGA and 5KGA was not observed when thosestrains were cultured at 37°C (Figure 1).
(2) Overexpression of 5KGR
The gno gene encoding 5KGR has been reported fromGluconobacter oxydans DSM 35036), and this is corresponding to GOX2178 in the genome of G. oxydans
ATCC621H7). Several plasmids were constructed and thefinal construct, pET-gno-NB, showed very good expressionin E. coli BL21 (DE3), 50% or more of proteins in the soluble fraction. It was found that the overexpressed strains

Fig. 1. Growth of thermotolerant Gluconobacter strains and production of 5KGA and 2KGA at different temperatures.
The strains were grown on G-GA medium. Growth (diamonds) was monitored using Klett-Summerson photometer with red filter. Small aliquots were takenfrom the culture and the amounts of 2KGA (white bar) and 5KGA (gray bar) in the culture supernatant were determined enzymatically using 2KGR and5KGR, respectively.

65

were unstable as the activity decreased after it was subcultured for several times or kept on plate more than 1 week.
Moreover, it was found that fresh cells transformed fromfreshly prepared competent cells give higher expression
level than the old one.
The soluble fraction of 5KGR-overexpressed E. coli
grown at 30°C was prepared in 10 mM KPB pH 6.0. This
enzyme solution was applied onto DEAE-cellulose column
chromatography and eluted with linear gradient of 0–0.3 M
KCl. The active fractions were eluted at the concentrationof KCl about 0.2 M. The partially purified 5KGR was obtained and it is ready to use for determination of 5KGA.
(3) Overexpression of 2KGR
The gene of 2KGR has not been identified yet. Therefore, N-terminal sequence of 2KGR was determined fromthe partially purified enzyme. The major 52 kDa protein hasN-terminal amino acid sequence as AYATTNPYTGETXXTFXEAT, matched to the sequence MAYATINPYTGETLKTFPEAT encoded by the locus GOX1122, aputative NAD-dependent aldehyde dehydrogenase gene, inthe genome of G. oxydans ATCC621H. In fact, the E. colitransformant which has GOX1122 showed no 2KGR activity but NADP-dependent acetaldehyde dehydrogenase activity. The 33 kDa protein has N-terminal amino acid sequence of SSXPDILAID which almost matched with thesequence MSSKPDILTID of the locus GOX0417, which isannotated as a putative 2-hydroxyacid dehydrogenase.
Several plasmids were constructed to express GOX0417
and the final construct, pET-GOX0417 M6, showed quite
good expression after the cells were grown to early-log
phase and induced by IPTG for 5 hours or more. It seemedthat 10% of proteins in the soluble fraction was 2KGR, although inclusion body of 2KGR observed in the precipitate
fractions after collecting the cell-free extracts. The expression level at 30°C was lower than that at 37°C. The instability of 2KGR expression was also observed as in the case of5KGR overexpression.
Partial purification of 2KGR was carried out from thesoluble fraction of 2KGR-overexpressed E. coli grown at37°C. After fractionated with (NH4)2SO4, the fraction wasapplied onto DEAE-cellulose column chromatography
which was equilibrated with 10 mM KPB pH 7.0. The active fractions obtained in passed fractions. The partially purified 2KGR thus obtained was ready to use for determination of 2KGA.
Conclusion(1) Four strains able to produce 5KGA at 37°C were obtained from thermotolerant Gluconobacter strains.
(2) 5KGR was successfully overproduced in E. coli.
(3) The gene of 2KGR was identified. 2KGR was also
succeeded to overexpress in E. coli.
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