U.S. patent application number 11/979022 was filed with the patent office on 2009-11-12 for cellooligosaccharide-fermentative zymobacter transformed microorganisms.
Invention is credited to Kenji Okamoto, Atsuko Sugiura, Takahide Takadera, Hideshi Yanase.
Application Number | 20090280547 11/979022 |
Document ID | / |
Family ID | 34385914 |
Filed Date | 2009-11-12 |
United States Patent
Application |
20090280547 |
Kind Code |
A1 |
Yanase; Hideshi ; et
al. |
November 12, 2009 |
Cellooligosaccharide-fermentative zymobacter transformed
microorganisms
Abstract
This invention provides transformed microorganisms which can
produce ethanol from cellooligosaccharide, by introducing
.beta.-glucosidase gene by recombinant DNA method, into
microorganisms belonging to genus Zymobacter which cannot utilize
cellooligosaccharide.
Inventors: |
Yanase; Hideshi;
(Tottori-shi, JP) ; Okamoto; Kenji; (Tottori-shi,
JP) ; Takadera; Takahide; (Naka-gun, JP) ;
Sugiura; Atsuko; (Yokohama-shi, JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
1030 15th Street, N.W.,, Suite 400 East
Washington
DC
20005-1503
US
|
Family ID: |
34385914 |
Appl. No.: |
11/979022 |
Filed: |
October 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10901119 |
Jul 29, 2004 |
7323322 |
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11979022 |
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Current U.S.
Class: |
435/165 ;
435/174; 435/289.1 |
Current CPC
Class: |
Y02E 50/16 20130101;
C12N 9/2445 20130101; C12P 7/10 20130101; C12P 7/065 20130101; Y02E
50/10 20130101; Y02E 50/17 20130101; C12Y 302/01021 20130101 |
Class at
Publication: |
435/165 ;
435/174; 435/289.1 |
International
Class: |
C12P 7/10 20060101
C12P007/10; C12N 11/00 20060101 C12N011/00; C12M 1/00 20060101
C12M001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 31, 2003 |
JP |
2003-284154 |
Claims
1-7. (canceled)
8. An immobilization carrier comprising a transformed
cellooligosaccharide-fermentative microorganism attached thereto,
wherein said microorganism belongs to genus Zymobacter palmae
strain and is transformed with an exogenous .beta.-glucosidase
gene.
9. A bioreactor equipped with the immobilization carrier of claim
8.
10. A process for producing ethanol, which comprises continuously
fermenting cellobiose-containing saccharification material using
the immobilization carrier of claim 8.
11. The immobilization carrier of claim 8, wherein said exogenous
gene is a polynucleotide encoding .beta.-glucosidase obtained from
Ruminococcus albus using PCR primers of SEQ ID No. 1 and SEQ ID No.
2.
12. The immobilization carrier of claim 8, wherein said Zymobacter
palmae strain is transformed with a polynucleotide encoding
.beta.-glucosidase obtained from Ruminococcus albus using PCR
primers of SEQ ID No. 1 and SEQ ID No. 2, and said transformed
strain having been deposited at National Institute of Advanced
Industrial Science and Technology, International Patent Organism
Depositary under deposit accession number FERM BP-10047.
Description
TECHNICAL FIELD
[0001] This invention relates to a recombinant DNA containing
.beta.-glucosidase exogenous gene and to transformed microorganisms
containing said recombinant DNA. Said transformed microorganisms
can be utilized for effective production of ethanol from
cellobiose-containing feedstocks.
BACKGROUND ART
[0002] Representative microorganisms used for ethanol production
are yeast belonging to genus Saccharomyces or bacteria belonging to
genus Zymomonas or Zymobacter. These microorganisms normally
produce ethanol efficiently from monosaccharide such as glucose,
but are incapable of producing ethanol from oligosaccharide or
polysaccharide. In carrying out ethanol production from cellulosic
biomass as the feedstock, therefore, it is necessary to first
degrade cellulose to monosaccharide which can be fermented by
microorganisms. Degradation and saccharification of cellulosic
biomass are normally carried out by enzyme process using cellulase
or acid saccharification process using sulfuric acid or the like.
Whereas, problems are present with these methods such that complete
degradation of cellulose to monosaccharide is occasionally found
difficult, or excessive reaction to raise the degradation ratio may
reduce the sugar recovery and in consequence aggravate ethanol
production efficiency.
[0003] Accordingly, therefore, for improving yield in ethanol
production from biomass feedstocks, it is necessary to introduce
.beta.-glucosidase gene into the microorganisms used for ethanol
production to construct transformed microorganisms which are
capable of producing ethanol on substrate of cellooligosaccharide,
a partial decomposition product of cellulose.
[0004] Genus Zymomonas and genus Zymobacter are known to show
higher fermentation speed than yeast of genus Saccharomyces, and
various attempts were made relating to construction of transformed
microorganisms using Zymomonas bacteria as host cells. For example,
U.S. Pat. No. 5,712,133 disclosed transformation of Zymomonas
bacteria to impart thereto pentose fermenting ability. However,
when .beta.-glucosidase gene is introduced into Zymomonas bacteria
by the method described in said US patent, .beta.-glucosidase is
not secreted exocellularly, and furthermore because
cellooligosaccharide cannot permeate through cell walls of
Zymomonas, fermentation of cellooligosaccharide to ethanol is
impossible. WO98/45451 disclosed transformation of
cellobiose-incorporating gene of bacteria belonging to genus
Klebsiella into Zymomonas to enable intracellular ethanol
production from cellobiose, but its ethanol production efficiency
is low.
DISCLOSURE OF THE INVENTION
[0005] A main object of the present invention is to provide
transformed microorganisms capable of producing ethanol from
cellooligosaccharide, by introducing .beta.-glucosidase into
microorganisms belonging to genus Zymobacter which are incapable of
utilizing cellooligosaccharide, by recombinant DNA method.
[0006] We noticed microorganisms which could produce
.beta.-glucosidase and carried out various screening procedures, to
successfully obtain enzymes exhibiting broad range of
cellooligosaccharide digesting characteristics. Because no
host-vector system with Zymobacter bacteria was established then,
we made concentrative studies on construction of vectors,
transformation method and cloning enzyme genes which participate in
cellooligosaccharide metabolism, to now discover that use of
Zymobacter bacteria as the host cells enabled extracellular
secretion of transformed .beta.-glucosidase, whereby it becoming
possible to exclude the influence of rate controlling by
incorporation of substrate and to produce ethanol effectively from
fermentation feedstocks containing cellooligosaccharide. The
present invention is whereupon completed.
[0007] Accordingly, therefore, the present invention provides a
transformed Zymobacter microorganism into which exogenus gene of
.beta.-glucosidase is introduced and which has
cellooligosaccharide-fermentative ability, i.e., an ability to
produce ethanol on cellooligosaccharide substrate.
[0008] The invention also provides recombinant DNA which is
constructed by ligating a DNA fragment with a vector, said DNA
fragment encoding .beta.-glucosidase derived from
.beta.-glucosidase-producing bacterial strain.
BRIEF EXPLANATION OF DRAWINGS
[0009] FIG. 1 shows a restriction enzyme cleavage map of a vector
plasmid.
[0010] FIG. 2 shows a restriction enzyme cleavage map of a
recombinant plasmid containing .beta.-glucosidase gene.
[0011] FIG. 3 is a graph showing ethanol productivity by
fermentation of cellobiose by recombinant Zymobacter palmae.
[0012] FIG. 4 is a graph showing ethanol productivity by batch
fermentation of cellobiose by recombinant Zymobacter palmae.
[0013] Hereinafter the present invention is explained in further
details.
[0014] In the present invention, a microorganism having
.beta.-glucosidase producing ability is used as DNA donor, from
which the DNA encoding .beta.-glucosidase is isolated and purified
and thereafter cleaved by various methods to provide a DNA fragment
containing .beta.-glucosidase gene. Ligating this
.beta.-glucosidase gene-containing DNA fragment with a vector-DNA
fragment by, for example, DNA ligase, to form a recombinant DNA
containing .beta.-glucosidase gene.
[0015] The microorganisms used as donors of .beta.-glucosidase
gene-containing DNAs are subject to no special limitation, and any
of those which can digest cellulose, partially decomposed cellulose
or cellooligasoccharide can be used. Whereas, microorganisms
belonging to genus Ruminococcus, inter alia, Ruminococcus albus,
are used with particular preference. Other Ruminococcus
microorganisms or those belonging to genera other than Ruminococcus
and having .beta.-glucosidase-producing ability, or those which do
not have .beta.-glucosidase-producing ability due to abnormality at
promoter site or ribosome linkage site but encode on their DNA
structural genes of .beta.-glucosidase, can also be used as
.beta.-glucosidase gene-containing DNA donors. Furthermore,
transformed microorganisms into which .beta.-glucosidase structural
genes have been introduced by such means as recombination of genes
also are useful as .beta.-glucosidase gene-containing DNA
donors.
[0016] .beta.-glucosidase gene-containing recombinant DNA can, as
introduced into host microorganisms belonging to genus Zymobacter,
construct transformed microorganisms having .beta.-glucosidase
producing ability. So introduced recombinant DNA may be
incorporated in the genome of the Zymobacter host cells in whole or
in part, or the whole or a part may be present on the vector used
for the transformation.
[0017] Separation and purification of the intended DNA from above
donor microorganisms can be effected by any means known per se, for
example, the method by Saito, Miura et al. (Biochem. Biophys.
Acta., Vol. 72, 619-629, 1963) or modifications thereof, or those
using commercially available DNA extraction kits. Hereinafter a
method following the one by Saito, Miura et al. is more
specifically explained.
[0018] First, the donor microorganism is inoculated into a suitable
liquid medium such as an yeast-starch medium containing 0.5%
glycine (composition: yeast extract, 0.2%; soluble starch, 1.0%; pH
7.3), followed by culture under agitation at 4-60.degree. C.,
preferably 30.degree. C., for 8-48 hours, preferably for an
overnight. After termination of the culture, the culture solution
is subjected to a solid-liquid separation means, for example,
centrifugation at 0-50.degree. C., preferably 4.degree. C., and at
a rotation rate of 3,000-15,000 rpm, preferably 10,000 rpm.
[0019] Thus collected microorganisms are then suspended in a VS
buffer (0.15M NaCl, 0.1M EDTA, pH 8.0). After addition of lysozyme,
the suspension is allowed to stand at 4-45.degree. C., preferably
37.degree. C., for 0.5-4 hours, preferably an hour, to provide a
protoplast liquid. To said liquid TSS buffer (0.1M TRIS, 0.1M NaCl,
1% SDS, pH 9.0) and 5M NaCl are added to dissolve the protoplast,
followed by addition of a TE solution (10 mM TRIS, 1 mM EDTA, pH
8.0)-saturated phenol, to effect mild and sufficient suspension.
The resultant suspension is centrifuged at 0-50.degree. C.,
preferably 4.degree. C., and at a rotation rate of 3,000-15,000
rpm, preferably 12,000 rpm, and the formed upper layer (aqueous
phase) is suspended in chloroform. The suspension is centrifuged at
0-50.degree. C., preferably 4.degree. C. and at a rotation rate of
3,000-15,000 rpm, preferably 12,000 rpm. Thus formed upper layer
(aqueous phase) is again suspended using phenol and chloroform.
[0020] Subsequently cold ethanol is added to the suspension, and
whereupon formed opaque crude chromosome DNA is recovered. Said DNA
is dissolved in SSC buffer (0.15M NaCl, 0.015M sodium citrate) and
the solution is dialyzed against SSC buffer for an overnight. To
the dialysate ribonuclease is added to a final concentration of
1-50 .mu.g/ml, preferably 10 .mu.g/ml, followed by standing at
4-45.degree. C., preferably 37.degree. C., for 0.5-16 hours,
preferably 2 hours. Protease is further added to a final
concentration of 0.1-10 .mu.g/ml, preferably 1 .mu.g/ml, followed
by standing at 4-45.degree. C., preferably at 37.degree. C., for 15
minutes-8 hours, preferably 30 minutes. Similarly to the above, the
system after the standing is treated with phenol and chloroform and
dialyzed against SSC buffer to provide a purified chromosome DNA
liquid of the donor microorganism.
[0021] Thus obtained donor microorganism's DNA is cleaved by, for
example, restriction enzyme, and from which DNA fragments of sizes
less than 1 kbp are removed by sucrose density gradient method. The
remnant can be used as the donor DNA fragment. The restriction
enzyme useful in that occasion is subject to no special limitation,
but any of various enzymes such as EcoRI which cleaves DNA can be
used. Besides the above enzymatic method, DNA can be cleaved by
ultrasonic treatment or physical shearing force. A treatment of the
donor DNA fragment ends with, for example, Klenow fragment or an
enzyme such as DNA polymerase or mung bean nuclease in that
occasion is preferred for improving subsequent binding efficiency
with vector DNA. Moreover, PCR-amplified products using donor
microorganism's DNA or a fragment thereof as a template can also be
used as the donor DNA fragments either as they are or after
treating them as described above.
[0022] On the other hand, while vector DNA fragments are subject to
no particular limitation, for example, pRK290, pMFY 40 or pMFY 31
derived from inter-Gram-negative bacterial broad host range
plasmid, which are cleaved with restriction enzymes are
conveniently used. Vectors other than above-named, for example,
broad host range plasmids of known Gram-negative bacteria, may be
suitably selected and used. Useful restriction enzymes are not
limited to those which produce adhesive ends but various other
enzymes which cleave DNAs can be used. Furthermore, vector DNAs can
also be cleaved by similar methods to those used for cleaving DNAs
of said donor microorganisms.
[0023] Thus obtained vector DNA fragments may be treated with
alkaline phosphatase in advance of their ligating reaction with
aforesaid donor DNA fragments, to improve ligation efficiency with
said donor DNA fragments. Furthermore, when a donor DNA fragment is
prepared by PCR amplification, its ligating efficiency can be
improved by applying in advance a restriction enzyme site-imparting
primer such as sal I to both ends of the amplified fragment, and by
using a vector fragment which is cleaved with the same restriction
enzyme which is used for cleaving the DNA fragment. The ligating
reaction between the donor DNA fragment and vector DNA fragment can
be conducted by conventionally practiced methods, for example, one
using known DNA ligase. For instance, a recombinant DNA can be
constructed in vitro by the action of a suitable DNA ligase, after
annealing the involved donor DNA fragment and vector DNA fragment.
Where necessary, furthermore, the annealed fragments may be
introduced into a host microorganism and converted to a recombinant
DNA, utilizing in vivo DNA-repairing ability.
[0024] As the host microorganism into which the recombinant DNA
containing a donor DNA fragment and a vector DNA fragment is to be
inserted, any that has ethanol fermentation ability and that can
stably retain said recombinant DNA can be used. Whereas,
microorganisms belonging to genus Zymobacter, generally Zymobacter
palmae, are conveniently used in the present invention. Method for
introducing such a recombinant DNA into the host microorganism is
not particularly limited, but when Zymobacter palmae or the like is
used as the host cell, introduction of the recombinant DNA
utilizing electrical stimulation such as electroporation is
preferred. Also as to ethanol-producing microorganisms other than
Zymobacter palmae, for example, Zymomonas mobilis, yeast and other
hosts, recombinant DNAs can be introduced thereinto by similar
methods.
[0025] As a growth medium for so obtained transformed
microorganisms, for example, where the host microorganism belongs
to Zymobacter, RM media are frequently used. Where host
microorganisms other than Zymobacter, such as Bacillus subtilis,
yeast or the like are used, cultivation in various media suitable
for individual host microorganisms can be conducted, and
cultivation conditions such as culture temperature can also be
suitably designed according to the properties of the used host
microorganism. When the vector DNA fragment codes various
antibiotic-resistant genes, addition of an adequate amount of a
corresponding antibiotic to the medium enables more stable
retention of the recombinant DNA which has been introduced.
Furthermore, when the used vector DNA is one which codes a gene
supplementing auxotrophicity of the host microorganism, stability
of the recombinant DNA can similarly be improved by using a medium
which contains none of the required nutrient.
[0026] The present invention provides a recombinant DNA which
enables imparting to Zymobacter microorganisms cellobiose
fermentation ability by recombinant DNA method; and transformed
microorganisms containing the recombinant DNA fragment(s). Use of
said transformed microorganisms enables efficient ethanol
production from cellobiose-containing sugar solution as the
feedstock.
[0027] Ethanol production from a cellobiose-containing sugar
solution as the feedstock can be conducted through the steps of
fermenting a saccharified feedstock containing cellobiose by the
action of said cellooligosaccharide-fermentative transformed
microorganism, and recovering ethanol from the resultant
fermentation liquid, according to, for example, alcoholic
fermentation method known per se, using a carrier on which said
transformed microorganisms are immobilized.
[0028] Immobilization of the transformed microorganisms on said
carrier can be effected by any of conventional techniques known per
se, for example, entrapping, physical adsorption or covalent
bonding.
[0029] As the carrier, those preferred have hollow, rugged or
porous forms having a large surface area per unit volume, or can
swell upon absorbing water, are fluidable and have particle sizes
and specific gravity values which do not allow the carrier's easy
flowing out of the reaction system. The carrier's configuration may
be versatile, for example, special forms of plates, fibers or
cylinders, sponge-like structures, particles, blocks or cubes. Of
those, fine particles which allow easy ensuring of fluidability and
sufficient surface area are preferred. As materials for the
carrier, various organic and inorganic materials heretofore used as
carrier materials for microorganisms or enzymes can be used,
examples of which include inorganic materials such as granular
activated carbon, crushed activated carbon, charcoal, zeolite, mica
and sand; resin materials such as photo-hardenable resin,
polyurethane, polyvinyl alcohol, polyethylene, polyacrylamide,
polyester, polypropylene, agar, alginic acid, carrageenan,
cellulose, dextran, agarose, ion-exchange resin and the like;
porous ceramics such as silica gel; anthracite; and activated
carbon or the like mixed in resinous material. These may be used
either alone or in combination of two or more.
[0030] Said immobilization carriers are normally used as being
filled in bioreactors. As bioreactors used for fermentation, there
are continuously stirred tank type, packed bed type, membrane type,
fluidized bed type and horizontal type, as classified by their
operation system. Use of such bioreactors allows continuous
fermentation and dispenses with supplying and recovery of the
microorganisms, etc. and, therefore, is preferred.
[0031] In the occasion of said alcoholic fermentation, various
nutrition sources for the microorganisms may be blended in the
sugar solutions where necessary. For example, as nitrogen source,
yeast extract, corn steep liquor, pepton, meat extract, bonito
extract and the like can be used.
[0032] Hereinafter the invention is still more specifically
explained referring to working examples, it being understood that
the invention is not limited thereto.
EXAMPLES
Example 1
Method for Introducing Zymobacter palmae Gene
[0033] Presence of self-transmissible, multi-drug resistant
plasmide DNA in Gram-negative bacteria such as Escherichia coli and
Pseudomonas in general has been reported, and these plasmids are
known to propagate among E. coli or Pseudomonas bacteria. These
broad host range multi-drug resistant plasmids and plasmids in
which the genic domain participating in the transmissibility and
self-replication of these broad host range multi-drug resistant
plasmids remains, are occasionally utilized as broad host range
vector plasmids (BIO/TECHNOLOGY, November, 784-791, 1983). Whereas,
vector plasmid of Zymobacter palmae and a method for introducing
its gene have not yet been developed. We, therefore, selected from
broad host range plasmids among Gram-negative bacteria the
following three kinds of plasmids of pRK290 and pMFY40 which have
Tc-resistance marker and pMFY31 having Cm-resistance marker (Agric.
Biol. Chem. Vol. 49(9), 2719-2724, 1985) (FIG. 1) as the vector
plasmids for introducing genes into Zymobacter palmae. Because no
gene-introducing method into Zymobacter palmae was known, we used
electroporation method among generally used methods for gene
introduction.
[0034] Zymobacter palmae (ATCC 51623) was statically cultured for
an overnight in RM medium (2.0% glucose, 1.0% Bacto-yeast extract,
0.2% KH.sub.2PO.sub.4, pH 6.0). Five (5) ml of the pre-cultured
liquid was subcultured in 50 ml of T medium (2.0% glucose, 1.0%
Bacto-yeast extract, 1.0% KH.sub.2PO.sub.4, 0.2%
(NH.sub.4).sub.2SO.sub.4, 0.05% MgSO.sub.4.7H.sub.2O, pH 6.0) at
30.degree. C. for 90 minutes. The cultured liquid was centrifuged
at 4.degree. C., 300 rpm and for 10 minutes to isolate the
microorganism cells to which 20 ml of cooled 10% glycerol was
added, followed by suspension and washing. Conducting another
centrifugation at 4.degree. C., 3000 rpm for 10 minutes, competent
cells were obtained. Two-hundred (200) .mu.l of the competent cells
and 10 .mu.l of vector-plasmid DNA solution were mixed on ice,
transferred into a cuvette attached to an electroporation device,
and electric pulse was applied thereto under such conditions as:
voltage 200V, capacitance 250 .mu.FD and resistance 200.OMEGA..
Immediately then 1 ml of T medium was added to the cuvette, the
cells were statically cultured at 30.degree. C. for an hour, and
caused to form a colony on a selective medium to which antibiotic
to cope with expression of the drug resistant gene in the used
broad host range plasmid vector had been added. The transformation
efficiency of Zymobacter palmae with said plasmid pMFY 40 by the
gene introducing method we have developed was about
1.times.10.sup.6/.mu.g DNA (Table 1).
TABLE-US-00001 TABLE 1 Transformation Efficiency of Zb. palmae
Transformation efficiency Plasmid used (number of bacteria/.mu.g)
pRK290 7.45 .times. 10.sup.3 pMFY40 1.01 .times. 10.sup.6 pMFY31
9.21 .times. 10.sup.5
Example 2
Preparation of Recombinant Plasmid Containing .beta.-Glucosidase
Gene
[0035] Ruminococcus albus-derived .beta.-glucosidase gene was
amplified by PCR using the genome DNA prepared from cells of said
bacterium as the template, and the amplified DNA fragment was
inserted in the vector plasmid to form a recombinant plasmid. As
the primers used in the PCR for amplifying .beta.-glucosidase gene,
the following two primers were used, which were so designed, based
on known base sequence of said gene (Nucleic Acids Res. Vol. 18,
671, 1990), to include the promoter domain conformed in the region
upstream of .beta.-glucosidase gene and to be imparted at its two
ends SalI site as restriction enzyme cleavage sites:
TABLE-US-00002 BGN primer: (SEQ ID NO: 1)
5'-GCGGTCGACATCAAGGTGTGATGTTGATTATACC-3' BGC primer: (SEQ ID NO: 2)
5-CGCGTCGACTCATGTTTGACAGCTTATCATCGAT-3'.
[0036] The DNA fragment of about 3.2 kbp containing the promoter
and .beta.-glucosidase gene as formed by the PCR was cleaved with
restriction enzyme SalI. The DNA fragment after the SalI cleavage
was given an alkali phosphatase-treatment and mixed with vector
plasmid pMFY31, and they were ligated, utilizing ligase. Ten (10)
.mu.l of this ligase reaction solution containing this recombinant
plasmid was mixed with 200 .mu.l of Zymobacter palmae competent
cells as formed in Example 1, and transformed by electroporation
method. The transformed strain was selected as blue colony on T
plate medium to which 100 .mu.g/ml of ampicillin and 20 .mu.g/ml of
5-bromo-4-chloro-3-indolyl-.beta.-D-glucopyranoside (x-glc) were
added as the chemicals. Thus obtained transformed strain has been
deposited with National Institute of Advanced Industrial Science
and Technology, International Patent Organism Depositary at AIST
Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken
305-8566, Japan under deposition number of FERM P-19450 (which has
been transferred to international deposition under Budapest Treaty
since Jun. 30, 2004 and given a deposition number of FERM
BP-10047). The recombinant plasmid into which .beta.-glucosidase
gene was inserted was named pMF31-.beta.g (FIG. 2).
Example 3
Cellooligosaccharide Fermentation Ability by Recombinant Zymobacter
palmae Strain
[0037] Expression and intracellular localized presence of
.beta.-glucosidase in the recombinant Zymobacter palmae prepared in
Example 2 were investigated.
[0038] Each of Zymobacter palmae/pMFY31-.beta.g strain, Zymobacter
palmae/pMFY31 strain and E. coli JM109/pMFY-31-.beta.g strain was
cultured and cell fractionation of recovered bacterium was
conducted (Science. Vol. 156(781), 1451-1455, 1967).
.beta.-glucosidase activity of each of the cell fractions, i.e.,
supernatant of culture solution corresponding to extracellular
fraction, bacterium washing corresponding to the cell cortex
fraction, hypertonic solution washing, osmotic shock solution
corresponding to periplasmic fraction, cell membrane fraction and
cytoplasm fraction, were measured (J. Bacteriol., Vol. 161(1),
432-434, 1985).
[0039] .beta.-glucosidase activity in Zymobacter
palmae/pMFY-31-.beta.g strain was of approximately the same level
with that of E. coli JM109/pMFY31-.beta.g strain. Furthermore, the
expressed .beta.-glucosidase was localized, as for Zymobacter
palmae/pMFY31-.beta.g, as 29.5% in the bacterium washing, 17.1% in
the osmotic shock solution, and 29.5% in the cell-free extract,
exhibiting higher secretion ability compared with E. coli (Table
2). That is, about 50% of the total expressed activity permeated
through the cell membrane and was secreted.
[0040] Recombinant bacterium Zymobacter palmae/pMFY31-.beta.g
strain was inoculated in culture media each comprising 2% glucose,
2% cellobiose, and 2% glucose+cellobiose as the respective carbon
source, and bacterial growth therein and ethanol production with
time were measured. In the medium wherein cellobiose was the sole
carbon source, the growth rate dropped compared with that in the
medium comprising glucose alone, but it consumed 2% cellobiose by
the 10th day of the culture to produce ethanol at the theoretical
yield (FIG. 3).
TABLE-US-00003 TABLE 2 Expression and Intracellular Localization of
.beta.- glucosidase in Zymobacter palmae Zb. palmae T109 Zb. palmae
T109 E. coli JM109 (pMFY31) (pMFY31-.beta.g) (pMFY31-.beta.g) Cell
Activity Localization Activity Localization Activity Localization
fraction (U/ml) (%) (U/ml) (%) (U/ml) (%) Supernatant of <0.01
-- 0.07 6.7 0.01 1.3 culture solution Bacterium <0.01 -- 0.31
29.5 0.06 7.6 washing Hypertonic <0.01 -- 0.05 4.8 0.01 1.3
solution washing Osmotic shock <0.01 -- 0.18 17.1 0.02 2.5
solution Cytoplasmic <0.01 -- 0.31 29.5 0.59 74.7 fraction
Membrane <0.01 -- 0.13 12.4 0.10 12.7 fraction Total activity
1.05 0.79 1 unit: amount of the enzyme to release p-nitrophenol
from 1 .mu.mole of p-nitrophenyl-.beta.-D-glucopyranoside per
minute
Example 4
[0041] Recombinant Zymobacter palmae FERM P-19450 (FERM BP-10047)
strain was inoculated in CB medium (2.0% cellobiose, 1.0% yeast
extract, 1.0% KH.sub.2PO.sub.4, 0.2% (NH.sub.4).sub.2SO.sub.4,
0.05% MgSO.sub.4.7H.sub.2O, pH 6.0) using biomass partially
saccharified liquid-derived cellobiose as the sole carbon source,
and statically cultured for five days to provide a pre-culture
solution. For the main culture the above CB medium was used, in
which 10% to the main culture CB medium of said pre-culture
solution was inoculated, followed by culturing under mild agitation
at 30.degree. C. Growth rate of the inoculated cells, cellobiose
concentration and ethanol concentration changes with time were
regularly measured, to confirm that substantially all of the
cellobiose was consumed by 7 days' culture and ethanol was produced
at the theoretical yield (FIG. 4).
Example 5
[0042] Using a medium prepared by adding yeast extract,
KH.sub.2PO.sub.4, (NH.sub.4).sub.2SO.sub.4 and MgSO.sub.4.7H.sub.2O
to a sugar solution (10% glucose, 1% cellobiose) formed by sulfate
saccharification of waste wood, in the amounts, respectively, of
1.0%, 1.0%, 0.2% and 0.05% to the sugar solution and adjusting pH
to 6.0, continuous fermentation was conducted. Recombinant
Zymobacter palmae FERM P-19450 (FERM BP-10047) was immobilized on
photo-hardenable resin ENTG.TM.-3800 (manufactured by Kansai Paint)
by entrapping. For the continuous fermentation a draft tube-formed
bioreactor (fluidized bed type) was used. After throwing the
immobilization carrier into the reactor at a fill ratio of 20%, the
medium was continuously poured into the reactor from a lower part.
A fluidized bed was formed by collecting the carbon dioxide formed
by the fermentation and recycling it into the reactor from a lower
part thereof. The continuous fermentation could be carried out at
30.degree. C. and at a dilution ratio D equaling 0.1 h.sup.-1
stably for more than a month, with the sugar consumption ratio not
lower than 99% and ethanol yield not less than 95%.
Example 6
[0043] Continuous fermentation was conducted using a medium
prepared by adding yeast extract, KH.sub.2PO.sub.4,
(NH.sub.4).sub.2SO.sub.4 and MgSO.sub.4.7H.sub.2O to a sugar
solution (8% glucose, 2% cellobiose) formed by enzymatic
saccharification of waste paper-derived cellulose with cellulase,
in the amounts, respectively, of 1.0%, 1.0%, 0.2% and 0.05% to the
sugar solution and adjusting pH to 6.0. Recombinant Zymobacter
palmae FERM P-19450 (FERM BP-10047) cells were immobilized on
cylindrical (2 mm.phi..times.3 mm) polypropylene carrier which was
thrown into the cell suspension. For the continuous fermentation a
fixed bed bioreactor (packed bed type) was used. After throwing the
immobilization carrier into the reactor at a fill ratio of 80%,
said medium was continuously supplied into the reactor from a lower
part. The continuous fermentation could be carried out at
30.degree. C. and at a dilution ratio D equaling 0.2 h.sup.-1
stably for more than a month, with the sugar consumption ratio not
lower than 99% and ethanol yield not less than 95%.
Sequence CWU 1
1
2134DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Construct 1gcggtcgaca tcaaggtgtg atgttgatta tacc
34234DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Construct 2cgcgtcgact catgtttgac agcttatcat cgat 34
* * * * *