U.S. patent application number 09/902693 was filed with the patent office on 2002-05-23 for novel microorganisms and method for producing xylitol or d-xylulose.
This patent application is currently assigned to AJINOMOTO CO., INC.. Invention is credited to Fudou, Ryosuke, Jojima, Yasuko, Mihara, Yasuhiro, Takeuchi, Sonoko, Tonouchi, Naoto, Yokozeki, Kenzo.
Application Number | 20020061561 09/902693 |
Document ID | / |
Family ID | 27279766 |
Filed Date | 2002-05-23 |
United States Patent
Application |
20020061561 |
Kind Code |
A1 |
Mihara, Yasuhiro ; et
al. |
May 23, 2002 |
Novel microorganisms and method for producing xylitol or
D-xylulose
Abstract
According to the present invention, there are provided
microorganisms having an ability to producing xylitol or D-xylulose
by fermentation, and a method for producing xylitol or D-xylulose
using the microorganisms. Osmophilic microorganisms were collected
from soil, and the obtained microorganisms were searched for a
bacterium having an ability to produce xylitol or D-xylulose from
glucose. Xylitol or D-xylulose is produced by culturing an isolated
bacterium in a suitable medium to accumulate xylitol or D-xylulose
in the medium, and collecting xylitol or D-xylulose from the
medium.
Inventors: |
Mihara, Yasuhiro; (Kanagawa,
JP) ; Takeuchi, Sonoko; (Kanagawa, JP) ;
Jojima, Yasuko; (Kanagawa, JP) ; Tonouchi, Naoto;
(Kanagawa, JP) ; Fudou, Ryosuke; (Kanagawa,
JP) ; Yokozeki, Kenzo; (Kanagawa, JP) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
AJINOMOTO CO., INC.
Tokyo
JP
|
Family ID: |
27279766 |
Appl. No.: |
09/902693 |
Filed: |
July 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09902693 |
Jul 12, 2001 |
|
|
|
09347001 |
Jul 2, 1999 |
|
|
|
Current U.S.
Class: |
435/72 ; 435/158;
435/200; 435/252.3; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12P 7/18 20130101; C12R
2001/01 20210501; Y02E 50/10 20130101; C12N 1/205 20210501; C12P
7/06 20130101; C12P 19/02 20130101 |
Class at
Publication: |
435/72 ; 435/158;
435/252.3; 435/69.1; 435/200; 536/23.2 |
International
Class: |
C12P 019/00; C07H
021/04; C12P 007/18; C12N 009/24; C12P 021/02; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 8, 1998 |
JP |
10-193472 |
Oct 30, 1998 |
JP |
10-310398 |
Jan 20, 1999 |
JP |
11-012244 |
Claims
What is claimed is:
1. 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.
2. The microorganism of claim 1 which belongs to the genus
Asaia.
3. The microorganism of claim 2 which is a strain of Asaia
ethanolifaciens.
4. A microorganism which has a 16S rRNA gene comprising a
nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence
equivalent to the nucleotide sequence of SEQ ID NO: 2 from the
viewpoint of molecular taxonomy based on the 16S rRNA sequence, and
has an ability to produce xylitol or D-xylulose from glucose.
5. The microorganism of claim 4 whose 16S rRNA gene comprises any
one of the nucleotide sequences of SEQ ID NOS: 3-5.
6. The microorganism of claim 4 which belongs to the genus
Zucharibacter.
7. The microorganism of claim 4 which is a strain of Zucharibacter
floricola.
8. A microbial strain P528 (FERM BP-6751) having an ability to
produce xylitol or D-xylulose from glucose.
9. A microbial strain S877 (FERM BP-6752) having an ability to
produce xylitol or D-xylulose from glucose.
10. A microbial strain S1009 (FERM BP-6753) having an ability to
produce xylitol or D-xylulose from glucose.
11. A microbial strain S1019 (FERM BP-6754) having an ability to
produce xylitol or D-xylulose from glucose.
12. A microbial strain S1023 having an ability to produce xylitol
or D-xylulose from glucose (FERM BP-6755).
13. A method for producing xylitol or D-xylulose, which comprises
culturing a microorganism having an ability to produce xylitol or
D-xylulose from glucose in a suitable medium to accumulate xylitol
or D-xylulose in the medium, and collecting xylitol or D-xylulose
from the medium.
14. The method of claim 13 wherein the microorganism is a
microorganism of any one of claims 1-12.
15. A method for producing ethanol, which comprises culturing a
microbial strain P528 (FERM BP-6751) in a suitable medium to
accumulate ethanol in the medium, and collecting ethanol from the
medium.
Description
TECHNICAL FIELD
[0001] 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
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] On the other hand, breeding of xylitol fermenting bacteria
has been attempted by using gene manipulation techniques.
International Publication WO94/10325 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
[0010] 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.
[0011] 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.
[0012] 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
[0013] 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.
[0014] 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.
[0015] Particular examples of the aforememtioned microorganisms
include, for example, strain P528 (FERM BP-6751), strain S877 (FERM
BP-6752), strain S1009 (FERM BP-6753), strain S1019 (FERM BP-6754),
and strain S1023 (FERM BP-6755)
[0016] The 16S rRNA gene of the strain P528 comprises the
nucleotide sequence of SEQ ID NO: 1, and the 16S rRNA gene of the
strain S877 comprises the nucleotide sequence of SEQ ID NO: 2.
Partial sequences of the 16S rRNA gene of the strains S1009, S1019,
and S1023 are of SEQ ID NOS: 3-5, respectively. These nucleotide
sequences are equivalent to the nucleotide sequence of SEQ ID NO: 2
from the viewpoint of molecular taxonomy based on the nucleotide
sequence of the 16S rRNA.
[0017] The present invention also provides a method for producing
xylitol or D-xylulose, which comprises culturing a microorganism
having an ability to produce xylitol or D-xylulose from glucose in
a suitable medium to accumulate xylitol or D-xylulose in the
medium, and collecting xylitol or D-xylulose from the medium.
[0018] Examples of the microorganism used for the above method
includes, for example, 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 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 belonging to the
family Acetobacteracea, which has a 16S rRNA gene comprising a
nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence
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.
[0019] Specific 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. Particular
examples of the aforementioned microorganisms include, for example,
the strains P528, S877, S1009, S1019, and S1023.
[0020] The present invention further provides a method for
producing ethanol, which comprises culturing the microbial strain
P528 (FERM BP-6751) in a suitable medium to accumulate ethanol in
the medium, and collecting ethanol from the medium.
[0021] According to the present invention, xylitol or D-xylulose
can be efficiently produced from inexpensive materials such as
glucose.
[0022] Further, ethanol can be produced by using the strain
P528.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows a molecular phylogenetic tree of the
microorganisms of the present invention and analogous bacteria
based on the nucleotide sequences of 16S rRNA.
[0024] FIG. 2 shows alignment of partial sequences of 16S rRNA of
xylitol producing microorganisms. It shows comparison of nucleotide
sequences of nucleotide numbers 1-691 of SEQ ID NO: 1 and SEQ ID
NO: 2 and the nucleotide sequences of SEQ ID NOS: 3-S. The dots
(.multidot.) indicate common nucleotides.
[0025] FIG. 3 is continuance of FIG. 2.
[0026] FIG. 4 is a graph representing influence of NaCl addition on
growth of microorganisms of the present invention.
[0027] FIG. 5 is a graph representing production of acetic acid
when the strains P528 and S877 are cultured in a medium added with
ethanol.
[0028] FIG. 6 is a graph representing consumption or production of
ethanol when the strains P528 and S877 are cultured in a medium
added with ethanol.
[0029] FIG. 7 is a graph representing ethanol production by the
strain P528.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention will be explained in detail
hereinafter.
[0031] <1> Microorganisms of the Present Invention
[0032] The present inventors extensively screened osmophilic
microorganisms as described in examples mentioned hereinafer, and
as a result, found novel microorganisms having an ability to
produce xylitol or D-xylulose from glucose. Those microorganisms
were designated as strains P528, S877, S1009, S1019, and S1023.
[0033] Microbiological characteristics of the above strains will be
mentioned below.
[0034] [1] Morphological and Cultural Characteristics
[0035] The aforementioned strains were cultured in YM medium (1%
glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, pH
6.0) supplemented with 11% (w/v) D-glucose at 30.degree. C. for 3
days, and then observed by a microscope. The results are shown in
Table 1.
1TABLE 1 Strain P528 S877 S1009 S1019 S1023 Cell 0.8-1 0.8-1 0.8-1
0.8-1 0.8-1 size .mu.m .times. .mu.m .times. .mu.m .times. .mu.m
.times. .mu.m .times. 4.5-5 .mu.m 2.5-3 .mu.m 2.5-3 .mu.m 2.5-4
.mu.m 2-2.5 .mu.m Shape Rod Rod Rod Rod Rod Motility None None None
None None Spore None None None None None
[0036] [2] Cultural Characteristics
[0037] (1) Agar Plate Culture
[0038] The strains were cultured on YM culture plates supplemented
with 11% (w/v) D-glucose at 30.degree. C. for 3 days, and observed
characteristics are shown in Table 2.
2TABLE 2 Strain P528 S877 S1009 S1019 S1023 Growth Good Good Good
Good Good Colony Round, Round, Round, Round, Round, smooth smooth
smooth smooth smooth for for for for for entire entire entire
entire entire periphery periphery periphery periphery periphery
Surface Low Low Low Convex Low convex convex convex convex Glisten
Lipid- Lipid- Lipid- Lipid- Lipid- like like like like like glisten
glisten glisten glisten glisten Color Lemon Slightly Slightly
Slightly Slightly yellow yellow yellow yellow yellow
[0039] (2) Broth Culture
[0040] The strains were cultured in YM culture broth supplemented
with 11% (w/v) D-glucose at 30.degree. C. for 3 days, and observed
characteristics are shown in Table 3.
3TABLE 3 Strain P528 S877 S1009 S1019 S1023 Surface None None None
None None growth Turbidity Strongly Strongly Strongly Strongly
Strongly turbid turbid turbid turbid turbid Precipi- Lot of Lot of
Lot of Lot of Lot of tates precipi- precipi- precipi- precipi-
precipi- tates tates tates tates tates
[0041] [3] Physiological Characteristics
[0042] (1) Test results for various physiological characteristics
are shown in Table 4.
4TABLE 4 Strain P528 S877 S1009 S1019 S1023 Gram Negative Negative
Negative Negative Negative stain Indole Negative Negative Negative
Negative Negative production Hydrogen Negative Negative Negative
Negative Negative disulfide production Oxidase Negative Negative
Negative Negative Negative Catalase Positive Positive Positive
Positive Positive O-F test Positive Negative Negative Negative
Negative
[0043] (2) Optimum Growth Condition
[0044] Optimum growth temperature and optimum pH when the strains
were cultured with the YM medium supplemented with 11% (w/v)
D-glucose are shown in Table 5.
5TABLE 5 Strain P528 S877 S1009 S1019 S1023 Optimum 30.degree. C.
27.degree. C. 27.degree. C. 27.degree. C. 27.degree. C. growth
temperature Optimum 5.0-7.0 5.0-7.0 5.0-7.0 5.0-7.0 5.0-7.0 growth
pH
[0045] (3) Growth Condition
[0046] Conditions which allow growth when the strains were cultured
with the YM medium supplemented with 11% (w/v) D-glucose are shown
in Table 6.
6TABLE 6 Strain P528 S877 S1009 S1019 S1023 Growth 10- 10- 10- 10-
10- temperature 37.degree. C. 37.degree. C. 32.degree. C.
32.degree. C. 32.degree. C. Growth pH 2.5-9.0 2.5-9.0 2.5-9.0
2.5-9.0 2.5-9.0
[0047] (4) Optimum sucrose concentrations when the strains were
cultured with the YM medium supplemented with sucrose are shown in
Table 7.
7TABLE 7 Strain P528 S877 S1009 S1019 S1023 Optimum 20% 10% 10% 10%
10% sucrose concentration
[0048] (5) The strains were cultured in a medium containing 20%
(w/v) D-glucose, 0.1% urea, and 0.5% yeast extract at 30.degree. C.
for 5 days. Saccharides detected in the medium after the
cultivation are mentioned in Table 8.
8TABLE 8 Strain P528 S877 S1009 S1019 S1023 Metabolite Xylitol,
Xylitol, Xylitol, Xylitol, Xylitol, from D- D- D- D- D- glucose
xylulose, xylulose, xylulose, xylulose, xylulose, D-arabitol,
D-arabitol D-arabitol D-arabitol D-arabitol sorbitol
[0049] Among the aforementioned strains, four of the strains S877,
S1009, S1019 and S1023 exhibit obligate osmophilicity, i.e., they
can grow only in a medium added with a saccharide at a high
concentration.
[0050] The major characteristic of those five microbial strains is
the ability to produce xylitol or D-xylulose from glucose. Since
any microorganism producing xylitol or D-xylulose from glucose has
not been reported at all to date, the strains having such
microbiological characteristics as mentioned above were determined
to be novel microorganisms.
[0051] [4] Molecular Taxonomic Analysis
[0052] In order to determine taxonomic positions of the strains
P528, S877, S1009, S1019 and S1023, nucleotide sequences of the 16S
rRNA gene of these strains were determined and a molecular
phylogenetic tree was prepared using those nucleotide sequences
together with nucleotide sequences of 16S rRNA gene of closest
microorganisms (FIG. 1). As a result, there has been suggested a
possibility that the strain P528 belongs to the family
Acetobacteracea, and is a new species belonging to the genus
Acetobacter or a new genus analogous to the genus Acetobacter. On
the other hand, there has been suggested a possibility that the
strain S877 belongs to the family Acetobacteracea, and is a
microorganism belonging to a new genus analogous to the genus
Acetobacter or the genus Gluconobacter. Three of the strains S1009,
S1019 and S1023 are considered to be the same species as the strain
S877.
[0053] A method for studying evolution of organisms or genes based
on a molecular phylogenetic tree has been established as molecular
taxonomy (see, for example, "Bunshi Shinka-gaku Nyumon
(Introduction of Evolutionary Molecular Biology)", Section 7,
Method for Preparation of Molecular Phylogenetic Tree and
Evaluation thereof, Ed. by T. Kimura, Baifukan, Japan,
pp.164-184).
[0054] A molecular phylogenetic tree based on the nucleotide
sequences of the 16S rRNA gene can be obtained by preparing a
phylogenetic tree based on data obtained through multiple sequence
alignment and calculation of evolution distance using nucleotide
sequences of the 16S rRNA gene of a microorganism of interest
together with those of known microorganisms estimated to be of the
same species or analogous to the microorganism of interest. The
nucleotide sequences of the 16S rRNA gene of known microorganisms
used for the preparation of the molecular phylogenetic tree can be
obtained by, for example, searching of available databases based on
homology. The term "evolution distance" herein used means a total
number of mutations per genetic locus (sequence length) for a
certain gene.
[0055] The multiple sequence alignment and evolution distance
calculation can be performed by, for example, using a commercially
available software such as CLUSTAL W included in the software
collection "Phylogeny Programs" (available from
http://evolution.genetics.washington.edu/phylip- /software. html,
see Thompson, D. J., et al., Nucleic Acids Res., 22, 4673-4680
(1994)) The phylogenetic tree can be prepared also by a generally
available software (e.g., Tree View, Tree drawing software for
Apple Machintosh: by Roderic D., Page 1995, Institute of Biomedical
and Life Sciences, University of Glasgow, UK). Specifically,
results obtained by computation on CLUSTAL W can be output as PHLYP
format data, and they can be processed by Tree View. PHLYP
(Felsenstein J. (1995) Phylogenetic inference package, version
3.5.7., Department of Genetics, University of Washington, Seatle
Wash., USA) is also included in the aforementioned Phylogeny
Programs.
[0056] [5] Other Biochemical and Physiological Characteristics
[0057] (1) Quinone Type and GC Content of DNA
[0058] The quinone type was ubiquinone-10 for all of the strains
P528, S877, S1009, S1019 and S1023, and the GC content of DNA was
56.5%, 52.3%, 52.3%, 51.9%, and 52.9%, respectively.
[0059] (2) Acid Production
[0060] Acid production from various carbon sources by the strains
was shown in Table 11.
[0061] (3) Influence of NaCl Addition on Growth
[0062] Growth of the strains cultured in a medium containing NaCl
at various concentrations is shown in FIG. 4. The strain P528 was
resistant to NaCl at least up to 2%.
[0063] (4) Consumption of Acetic Acid and Lactic Acid
[0064] When the strains were cultured in a medium containing
glucose as a carbon source and supplemented with acetic acid or
lactic acid, all of the strains exhibited lactic acid decomposition
ability, but weak or substantially no acetic acid decomposition
ability.
[0065] (5) Influence of Acetic Acid or Ethanol Addition on
Growth
[0066] All of the strains showed active growth in a medium added
with up to 1% of acetic acid or up to 3% of ethanol. All of the
strains did not grow in a medium added with 4% or more of acetic
acid or 5% or more of ethanol.
[0067] (6) Production of Acetic Acid and Consumption of Ethanol
[0068] When cultured in a medium containing glucose as a carbon
source, the strains showed weak acetic acid productivity. The
strains did not show significant ethanol consumption, and the
strain P528 showed ethanol production.
[0069] [6] Phenotypic Comparison of Microorganisms of the Present
Invention and Other Acetic Acid Bacteria
[0070] The results of phenotypic comparison of the strains P528,
S877, S1009, S1019 and S1023 and previously reported known acetic
acid bacteria, Asaia bogorensis, Acetobacter aceti, Gluconobacter
oxydans, Gluconacetobacter liquefaciens, and Acidomonas methanolica
(The Congress of the Japan Society for Bioscience, Biotechnology,
and Agrochemistry, 1999, Lecture Abstracts, p. and p.66) are shown
in Table 9. Asaia bogorensis is a microorganism belonging to a new
genus (gen. nov.), and a new species (sp. nov.) reported in the
meeting by Yamada et al. As for the strains P528, S877, S1009,
S1019 and S1023, acetic acid production, ethanol production, DNA
nucleotide composition, and major quinone were determined as
described in Examples 5 and 6. The other characteristics are
determined by the method of Asai et al. (Asai, T. et al., J. Gen.
Appl. Microbiol., 10 (2), p.95, 1964).
9 TABLE 9 S-877, S-1009, Glucono- Glucono- S-1019, S-1023 P-528
Asaia Acetobacter bacter acetobacter Acidomonas Motility - - +/-
+/- +/- +/- - Production of acetic W W - + + + + acid from ethanol
Production of acetic - + nd - - nd nd acid from glucose Oxidation
of acetic - W + + - +/- + acid Oxidation of lactic + + + + - +/- -
acid Growth Mannitol agar medium + + + +/- + +/- - Glutamic acid
agar - + + +/- - +/- - medium (1%) Glutamic acid agar + + nd + - -
nd medium (7%) Growth with 30% + + nd - - +/- - glucose Acid
production From mannitol + + + - + +/- - From sorbitol - + + - + -
- From glycerol - + + - + + - From ethanol - - W/- + + + + DNA base
composition 52-53 56.5 59-61 53-63 56-64 55-66 63-66 (mol % G + C)
Major quinone UQ-10 UQ-10 UQ-10 UQ-9 UQ-10 UQ-10 UQ-10 nd: Not
determined, W: Weak
[0071] As shown in Table 9, the strain P528 resembles Asaia
bogorensis, but it is different from Asaia bogorensis in that the
strain showed acetic acid production from ethanol though it was
weak and that GC content in a nucleotide composition of DNA is 56.5
which is significantly lower than that of Asaia bogorensis (59-61).
The strains S877, S1009, S1019 and S1023 were different from the
other acetic acid bacteria in that their acetic acid production
from ethanol was weak, they could grow in the presence of 30%
glucose, and they did not show acid production from ethanol.
[0072] Based on the above results, the strain P528 was identified
as a new species belonging to the genus Asaia, and provisionally
designated as Asaia ethanolifaciens sp. nov. The strains S877,
S1009, S1019 and S1023 all were identified as a new species
belonging to a new genus, and provisionally designated as
Zucharibacter floricola gen. nov., sp. nov.
[0073] <2> Production Method of Xylitol and D-xylulose
[0074] Xylitol and/or D-xylulose can be produced by culturing a
microorganism having an ability to produce xylitol or D-xylulose
from glucose in a suitable medium so that xylitol or D-xylulose or
the both should accumulate in the medium, and collecting xylitol
and/or D-xylulose from the medium.
[0075] While the microorganism is not particularly limited so long
as it has the ability to produce xylitol or D-xylulose from
glucose, specific examples thereof include the aforementioned
strains P528, S877, S1009, S1019 and S1023. Those microorganisms of
the same species or belonging to the same genus as the
aforementioned strains and having the ability to produce xylitol or
D-xylulose from glucose can also be used for the present invention.
Examples of such microorganisms include, for example, those
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 from the viewpoint
of molecular taxonomy based on the 16S rRNA sequence, or a
nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence
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.
Specifically, those belonging to the genus Asaia or the genus
Zucharibacter, more specifically strains of Asaia ethanolifaciens
or Zucharibacter floricola can be mentioned.
[0076] The target product produced by the method of the present
invention may be one of xylitol or D-xylulose, or both of them.
[0077] According to the present invention, any of mutant strains
obtained from microbial strains having an ability to produce
xylitol or D-xylulose from glucose by UV exposure,
N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment, ethyl
methanesulfonate (EMS) treatment, nitrous acid treatment, acridine
treatment and the like, or genetic recombinant strains and the like
obtained by cell fusion or genetic engineering techniques such as
genetic recombination can also be used.
[0078] The medium for culturing the aforementioned microorganisms
may be a usual medium containing usual carbon source, nitrogen
source, inorganic ions, as well as organic nutrients as required.
While the microorganisms of the present invention grow under high
osmotic stress condition, they may also grow under normal osmotic
condition as the case may be. For example, the strain P528 grows
under normal osmotic condition.
[0079] As the carbon source, carbohydrates such as glucose,
alcohols such as glycerol, organic acids and the like can be
suitably used. In view of the preference observed in the known
methods for the production of xylitol, for example, the method for
producing xylitol from pentitols such as D-xylose or D-arabitol,
preferred are hexoses such as fructose and sucrose, disaccharides
such as sucrose and lactose, and polysaccharides such as starch.
These materials are used as a main carbon source in the medium in
an amount of 10-60%, preferably 20-50%. These carbon sources may be
added to the medium at a time, or in parts according to the
cultivation time course.
[0080] As the nitrogen source, ammonia gas, aqueous ammonia,
ammonium salts and the like are used. As the inorganic ions,
magnesium ions, phosphate ions, potassium ions, iron ions,
manganese ions and the like are used as required. As the organic
nutrient, vitamins, amino acids and materials containing them such
as lever extract, yeast extract, malt extract, peptone, meat
extract, corn steep liquor, casein decomposition product and the
like are used as required.
[0081] The culture conditions are also not particularly limited.
However, the microorganisms may be cultured at limited pH and
temperature selected within a pH range of 5-8 and temperature range
of 25-40.degree. C. The cultivation is performed under an aerobic
condition by, for example, stirring or shaking for aeration. As for
the culture period, the microorganisms are desirably cultured until
the main carbon source is consumed, i.e., usually for 3-8 days.
[0082] Xylitol and/or D-xylulose produced in the medium during such
cultivation as described above is separated and collected from the
culture in a conventional manner. Specifically, for example, after
the solid matter is removed from the culture by centrifugation,
filtration or the like, the residual solution can be decolorized
and desalted by using activated carbon, ion-exchange resin or the
like, and xylitol and/or D-xylulose can be crystallized from the
solution. The procedures of the separation and the collection of
xylitol and/or D-xylulose from culture are easier than the
separation from plant material hydrolysate because of lower content
of impurities.
[0083] The produced D-xylulose can be converted into xylitol by
hydrogenation, which can be performed in a known manner.
[0084] <3> Production Method of Ethanol
[0085] Ethanol can be produced by culturing the microbial strain
P528 (FERM BP-6751) in a medium containing glucose so that ethanol
should accumulate in the medium, and collecting ethanol from the
medium. Other than the strain P528, microorganisms having 16S rRNA
gene comprising a nucleotide sequence of SEQ ID NO: 1 or a
nucleotide sequence equivalent to the nucleotide sequence from the
viewpoint of molecular taxonomy based on the 16S rRNA sequence, and
has an ability to produce ethanol from glucose, or mutant strains
thereof can similarly be used for the production of ethanol.
[0086] The medium and culture conditions can be similar to those
explained above for the method for producing xylitol and
D-xylulose. Ethanol produced in the medium can be concentrated and
purified in such a manner as used in usual ethanol
fermentation.
BEST MODE FOR CARRYING OUT THE INVENTION
[0087] The present invention will be explained more specifically
with reference to the following examples. However, the present
invention is not limited to these examples.
[0088] In the examples, the produced xylitol and D-xylulose were
analyzed by high performance liquid chromatography (HPLC) under the
following conditions.
[0089] Column: Shodex SC1211 (product of Showa Denko)
[0090] Mobile phase: 50% acetonitrile/50% 50 ppm aqueous solution
of Ca-EDTA
[0091] Flow rate: 0.8 ml/minute
[0092] Temperature: 60.degree. C.
[0093] Detection: RI detector
EXAMPLE 1
Isolation of Microorganisms Producing Xylitol or D-xylulose
[0094] First, osmophilic microorganisms were collected from nature
by enrichment culture. A medium containing 20% D-glucose, 1% yeast
extract (Difco), and 0.1% urea was introduced into test tubes in an
amount of 4 ml each, and sterilized at 120.degree. C. for 20
minutes. Soil samples collected from various locations were
inoculated to the medium, and cultured at 30.degree. C. for 4 to 7
days with shaking. When bacterial growth was observed, the culture
was plated on an agar plate having the same composition, and
incubated at 30.degree. C. for 1 to 3 days. Then, formed colonies
were picked up.
[0095] Then, about 3000 strains of osmophilic bacteria obtained as
described above were cultured in a medium containing 20% (w/v)
D-glucose, 0.1% urea, and 0.5% yeast extract at 30.degree. C. for 5
days, and the medium was analyzed by HPLC to screen for a strain
having the xylitol or D-xylulose producing ability. As a result,
five bacterial strains separated from soil collected from the bank
of Tama river, Kawasaki-shi, Kanagawa-ken, were found to have the
ability to produce xylitol from glucose. These strains were each
designated as strains P528, S877, S1009, S1019 and S1023. These
five strains were assigned private numbers of AJ14757, AJ14758,
AJ14759, AJ14760, and AJ14761 in this order, and have been
deposited since Jun. 18, 1998 at the National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology (zip code: 305-8566, 1-3 Higashi 1-Chome, Tsukuba-shi,
Ibaraki-ken, Japan), as deposition numbers of FERM P-16848, FERM
P-16849, FERM P-16850, FERM P-16851, and FERM P-16852 in this
order, and transferred from the original deposition to
international deposition based on Budapest Treaty on June ______,
1999, and has been deposited as deposition numbers of FERM BP-6751,
FERM BP-6752, FERM BP-6753, FERM BP-6754, and FERM BP-6755.
EXAMPLE 2
Production of Xylitol and D-xylulose from Glucose
[0096] A medium containing 0.5% yeast extract (Difco), and 0.1%
urea (pH 6.0) was introduced into a 500 ml Sakaguchi flask in an
amount of 50 ml, and sterilized by heating at 120.degree. C. for 20
minutes. Separately sterilized glucose was added to this medium in
such an amount that the medium should contain 20% (w/v) glucose.
The strains P528, S877, S1009, S1019 and S1023 were each inoculated
to this medium, and cultured at 30.degree. C. for 5 days with
shaking. Then, after the bacterial cells were removed by
centrifugation, xylitol and D-xylulose formed in the medium were
mesured by HPLC. The results are shown in Table 10.
10TABLE 10 Production amount of xylitol and D-xylulose
Concentration of produced xylitol Concentration of produced Strain
(g/l) D-xylulose (g/l) P528 5.3 3.3 S877 1.9 9.7 S1009 1.6 9.0
S1010 1.7 9.2 S1023 1.5 5.0
EXAMPLE 3
Molecular Taxonomic Analysis of Strains P528, S877 S1009, S1019 and
S1023
[0097] The strains P528 and S877 were analyzed from the viewpoint
of molecular taxonomy by nucleotide sequence analysis of 16S rRNA
in a conventional manner.
[0098] A bacterial cell suspension of each strain was treated with
protease at 60.degree. C. for 20 minutes, then heated in boiling
water for 5 minutes, and centrifuged. The obtained supernatant was
directly used as template for PCR.
[0099] Using universal primers corresponding to the positions 8-27
and 1492-1510 of the 16S rRNA of E. coli (SEQ ID NOS: 6 and 7), 30
cycles of PCR was performed in a conventional manner, and the
product was collected by PEG precipitation. The PCR product was
directly sequenced by fluorescence cycle sequencing, and the
reaction product was analyzed by a DNA sequencer (Pharmacia). The
determined nucleotide sequences are shown in SEQ ID NO: 1 (strain
P528) and SEQ ID NO: 2 (strain S877). Any sequence corresponding to
these nucleotide sequences was not found in databases. The
bacterial group having the closest nucleotide sequences of the 16S
rRNA gene for each strain was bacteria belonging to the genus
Gluconobacter and the genus Acetobacter.
[0100] The obtained nucleotide sequence data were processed by
GENETYX (Software Development, Tokyo), and multiple alignment and
evolution distance calculation were performed by CLUSTAL W for the
obtained sequences and analogous sequences available from databases
(16S rRNA gene sequences of 13 kinds of acetic acid bacteria
currently considered valid names) The obtained PHYLIP format data
were read and processed by Tree View to prepare a molecular
phylogenetic tree. The result is shown in FIG. 1. The alignment of
16S rRNA of the xylitol producing bacteria is shown in FIGS. 2 and
3. The aforementioned 13 kinds of acetic acid bacteria are
mentioned below.
[0101] Gluconobacter asaii
[0102] Gluconobacter cerinus
[0103] Gluconobacter frateurii
[0104] Gluconobacter oxydans subsp. oxydans
[0105] Acetobacter aceti
[0106] Acetobacter pasteurianus
[0107] Acetobacter methanolicus
[0108] Gluconobacter europaeus
[0109] Gluconobacter xylinus subsp. xylinus
[0110] Gluconobacter intermedicus
[0111] Gluconobacter hansenii
[0112] Gluconobacter liquefaciens
[0113] Gluconobacter diazotrophicus
[0114] Rhodophila globiformis
[0115] As a result, known strains exhibiting a close evolution
distance with respect to the strain P528 were Gluconobacter
intermedicus, Gluconobacter liquefaciens, Acetobacter aceti,
Acetobacter methanolicus and Acetobacter pasteurianus, whose
evolution distance was 0.0345, 0.0359, 0.0403, 0.0419 and 0.0499,
and homology of the 16S rRNA gene was 96.5%, 96.3%, 96.0%, 95.9%
and 95.1%, respectively. Further, known strains exhibiting a close
evolution distance with respect to the strain S877 were
Gluconobacter cerinus and Gluconobacter oxydans, whose evolution
distance was 0.0622 and 0.0629, and homology of the 16S rRNA gene
was 94.0% and 93.9%, respectively. While the strain P528 is
included in the cluster of the genus Acetobacter, it was far away
from three strains of the known species, and hence considered a new
species. Acetobacter methanolicus has also been reported to belong
to another genus (genus Acidomonas). If Acetobacter methanolicus is
considered to belong to another genus, the strain P528 may belong
to a new genus, since the strain is located outside the cluster of
the genus Acetobacter.
[0116] On the other hand, the strain S877 is located outside the
cluster of the genus Gluconobacter, and far away from any known
species belonging to the genus Gluconobacter. The evolution
distance from the strain S877 to the closest strain (Gluconobacter
cerinus) is 0.066, and this value is significantly larger than the
distance between the genus Gluconobacter and the genus Acetobacter
(0.044). Therefore, it is reasonable to consider that this strain
belongs to a new genus.
[0117] When partial nucleotide sequences of the 16S rRNA of the
strains S1009, S1019 and S1023 were determined (SEQ ID NOS: 3 to 5,
respectively), they showed substantially the same sequence as that
of the strain S877, and hence they were found to be of the same
species.
[0118] From the above molecular taxonomic analysis and the
phenotypes shown in Table 9, the strain P528 was identified as a
new species belonging to the genus Asaia, and provisionally
designated as Asaia ethanolifaciens sp. nov. The strains S877,
S1009, S1019 and S1023 strain were all identified as a
microorganism of a new species belonging to a new genus, and
provisionally designated as Zucharibacter floricola gen. nov., sp.
nov.
EXAMPLE 4
Production of Xylitol and D-xylulose from Glucose
[0119] A medium containing 0.2% ammonium acetate, 0.3% potassium
dihydrogenphosphate, 0.05% magnesium sulfate heptahydrate, 0.5%
yeast extract (Difco), and 4% calcium carbonate was introduced into
a 500 ml Sakaguchi flask in an amount of 50 ml, and sterilized by
heating at 120.degree. C. for 20 minutes. Separately sterilized
glucose was added to the medium in such an amount that the medium
should contain 20% (w/v) glucose. The strain P528 was inoculated to
this medium, and cultured at 30.degree. C. for 4 days with shaking.
Then, after the bacterial cells were removed by centrifugation,
xylitol and D-xylulose formed in the medium were mesured by HPLC.
As a result, it was found that 6.4 g/L of xylitol and 17.5 g/L of
D-xylulose was formed.
EXAMPLE 5
Biochemical and Physiological Characteristics of Strains P528, S877
S1009, S1019 and S1023
[0120] (1) Analysis of Quinone and GC Content of DNA
[0121] Quinone and GC content of DNA of the aforementioned strains
were analyzed by high performance liquid chromatography (HPLC) in a
usual manner (see Saikingaku Gijutsu Sosho (Library of Techniques
in Bacteriology), Vol. 8 "Method for Microbial Identification
Following New Taxonomy", pp.61-73, pp.88-97, Saikon Shuppan,
Japan). The results are shown in Table 11.
11TABLE 11 Quinone type and GC content of DNA Strain P528 S877
S1009 S1019 S1023 Quinone UQ-10 UQ-10 UQ-10 UQ-10 UQ-10 GC (%) 56.5
52.3 52.3 51.9 52.9 UQ: Ubiquinone
[0122] (2) Acid Production from Various Carbon Sources
[0123] The aforementioned strains were each cultured in a medium
containing one of various carbon sources (1%), and presence of
formed acid was determined. The strains were pre-cultured in the
YPG medium at 28.degree. C. for one day, and the bacterial cells
were washed with 0.5% yeast extract solution, inoculated to the YPC
medium, and cultured at 28.degree. C. for 4 to 7 days with shaking.
Then, production of acid was determined by color variation
(purplish red to yellow) of pH indicator in the medium.
[0124] The YPG medium was prepared as follows. A medium containing
1% yeast extract (Difco), and 1% peptone was sterilized by heating
at 120.degree. C. for 20 minutes. To this medium, separately
sterilized D-glucose was added in such an amount that the medium
should contain 7% D-glucose.
[0125] The YPC medium was prepared as follows. A medium containing
0.5% yeast extract (Difco), 0.012% bromocresol purple, and 1% of
one of various carbon sources was sterilized by heating at
120.degree. C. for 20 minutes.
[0126] The results are shown in Table 12.
12TABLE 12 Acid formation from various carbon sources Strain P528
S877 S1009 S1019 S1023 Xylose + - - - - Arabinose + - - - - Glucose
+ + + + + Galactose + - - - - Mannose + - + + + Fructose + + - - -
Sorbose .+-. - - - - Sucrose .+-. + + + + Maltose - - - - -
Rhamnose + - - - - Glycerol .+-. - - - - Mannitol .+-. + + + +
Sorbitol .+-. - - - - Lactose + - - - - Starch - - - - - Ethanol -
- - - - +: Presence of acid production, .+-.: weak acid production,
-: no acid production
[0127] (3) Influence of NaCl Addition on Growth
[0128] Influence of NaCl addition on growth of the aforementioned
strains was examined by culture in the YPM medium. The
aforementioned strains and Acetobacter aceti strain NCIB 8621 as a
control were pre-incubated in the aforementioned YPG medium at
28.degree. C. for one day, and the bacterial cells were washed with
the YPG medium not added with D-glucose, and suspended in the YPG
medium not added with D-glucose. The obtained bacterial suspension
was inoculated (1.6% v/v) to YPM medium added with NaCl at one of
various concentrations, and cultured at 28.degree. C. for two days
with shaking. Then, turbidity of the medium was measured by a
spectrophotometer ANA-75A from Tokyo Koden (OD 660 nm) to determine
the growth.
[0129] The YPM medium was prepared as follows. A medium containing
1% yeast extract (Difco), 1% peptone, and 1% mannitol was
sterilized by heating at 120.degree. C. for 20 minutes.
[0130] The results are shown in FIG. 4. The strain P528 showed
active growth in a medium added with up to 2% of NaCl, i.e., showed
NaCl resistance.
[0131] (4) Consumption of Acetic Acid and Lactic acid
[0132] The aforementioned strains were cultured in the YG medium
added with acetic acid or lactic acid to examine consumption of
acetic acid and lactic acid.
[0133] The aforementioned strains and Acetobacter aceti strain NCIB
8621 as a control were pre-cultured in the aforementioned YPG
medium at 28.degree. C. for one day with shaking. The obtained
pre-medium was inoculated (1.6%, v/v) to YG medium added with 1%
acetic acid or lactic acid, and incubated at 28.degree. C. for
seven days. The consumption of acetic acid and lactic acid was
examined by time course sampling of the medium. The measurement of
acetic acid and lactic acid was performed by HPLC under the
following conditions.
[0134] Column: ULTTRON PS-80 (product of Shinwa Kagaku Kogyo)
[0135] Mobile phase: Perchloric acid solution (pH 2.1)
[0136] Flow rate: 0.9 ml/minute
[0137] Temperature: 60.degree. C.
[0138] Detection: UV detector (210 nm)
[0139] The YG medium added with acetic acid or lactic acid was
prepared as follows. A medium containing 1% yeast extract (Difco),
and 1% acetic acid or lactic acid was adjusted to pH 6.0, and
sterilized by heating at 120.degree. C. for 20 minutes. Separately
sterilized D-glucose was added to the medium in such an amount that
the medium should contain 7% D-glucose.
[0140] The results are shown in Table 13 (the data were represented
in consumed amount (%)). The strains P528, S877, S1009, S1019 and
S1023 all showed lactic acid decomposition ability, whereas they
showed weak or substantially no acetic acid decomposition
ability.
13TABLE 13 Consumption of acetic acid and lactic acid Strain P528
S877 S1009 S1019 S1023 A. aceti Acetic 87.8 100.0 88.6 100.0 96.6
7.7 acid (%) Lactic 0.0 4.7 29.7 30.7 24.3 0.0 acid (%) A. aceti:
Acetobacter aceti strain NCIB8621
[0141] (5) Influence of Acetic Acid or Ethanol Addition on
Growth
[0142] Influence of addition of acetic acid on growth of the
aforementioned strains was examined in the YG medium added with
acetic acid. Influence of addition of ethanol on growth of the
aforementioned strains was also examined in the YPG medium added
with ethanol.
[0143] The aforementioned strains were each pre-cultured in the
foregoing YPG medium at 28.degree. C. for one day, and each medium
was inoculated (1.6% v/v) to the YG medium added with lactic acid
at one of various concentrations, and the YPG medium added with
ethanol at one of various concentrations, and incubated at
28.degree. C. for ten days with shaking. Then, turbidity of the
medium was measured by a spectrophotometer ANA-75A from Tokyo Koden
(OD 660 nm) to determine the growth.
[0144] All of the strains P528, S877, S1009, S1019 and S1023 showed
active growth in the medium added with up to 1% acetic acid or 3%
ethanol. All of the strains did not grow in the medium added with
4% or more of acetic acid or 5% or more of ethanol.
[0145] (6) Production of Acetic Acid and Consumption of Ethanol
[0146] The aforementioned strains were cultured in the YPG medium
added with ethanol to examine production of acetic acid and
consumption of ethanol. The aforementioned strains and Acetobacter
aceti strain NCIB 8621 as a control were pre-cultured in the
aforementioned YPG medium at 28.degree. C. for two days. Each
medium was inoculated (1%, v/v) to the YPG medium added with 1%
ethanol and incubated at 28.degree. C. The concentrations of acetic
acid and ethanol in the medium were examined by time course
sampling of the medium. The measurement of acetic acid and ethanol
concentrations was performed by using F-kit (Roche
Diagnostics).
[0147] The results are shown in FIGS. 5 and 6. All of the strains
P528, S877, S1009, S1019 and S1023 showed weaker acetic acid
productivity compared with the control bacteria, Acetobacter aceti
strain NCIB 8621 (the figure indicates the data only for the
strains P528 and S877). Further, strains S877, S1009, S1019 and
S1023 did not show ethanol consumption in contrast to the control
bacteria, Acetobacter aceti strain NCIB 8621. The strain P528
showed, to the contrary, showed increase of ethanol amount.
EXAMPLE 6
Production of Ethanol by Strain P528
[0148] The strain P528 was cultured by using the YPG medium in a
manner similar to that mentioned above, and ethanol concentration
in the medium was measured over time. The results are shown in FIG.
7. The strain P528 showed ethanol productivity.
Sequence CWU 1
1
5 1 1438 DNA Unknown Organism Description of Unknown Organismstrain
P528 1 tgatcctggc tcagagcgaa cgctggcggc atgcttaaca catgcaagtc
gcacggacct 60 ttcggggtga gtggcggacg ggtgagtaac gcgtagggat
ctatccacgg gtgggggata 120 acactgggaa actggtgcta ataccgcatg
atacctgagg gtcaaaggcg cgagtcgcct 180 gtggaggagc ctgcgttcga
ttagcttgtt ggtggggtaa aggcctacca aggcgatgat 240 cgatagctgg
tctgagagga tgatcagcca cactgggact gagacacggc ccagactcct 300
acgggaggca gcagtgggga atattggaca atgggcgcaa gcctgatcca gcaatgccgc
360 gtgtgtgaag aaggtcttcg gattgtaaag cactttcgac ggggacgatg
atgacggtac 420 ccgtagaaga agccccggct aacttcgtgc cagcagccgc
ggtaatacga agggggctag 480 cgttgctcgg aatgactggg cgtaaagggc
gtgtaggcgg ttgttacagt cagatgtgaa 540 attccagggc ttaaccttgg
ggctgcattt gatacgtagc gactagagtg tgagagaggg 600 ttgtggaatt
cccagtgtag aggtgaaatt cgtagatatt gggaagaaca ccggtggcga 660
aggcggcaac ctggctcatg actgacgctg aggcgcgaaa gcgtggggag caaacaggat
720 tagataccct ggtagtccac gctgtaaacg atgtgtgctg gatgttgggt
aacttagtta 780 ctcagtgtcg aagctaacgc gctaagcaca ccgcctggga
agtacggccg caaggttgaa 840 actcaaagga attgacgggg gcccgcacaa
gcggtggagc atgtggttta attcgaagca 900 acgcgcagaa ccttaccagg
gcttgacatg gggaggctgt actcagagat gggtatttcc 960 cgcaagggac
ctcctgcaca ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt 1020
gggttaagtc ccgcaacgag cgcaaccctc gcctttagtt gccagcacgt ttgggtgggc
1080 actctagagg aactgccggt gacaagccgg aggaaggtgg ggatgacgtc
aagtcctcat 1140 ggcccttatg tcctgggcta cacacgtgct acaatggcgg
tgacagtggg aagctagatg 1200 gtgacatcat gccgatctca aaaagccgtc
tcagttcgga ttgtactctg caactcgagt 1260 acatgaaggt ggaatcgcta
gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg 1320 gccttgtaca
caccgcccgt cacaccatgg gagttggttt gaccngaagc cggtgagcga 1380
accgcaagga cgcagccgac cacggtcggg tcagcgactg gggtgaagtc gtaacaag
1438 2 1436 DNA Unknown Organism Description of Unknown
Organismstrain S877 2 tgatcctggc tcagagcgaa cgctggcggc atgcttaaca
catgcaagtc gcacgaacct 60 ttcggggtta gtggcggacg ggtgagtaac
gcgtaggaac ctatccagag gtgggggata 120 acaccgggaa actggtgcta
ataccgcatg atacctgagg gttaaaggct tttgttgcct 180 ttggaggggc
ctgcgtttga ttagctagtt ggttgggtaa aggctgacca aggcgatgat 240
caatagctgg tttgagagga tgatcagcca cactgggact gagacacggc ccagactcct
300 acgggaggca gcagtgggga atattggaca atgggggcaa ccctgatcca
gcaatgccgc 360 gtgtgtgaag aaggtcttcg gattgtaaag cactttcact
agggaagatg atgacggtac 420 ctagagaaga agccccggct aacttcgtgc
cagcagccgc ggtaatacga agggggctag 480 cgttgctcgg aatgactggg
cgtaaagggc gcgtaggcgg tttatacagt cagatgtgaa 540 atccccgggc
ttaacctggg aactgcattt gatacgtata gactagagtc cgagagagga 600
ttgcggaatt cccagtgtag aggtgaaatt cgtagatatt gggaagaaca ccagttgcga
660 aggcggcaat ctggctcgga actgacgctg aggcgcgaaa gcgtggggag
cgaacaggat 720 tagataccct ggtagtccac gctgtaaacg atgtgtgctg
gatgttggga aacttagttt 780 ttcagtgtcg aagctaacgt gttaagcaca
ccgcctgggg agtacgaccg caaggttgaa 840 actcaaagaa attgacgggg
gcccgcacaa gcggtggagc atgtggttta attcgaagca 900 acgcgcagaa
ccttaccagg tcttgtatgg ggaggacgtg ctcagagatg agtatttctt 960
cggacctccc gcacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt
1020 aagtcccgca acgagcgcaa cccctgtctt tagttgccat cacgtttggg
tgggcactct 1080 agagagactg ccggtgacaa gccggaggaa ggtggggatg
acgtcaagtc ctcatggccc 1140 ttatgacctg ggctacacac gtgctacaat
ggcggtgaca atgggaagct acatggtgac 1200 atgatgccga tctcaaaaaa
ccgtctcagt tcggattgca ctctgcaact cgagtgcatg 1260 aaggtggaat
cgctagtaat cgtggatcag catgccacgg tgaatacgtt cccgggcctt 1320
gtacacaccg cccgtcacac catgggagtt ggtttgacct taagccggtg agcgaaccgc
1380 aagggcgcag cgacccacgg tcgggtcagc gactggggtg aagtcgtaac aaggta
1436 3 691 DNA Unknown Organism Description of Unknown
Organismstrain S1009 3 tgatcctggc tcagagcgaa cgctggcggc atgcttaaca
catgcaagtc gcacgaacct 60 ttcggggtta gtggcggacg ggtgagtaac
gcgtaggaac ctatccagag gtgggggata 120 acaccgggaa actggtgcta
ataccgcatg atacctgagg gttaaaggct tttgttgcct 180 ttggaggggc
ctgcgtttga ttagctagtt ggttgggtaa aggctgacca aggcgatgat 240
caatagctgg tttgagagga tgatcagcca cactgggact gagacacggc ccagactcct
300 acgggaggca gcagtgggga atattggaca atgggggcaa ccctgatcca
gcaatgccgc 360 gtgtgtgaag aaggtcttcg gattgtaaag cactttcact
agggaagatg atgacggtac 420 ctagagaaga agccccggct aacttcgtgc
cagcagccgc ggtaatacga agggggctag 480 cgttgctcgg aatgactggg
cgtaaagggc gcgtaggcgg tttatacagt cagatgtgaa 540 atccccgggc
ttaacctggg aactgcattt gatacgtata gactagagtc cgagagagga 600
ttgcggaatt cccagtgtag aggtgaaatt cgtagatatt gggaagaaca ccagttgcga
660 aggcggcaat ctggctcgga actgacgctg a 691 4 691 DNA Unknown
Organism Description of Unknown Organismstrain S1019 4 tgatcctggc
tcagagcgaa cgctggcggc atgcttaaca catgcaagtc gcacgaacct 60
ttcggggtta gtggcggacg ggtgagtaac gcgtaggaac ctatccagag gtgggggata
120 acaccgggaa actggtgcta ataccgcatg atacctgagg gttaaaggct
tttgttgcct 180 ttggaggggc ctgcgtttga ttagctagtt ggttgggtaa
aggctgacca aggcgatgat 240 caatagctgg tttgagagga tgatcagcca
cactgggact gagacacggc ccagactcct 300 acgggaggca gcagtgggga
atattggaca atgggggcaa ccctgatcca gcaatgccgc 360 gtgtgtgaag
aaggtcttcg gattgtaaag cactttcact agggaagatg atgacggtac 420
ctagagaaga agccccggct aacttcgtgc cagcagccgc ggtaatacga agggggctag
480 cgttgctcgg aatgactggg cgtaaagggc gcgtaggcgg tttatacagt
cagatgtgaa 540 atccccgggc ttaacctggg aactgcattt gatacgtata
gactagagtc cgagagagga 600 ttgcggaatt cccagtgtag aggtgaaatt
cgtagatatt gggaagaaca ccagttgcga 660 aggcggcaat ctggctcgga
actgacgctg a 691 5 691 DNA Unknown Organism Description of Unknown
Organismstrain S1023 5 tgatcctggc tcagagcgaa cgctggcggc atgcttaaca
catgcaagtc gcacgaacct 60 ttcggggtta gtggcggacg ggtgagtaac
gcgtaggaac ctatcctgag gtgggggata 120 acactgggaa actggtgcta
ataccgcatg atacctgagg gtcaaaggct tttgttgcct 180 taggaggggc
ctgcgtttga ttagctagtt ggttgggtaa aggctgacca aggcgatgat 240
caatagctgg tttgagagga tgatcagcca cactgggact gagacacggc ccagactcct
300 acgggaggca gcagtgggga atattggaca atgggggcaa ccctgatcca
gcaatgccgc 360 gtgtgtgaag aaggtcttcg gattgtaaag cactttcact
agggaagatg atgacggtac 420 ctagagaaga agccccggct aacttcgtgc
cagcagccgc ggtaatacga agggggctag 480 cgttgctcgg aatgactggg
cgtaaagggc gcgtaggcgg tttatacagt cagatgtgaa 540 atccccgggc
ttaacctggg aactgcattt gatacgtata gactagagtc cgagagagga 600
ttgcggaatt cccagtgtag aggtgaaatt cgtagatatt gggaagaaca ccagttgcga
660 aggcggcaat ctggctcgga actgacgctg a 691
* * * * *
References