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Microsoft powerpoint - fzj_ibt1_straincharacterization_goxydans_genomic_tagung_2003.ppt

Characterization of the recently sequenced Gluconobacter oxydans
DSM 2343 in comparison to other G. oxydans wild type strains
Christoph Bremus, Cornelia Gätgens, Ute Herrmann, Stephanie Bringer-Meyer and Hermann Sahm
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
Introduction
G. oxydans is an obligate aerobic Gram-negative bacterium belonging to the family of Acetobacteraceae [1]. Its incomplete oxidation of many carbohydrates and alcohols is interesting for several biotechnical applications
[2-4]. Examples of the industrial use are the production of L-sorbose (vitamin C synthesis), 6-amino-L-sorbose (synthesis of the antidiabetic drug miglitol), gluconate, dihydroxyacetone, and 5-ketogluconate (5-KGA).
5- KGA is of considerable interest as a precursor for tartaric acid production [5, 6].
The substrates are oxidized with high regio- and stereoselectivity and the products can be found with nearly quantitative yields in the medium. For the manifold oxidation steps G. oxydans
possesses several soluble
dehydrogenases and many membrane bound dehydrogenases coupled to the respiratory chain. Since the reactive centres of the membrane bound enzymes are oriented towards the periplasmic space, transport of the
substrates and products into respectively out of the cell is not necessary. Thus, the incompletely oxidized products accumulate in the medium and cell disruption is not needed. G. oxydans
is able to grow in highly
concentrated sugar solutions, at low pH-values and generates only low biomass concentrations. All these features make G. oxydans
an interesting organism for large-scale fermentation processes. Further studies of the
multiplicity of dehydrogenases with their possible substrates and corresponding products in combination with genetic engineering will reveal new application areas for biotechnology.

grow th w ith 25 g/l sorbitol
conversion of sorbitol
Objectives
In order to establish G. oxydans for new applications in
vitamin C and 5-KGA production we elucidated the function
and importance of the naturally occurring enzyme
accoutrement by analyzing the growth and product spectra of
the four different G. oxydans strains DSM 2343, DSM 3503,
DSM 3504 and NCIMB 8084. The growth and conversion
parameters were determined after cultivation with four
growth w ith 25 g/l mannitol
conversion of mannitol
carbon sources that are most frequently described for
cultivation of G. oxydans in literature (sorbitol, mannitol,
sorbitol
mannitol
glycerol
glucose, glycerol).
Figure 2: Comparison of the strains concerning growth
DSM3504 has the highest biomass yields with all tested carbon sources
Methods applied
grow th w ith 25 g/l glucose - DSM2343 and DSM3503
conversion of glucose DSM2343
G. oxydans DSM2343, DSM3503, DSM3504 and NCIMB8084
were cultivated in a complex medium with corn steep liquor
(composition kindly provided by BASF AG, Ludwigshafen) at
Gl uk on a t
30°C, pH 5,5 and 25 g/l carbon source concentration. Growth
experiments with sorbitol, mannitol and glycerol were carried
out in shaking flasks. Due to the formation of acids during the
conversion of glucose, acidification was compensated by pH
conversion of glucose DSM3503
Gl uk on a t
regulation with the fedbatch-pro multi fermentation system
grow th w ith 25 g/l glucose - DSM3504 and NCIMB8084
(DASGIP AG, Jülich). The quantification of substrates and
products was done by HPLC analysis.
sorbitol -> sorbose
mannitol -> fructose
glucose -> 5-KGA
conversion of glucose DSM3504
Gl u k on a t
Results 1: Growth parameters of G. oxydans
2 - / 2 , 5 - K GA
Growth rates and biomass production of the four tested
strains on the various carbon sources diverge largely as shown
grow th w ith 25 g/l glycerol
in growth curves (fig. 1A) and in biomass yields (fig. 2). As
presented in table 1, where all growth parameters are
conversion of glucose DSM8084
summarized, growth rates ranged from 0.02 h-1 (cultivation of
2 - / 2 , 5 - K GA
DSM2343 on glycerol) to 0.23 h-1 (cultivation of DSM3504 on
sorbitol) and biomass concentrations from 0.11 g cdw/l
Gl u k o na t
(cultivation of DSM2343 on glycerol) to 1.00 g cdw/l
sorbitol -> sorbose
mannitol -> fructose
glucose -> 5-KGA
(fermentation of DSM3504 on glucose in the fedbatch-pro
NCIMB8084
system) were achieved.
Fig. 1: Growth curves (panel A) and substrate conversion (panel B)
Whereas DSM3504 produces the highest biomass
Figure 3: Comparison of the strains concerning conversion
Cultivation of each strain was carried out in duplicates (“-1” and “-2”) and HPLC analysis was carried out for both fermentations. For calculating the growth and production parameters (table 1) mean values were taken. In case of glucose fermentation with DSM3504 and concentration with all carbon sources tested, DSM2343,
A: the conversion of DSM2343 with lower biomass production is equal or even higher than the conversion of the NCIMB8084 cultivation was carried out for 60 hours as growth and conversion were not finished after 30 hours. Due to comparability, other strains, resulting in higher Yp/x values.
growth and production parameters were calculated after 30 hours. Possibly, the longer growth and higher biomass yields of DSM3504 DSM3503 and NCIMB8084 produce significantly minor
B: whereas sorbitol and mannitol were nearly quantitatively oxidized to sorbose and fructose, respectively, the and NCIMB8084 with glucose were caused by the formation of 2.5-di-KGA, which is only produced by these strains and can be judged by Yp/s values for the conversion of glucose into 5-KGA are smaller due to the formation of coproducts or the colour change of the fermentation broth. As a result of the additional oxidation step, necessary for 2.5-di-KGA formation, cells are amounts of biomass with smaller growth rates.
Table 1: Summary of the growth parameters
gluconate
gluconate
gluconate
/ carbon source]
2/2,5-KGA
2/2,5-KGA
sorbitol
sorbitol
sorbitol
NCIMB8084
Fig. 4: Product spectra of the different strains using glucose as carbon source
All strains investigated convert glucose completely into gluconate, 2-KGA, 5-KGA or 2.5-di-KGA, respectively, so that
the singel Y
values add up to a total Y
of about 1.
Results 2: Conversion parameters of G. oxydans
The conversion of the carbon sources tested diverges also with the different strains as shown in the conversion curves (fig. 1B) and in product yields (fig. 3). The strain DSM2343 produces at least equal amounts of the
different products faster and with significantly lower biomass concentrations than DSM3504 or the other strains investigated. Whereas DSM2343 produces 17.2 g sorbose/(g cdw *h) and 10.9 g fructose/(g cdw*h),
DSM3504 produces 13.7 g sorbose/(g cdw*h) and 9.0 g fructose/(g cdw*h). Therefore, in all conversions tested the highest product yields (Y

) were determined for DSM2343 (fig. 3a).
Remarkable differences were also found concerning the product spectra of the strains tested. Whereas all strains nearly quantitatively oxidize mannitol to fructose and sorbitol to sorbose (YP/S ~ 1) in three of four cases
only a small part of the glucose was converted into 5-KGA (YP/S << 1, fig 3b). As demonstrated in fig. 4 this is caused by the formation of coproducts and intermediates. Thus, G. oxydans
DSM3503 accumulates high
amounts of gluconate (1.04 mol gluconate/mol glucose) and G. oxydans
DSM3504 and NCIMB8084 primarily produce 2-KGA and 2,5-di-KGA, which were not separated in the HPLC column and are therefore presented
combined in fig. 1B and fig. 4. In contrast to these strains, G. oxydans
DSM2343 converts glucose into 5-KGA (0.52 mol 5-KGA/mol glucose) and 2-KGA (0.52 mol 2-KGA/mol glucose), one half each (fig. 4). For the first
time such striking amounts of 5-KGA were obtained with DSM2343 during glucose fermentation enabled by the fedbatch-pro system.

Conclusions
Although G. oxydans DSM3504 produces the highest biomass concentrations with the highest growth rates, DSM2343 turned out to be the most appropriate strain regarding conversion of the carbon sources investigated
(obviously in case of glucose and sorbitol, less significantly in case of mannitol). The correlation of high oxidation rates with low biomass production in case of DSM2343 makes it most suitable for biotechnical applications,
because of low waste water production. Especially the amount of 5-KGA depends strongly on the wild type strain. Due to the high basal level of 5-KGA production, DSM2343 is apparently the most suitable strain to
generate a 5-KGA producing biotransformation system [7]. Further investigations in addition to studies of the recently sequenced G. oxydans
DSM 2343 genome should be carried out to reveal the genetic reason of the
observed differences in growth, conversion and product spectra.

References
Acknowledgement
[1] De Ley et al. (1984), Bergey’s manual of systematic bacteriology, vol 1, pp 267– 278.
[5] Matzerath et al. (1995), Inorg. Chim. Acta, 237: 203-205 This work was supported by the BASF AG, Ludwigshafen/ Germany.
[2] Deppenmeier et al. (2002), Appl. Microbiol. Biotechnol., 3: 233-42 [6] Klasen et al. (1992), Biotechnol. Bioeng., 40: 183-186 [3] Gupta et al. (2001), J. Mol. Microbiol. Biotechnol., 3: 445-56 [7] Merfort et al. (2003), poster presentation I.25, PROKAGEN 2003, Göttingen [4] Macauley et al. (2001), Crit. Rev. Biotechnol., 21: 1-25

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