Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
1999-07-02
2002-01-01
Nguyen, Dave T. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S072000, C435S105000, C435S161000, C435S243000, C435S252100
Reexamination Certificate
active
06335177
ABSTRACT:
TECHNICAL FIELD
The present invention relates to novel microorganisms having an ability to produce xylitol or D-xylulose, and a method for producing xylitol or D-xylulose by using a microorganism having an ability to produce xylitol or D-xylulose. D-Xylulose is useful as a material for the production of xylitol, and xylitol is useful as a sweetener in the field of food industry and the like.
BACKGROUND ART
The demand of xylitol which is a naturally occurring sugar alcohol is expected to increase in future. Xylitol is a promising low-calorie sweetener because it has lower calories and exhibits comparable sweetness compared with sucrose. In addition, because of its anti-dental caries property, it is utilized as a dental caries preventive sweetener. Furthermore, because xylitol does not elevate glucose level, it is utilized for fluid therapy in the treatment of diabetes. For these reasons, it is expected that the demand of xylitol will increase in future.
The current industrial production of xylitol mainly relies on hydrogenation of D-xylose as disclosed in U.S. Pat. No. 4,008,285. D-Xylose used as a raw material is obtained by hydrolysis of plant materials such as trees, straws, corn cobs, oat hulls and other xylan-rich materials.
However, such D-xylose produced by hydrolysis of plant materials suffers a drawback that it is rather expensive, and it is arisen from high production cost. For example, the low yield of the hydrolysis treatment of plant materials leads to low purity of the produced D-xylitol. Therefore, the acid used for the hydrolysis and the dyes must be removed by ion exchange treatment after the hydrolysis treatment, and the resulting D-xylose must be further crystallized to remove other hemicellulosic saccharides. In order to obtain D-xylose suitable for foodstuffs, further purification would be required. Such ion exchange treatment and crystallization treatment invite the increase of production cost.
Therefore, several methods for producing xylitol have been developed, which utilize readily available raw materials and generate little waste. For example, there have been developed methods for producing xylitol utilizing other pentitols as a starting material. One of such readily available pentitols is D-arabitol, and D-arabitol can be produced by using yeast (
Can. J. Microbiol.,
31, 1985, 467-471
; J. Gen. Microbiol.,
139, 1993, 1047-54). As a method for producing xylitol by utilizing D-arabitol as a raw material, there can be mentioned the method reported in
Applied Microbiology.,
18, 1969, 1031-1035, which comprises producing D-arabitol from glucose by fermentation using
Debaryomyces hansenii
ATCC20121, then converting the D-arabitol into D-xylulose using
Acetobacter suboxydance
, and converting D-xylulose into xylitol by the action of
Candida guilliermondii
var.
soya.
EP 403 392A and EP421 882A disclose methods comprising producing D-arabitol by fermentation using an osmosis-resistant yeast, then converting D-arabitol into D-xylulose using a bacterium belonging to the genus Acetobacter, the genus Gluconobacter, or the genus Klebsiella, forming a mixture of xylose and D-xylulose from the D-xylulose by the action of glucose (xylose) isomerase, and converting the obtained mixture of xylose and D-xylulose into xylitol by hydrogenation. There is also disclosed the production of xylitol comprising preliminarily concentrating xylose in the mixture of xylose and D-xylulose and converting the xylose into xylitol by hydrogenation.
However, those methods for the production of xylitol mentioned above utilize D-arabitol produced by fermentation as a starting material, and convert it by multiple process steps. Therefore, the processes are complicated, and less satisfactory ones in view of process economy compared with the methods based on extraction.
Accordingly, there has been desired a microorganism which has an ability to produce xylitol or D-xylulose through a single step by fermentation starting from glucose as used in the production of other saccharides and sugar alcohols. However, such a bacterium having an ability to produce xylitol or D-xylulose has not been reported so far.
On the other hand, breeding of xylitol fermenting bacteria has been attempted by using gene manipulation techniques. International Publication W094/10325 discloses production of xylitol from glucose by fermentation by using a recombinant microorganism obtained by introducing an arabitol dehydrogenase gene derived from a bacterium belonging to the genus Klebsiella and a xylitol dehydrogenase gene derived from a bacterium belonging to the genus Pichia into an arabitol fermenting microorganism (yeast belonging to the genus Candida, the gunus Torulopsis, or the genus Zygosaccharomyces). However, while production of 15 g/L of xylitol from 400 g/L of glucose has been reported for the aforementioned recombinant microorganism, it does not reach a practically useful accumulation level. Moreover, the aforementioned recombinant microorganism is introduced with a gene derived from a different species, and therefore information about its safety cannot be considered sufficient.
SUMMARY OF THE INVENTION
The present invention has been accomplished in view of the aforementioned state of the art, and its object is to provide a microorganism having an ability to produce xylitol or D-xylulose from glucose by fermentation, as well as a method for producing xylitol or D-xylulose utilizing such a microorganism.
In order to achieve the aforementioned object, the present inventors searched a microorganism having an ability to produce xylitol or D-xylulose from glucose by fermentation. As for direct production of sugar alcohols by fermentation of microorganisms such as yeasts, there have also been reported production of glycerol by using
Zygosaccharomyces acidifaciens
(
Arch. Biochem.,
7, 257-271 (1945)), production of erythritol by using a yeast belonging to the genus Trychosporonoides (Trychosporonoides sp.,
Biotechnology Letters,
15, 240-246 (1964)) and the like, in addition to the aforementioned arabitol fermentation. All of these yeasts having sugar alcohol producing ability show osmophilicity, i.e., good growth in a culture medium of high osmotic pressure. Therefore, while any microbes having xylitol producing ability have not found among the osmophilic yeasts, the present inventors considered that a novel microorganism having xylitol producing ability may exist among osmophilic microorganisms, and extensively screened osmophilic microorganisms. As a result, they found microorganisms having an ability to produce xylitol and D-xylulose from glucose among osmophilic microorganisms. Those microorganisms were estimated to be novel bacteria from the viewpoint of taxonomic phylogeny based on the nucleotide sequence of 16S rRNA gene. The present invention has been accomplished based on the aforementioned finding.
Accordingly, the present invention provides a microorganism belonging to the family Acetobacteracea, which has a 16S rRNA gene comprising a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence equivalent to the nucleotide sequence of SEQ ID NO: 1 from the viewpoint of molecular taxonomy based on the 16S rRNA sequence, and has an ability to produce xylitol or D-xylulose from glucose, and
A microorganism which has a 16S rRNA gene comprising a nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence of SEQ ID NO: 2 equivalent to the nucleotide sequence from the viewpoint of molecular taxonomy based on the 16S rRNA sequence, and has an ability to produce xylitol or D-xylulose from glucose.
Examples of the aforementioned microorganisms include, for example, those microorganisms belonging to the genus Asaia or the genus Zucharibacter, more specifically strains of
Asaia ethanolifaciens
or
Zucharibacter floricola. Asaia ethanolifaciens
is a new species (sp. nov.) provisionally designated by the present inventors. The genus Zucharibacter and
Zucharibacter floricola
are a new genus (gen. nov.) and new species, respectively, which were provisionally designated by the present inventors.
Pa
Fudou Ryosuke
Jojima Yasuko
Mihara Yasuhiro
Takeuchi Sonoko
Tonouchi Naoto
Ajinomoto Co. Inc.
Nguyen Dave T.
Nguyen Quang
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