KR101801786B1 - Paenibacillus sp. CAA11 capable of saccharification and fermentation of cellulose and transformed strain thereof - Google Patents

Abstract

The present invention relates to a novel strain capable of saccharifying and fermenting biomass-derived cellulose and a recombinant strain having improved biomass glycosylation ability. The present invention also relates to a method for producing a substance used as a raw material of bioenergy such as ethanol, acetic acid and formic acid by using the strain and the recombinant strain, and the strain and the recombinant strain are useful for the bioenergy industry have.

Description

Fannibacillus CAA11 and Fannibacillus CAA11 transforming strains {Paenibacillus sp. CAA11 capable of saccharification and fermentation of cellulose and transformed strain thereof < RTI ID = 0.0 >

Disclosed herein are novel strains capable of saccharifying and fermenting biomass-derived cellulose and genetic recombinant strains thereof.

Recently, research on alternative energy has been increasing due to the restriction of the use of fossil fuels due to the surge in oil prices and strengthening of the implementation of the Convention on Climate Change. The proportion of energy sources using biomass is expected to increase significantly, Interest in utilization is increasing rapidly.

The process using biomass is divided into pretreatment, saccharification and fermentation processes, and the cost of biofuels is low due to the high cost of saccharification enzymes used in the saccharification process.

Recently, a lot of research has been concentrated on the development of an integrated bioprocess for simultaneous saccharification and fermentation of a single microorganism for economical utilization of biomass. In order to develop the integrated bioprocess, research on the recombination of glycosidase genes into monosaccharide-degrading E. coli or yeast in which genetic recombination technology has been established is underway, but the glycosylation efficiency is low and the result is low biofuel production. In order to develop microorganisms optimized for integrated bioprocess development, it is necessary to study not only monosaccharide degrading microorganisms, but also microorganisms capable of decomposing polysaccharides and glycosylation improvement studies through gene recombination technology.

  Current Opinion in Biotechnology (2012) 23: 364   Applied and Environmental Microbiology (2010) 76: 6360   Journal of Biomedicine and Biotechnology (2012) article ID405842

It is an object of the present invention to provide a novel strain capable of efficiently producing a raw material of bioenergy by allowing saccharification of biomass and a fermentation process using the biomass by a single process.

It is also an object of the present invention to provide a genetically modified organism strain having improved biomass glycosylation ability of the novel strain.

One aspect of the present invention for achieving the above object provides a Companion Bacillus CAA11 (Paenibacillus sp. CAA11) or its culture.

One aspect of the present invention provides a Bacillus subtilis 168 (Bacillus subtilis 168) genetically modified strain of the transformed by the vector containing the cellulase gene, Companion Bacillus CAA11 (Paenibacillus sp. CAA11-Cel ) , and its culture .

One aspect of the present invention is a method for producing a recombinant strain of Paenibacillus sp. CAA11-Cel of Fanny Bacillus CAA11, comprising the steps of: preparing an expression vector by inserting a promoter into a shuttle vector; Adding the promoter, the signal peptide and the cellulase encoding gene to the expression vector by overlap PCR, and cloning the recombinant vector; And introducing the recombinant vector into a FannBacillus CAA11 strain, thereby transforming the transformant.

One aspect of the present invention is Bacillus Waist CAA11 or gene recombinant strain of the Bacillus CAA11 Companion (Paenibacillus sp . CAA11-Cel) in the presence of woody biomass or cellulose, and a method of producing the fermented material containing the same.

One novel Bacillus Waist CAA11 (Paenibacillus sp. CAA11) strain in accordance with an embodiment of the present invention can decompose the cellulose without the need for adding enzymes such as cellulose-raised, the culture is possible as well as in anaerobic conditions, aerobic conditions.

The Companion (Paenibacillus sp. CAA11-Cel) genetically modified strain of Bacillus CAA11 according to one embodiment of the present invention is to improve the biomass saccharification ability of Companion Bacillus CAA11 strain of the Companion Bacillus CAA11 being is a glycosylated enzyme cellulase excess expression .

Recombinant strains of the Bacillus CAA11 Companion and Companion Bacillus CAA11 according to one embodiment of the present invention (Paenibacillus sp . CAA11-Cel) can not only perform the saccharification reaction of cellulose but also can produce a material used as a raw material of bio energy by using glucose, xylose and cellobiose, which are the reducing sugars obtained therefrom, as carbon sources, , Particularly in the field of woody biomass. Biomass can be decomposed to produce useful products such as formic acid, acetic acid, and ethanol from the resulting sugars.

In order to confirm the cellulose decomposing activity of Fanny Bacillus strain CAA11, the strain was cultured on a medium containing cellulose, and then the strain was cultured. Fig. 1 (a) , And Fig. 1 (b) shows after dyeing.
Fig. 2 is a diagram showing phylogenetic positions of Fanny Bacillus CAA11 strain.
FIGS. 3, 4A to 4C, and 5A to 5C are graphs showing the degree of growth according to culture conditions in the culture of Fanny Bacillus CAA11 strain.
6A to 6C are diagrams showing the degree of sugar consumption and the amount of product produced by culturing Fanny Bacillus strain CAA11 together with disaccharides or monosaccharides.
Figures 7a and 7b is a view showing a result of analysis of fermentation products produced and the degree of sugar consumption when using simultaneously the Companion Bacillus CAA11 strain yuktan saccharide glucose and disaccharide cellobiose or pentose is xylose and the disaccharide cellobiose.
8 is a graph showing the degree of growth of the Fanny Bacillus CAA11 strain in a medium to which soluble cellulose dissolved in a liquid is added.
9 is a diagram showing by measuring the growth degree of the Bacillus CAA11 Waist strain in the water-insoluble cellulose is added to the medium.
10 (a) and 10 (d) show the growth of strains (Figs. 10A and 10B) and the bioenergy feedstock (Figs. 10C and 10D) when the Fanny Bacillus CAA11 strain was divided into an aerobic condition and an anaerobic condition in an insoluble cellulose medium. .
FIGS. 11A and 11B are a diagram (FIG. 11A) and a result of promoter intensity measurement (FIG. 11B) of a vector (pAD123-promoter-GFP) for measuring the promoter strength according to an embodiment of the present invention.
FIG. 12 is a diagram showing a production chart of a vector for expressing saccharifying enzyme (pNW33N P43 P43 nprB 168cel5) using the promoter developed in FIGS. 11A and 11B according to an embodiment of the present invention.
FIG. 13 is a graph showing the results of enhancing the saccharifying enzyme activity of the Fanny Bacillus CAA11 recombinant strain. FIG.
Figs. 14A to 14E are diagrams showing the results of analysis of the products produced by culturing the recombinant strains of Fanny Bacillus CAA11 in a cellulose culture medium. Fig.

The present invention, the Bacillus CAA11 Waist strain on Companion Bacillus CAA11 (s Paenibacillu sp. CAA11) In one aspect is the separated first identified by the inventors. The present inventors named the newly isolated strain as Fanny Bacillus CAA11 ( Paenibacillus sp. CAA11) and deposited it on the Korean Microorganism Preservation Center on Nov. 6, 2014 and received the deposit number KCCM11602P. Therefore, the present invention is the accession number KCCM11602P In another aspect, the present invention relates to a Bacillus Waist CAA11 (Paenibacillus sp. CAA11). One aspect of the Companion CAA11 Bacillus strain of the present invention may comprise a sequence of SEQ ID NO: 3.

Waist Bacillus is a gram-positive bacteria of the bars, the Companion Bacillus CAA11 of the present invention is a novel strain of accession number KM275937 (Genebank accession number KM275937) of a gene bank belonging to the phylogenetic tree in the Companion Bacillus. Generally, sequence similarity is 98% or less if there is classified as a novel strain, the Bacillus Waist CAA11 is conventional Waist Bacillus Varennes troublesome SAFN ^016-T ( Paenibacillus barengoltzii SAFN-016 ^T ) and 97.01%, Fanny Bacillus sp. 3PO2SA ^T ( Paenibacillus phoenicis 3PO2SA ^T ) and 96.16%.

This invention also relates in another aspect to a culture in which the culture of Bacillus CAA11 Companion (s Paenibacillu sp. CAA11) or Bacillus Waist CAA11 (Paenibacillus sp. CAA11) the accession number KCCM11602P is as described above. In addition, the present invention relates to a culture method comprising culturing in a further aspect, the Bacillus CAA11 Companion (s Paenibacillu sp. CAA11) or phosphorus, Companion Bacillus CAA11 (Paenibacillus sp. CAA11) Accession number KCCM11602P.

As used herein, the term "cultivation" is not limited as long as it is a culturing method known in the art, and includes a fermentation substance obtained by culturing the strain isolated and identified first by the present inventors or a method of culturing the strain do. Specifically, the culture of the Bacillus CAA11 Waist strain herein be carried out in the presence of sodium chloride, but is not limited thereto. The concentration of the sodium chloride in one aspect of the present invention may be in the range of 0.1 to 10% (w / v), 1 to 3% (w / v) for example, but are not limited to, if possible, the incubation of Companion Bacillus CAA11 strain.

In one aspect, said Waist Bacillus culture of CAA11 strain is pH 5.5 to 8 and / or 25 to be carried out under the conditions of 45 ℃, but not limited to not create a further excellent strain growth, and a by-product in the condition in this specification Can be observed. In view of the above, the cultivation of the Fanny Bacillus CAA11 strain of the present invention can be carried out under the conditions of pH 5.5 to 8, pH 5.7 to 7.8, pH 5.9 to 7.6, pH 6.1 to 7.4 or pH 6.3 to 7.2 and / or 26 to 44 ° C, 27 to 43 占 폚, 28 to 42 占 폚, 29 to 41 占 폚, or 30 to 40 占 폚.

The present invention relates to a culture method In still another aspect, which comprises Waist CAA11 Bacillus or to the cultured under accession number KCCM11602P the Companion Bacillus CAA11 wood-based biomass or cellulose presence under aerobic or anaerobic conditions. In one aspect, the term "cultivation" as used herein may occur in both aerobic and anaerobic conditions, but is not limited thereto.

Conventionally known bacteria to break down cellulose, such as Clostridium thermo selreom (Clostridium thermocellum) or Clostridium cellular Robo lance (Clostridium cellulovorans) had a difficulty in the culture, because in most of the anaerobic bacteria. Therefore, there is a problem that the recombinant DNA technology is subject to a lot of restrictions. However, the Companion Bacillus according to one aspect of the present invention is CAA11 anaerobic as well as aerobic culture with up to culture, it is possible to produce a bio-energy material such as formic acid, acetic acid, ethanol from the culture. That is, the Companion Bacillus CAA11 may decompose the cellulose to react quickly to changes in the dissolved oxygen conditions and the oxidation-reduction condition. In one embodiment the Bacillus Waist CAA11 is aerobic and can produce acetic acid through culture under conditions, can produce formic acid, acetic acid, ethanol via acid culture under anaerobic conditions.

The invention also relates to a method for producing fermentation products, which comprises culturing the Bacillus CAA11 Companion (s Paenibacillu sp. CAA11) or Bacillus Waist CAA11 deposited with number KCCM11602P from another point of view. The "fermentation substance" is the broadest meaning including not only substances obtained by culturing the novel strain of the present invention, i.e., fermentation, but also substances obtained by culturing a genetically modified microorganism having the novel strain as a parent strain. Examples of the fermentation material that can be produced include raw materials capable of producing bioenergy. In one aspect the method of production may comprise cultured in the wood-based biomass or cellulose present the Companion Bacillus CAA11. Specifically, the production method is characterized in that the Fani Bacillus CAA11 decomposes the woody biomass or cellulose to produce at least one of glucose, xylose and cellobiose, and at least one of the produced glucose, xylose and cellobiose It may include the culture of the Bacillus Waist CAA11 used. In another aspect, the culture may be an anaerobic culture or an aerobic culture.

The invention in another aspect relates to a method of production of formic acid which comprises culturing the Bacillus CAA11 Companion or a Companion Bacillus CAA11 Accession No. KCCM11602P under the cellulose exists. In another aspect, the present invention relates to a method for producing acetic acid, comprising culturing Fanny Bacillus CAA11 in the presence of cellulose. In another aspect, relates to a method for producing ethanol which comprises culturing the Bacillus penny CAA11 under the cellulose exists.

In the present specification, formic acid, acetic acid, and ethanol are used as raw materials for bioenergy, respectively, and may refer to bioformic acid, bioacetic acid, and bioethanol derived from biomass, which is widely used in the art.

It is possible to in a method of one aspect of the present invention, a Companion Bacillus CAA11 or deposit number KCCM11602P the Companion Bacillus CAA11 using the thus obtained reducing sugar together and having a cellulose raise active producing a raw material for bio-energy, the culture of the strain The saccharification of cellulosic material derived from woody biomass and the fermentation process of reducing sugar can be performed. Therefore, according to the present invention, since the cellulase is not added, a substance used as a raw material for bio energy such as formic acid, acetic acid and ethanol can be obtained more simply and economically, and therefore, it is very useful in the production of environmentally friendly bioenergy Lt; / RTI >

In another aspect of the present invention, there is provided a method for producing bioenergy comprising producing bioenergy using a byproduct obtained by culturing Fanny Bacillus CAA11 in the presence of Fanny Bacillus CAA11 or Accession Number KCCM 11602P by the above method in the presence of cellulose to provide. In one aspect, the byproduct may include at least one selected from the group consisting of acetic acid, formic acid, and ethanol. The bio-energy refers to energy obtained by using biomass as a fuel. Any energy produced using a fermentation material such as acetic acid, formic acid, and / or ethanol obtained by a method of the present invention can be used without limitation And can be used without limitation as long as it is known in the art as a method for producing bioenergy using a fermentation substance such as acetic acid, formic acid and / or ethanol.

The present invention also as a medium for culturing the Bacillus CAA1 Companion or a Companion Bacillus CAA11 Accession No. KCCM11602P in a further aspect, the present invention relates to a culture medium containing sodium chloride.

Another aspect of the present invention is Bacillus subtilis 168 (Bacillus subtilis 168) genetically modified strain of the transformed by the vector containing the cellulase gene, the Bacillus Waist CAA11 (Paenibacillus sp. CAA11-Cel) relates to.

Alternatively, another aspect of the present invention is Bacillus subtilis 168 (Bacillus subtilis 168) genetically modified strain of the transformed by the vector containing the cellulase gene, a bar sealer's Companion CAA11 (Paenibacillus sp . CAA11-Cel). ≪ / RTI >

In one aspect of the present invention, a gene recombinant strain of the Bacillus Waist CAA11 may be a strain Accession No. KCCM11825P.

One aspect the steps of: by inserting the promoter in the production method, a shuttle vector (shuttle vector) of the genetically modified strain (Paenibacillus sp CAA11-Cel.) Of the Companion Bacillus CAA11 producing the expression vector of the present invention; Adding the promoter, the signal peptide and the cellulase encoding gene to the expression vector by overlap PCR, and cloning the recombinant vector; And introducing the recombinant vector into FannBacillus CAA11 strain to transform.

Specifically, one aspect of the present invention includes a method for preparing the expression vector, the transfection method of building Companion Bacillus CAA11; Inserting a promoter into E. coli and a Bacillus shuttle vector to search for a strong promoter capable of overexpressing the target protein in Fanny Bacillus CAA11 as a target strain; And inserting the finally selected promoter into Escherichia coli and Bacillus shuttle vector and then cloning the endo-cellulase cel5 of Bacillus subtilis 168 to prepare a recombinant vector.

The expression vector used in one aspect of the present invention may include a promoter, a target gene, and a termination coding sequence. The expression vector may include an antibiotic resistance gene commonly used in the art as a selection marker. For example, the expression vector may include an ampicillin, a gentamycin, a carbenicillin, a streptomycin, a kanamycin, a geneticin, neomycin and tetracycline And may contain a resistance gene for resistance. In one aspect, the expression vector includes a promoter sequence, a nucleotide sequence and a terminator sequence of a gene to be expressed, and the sequences may be connected in the order of 5'-3 '.

In one aspect of the present invention, the gene to be expressed is the cellulase-encoding gene, for example, the cel5 gene. The gene is introduced into the expression vector is expressed in the Companion CAA11 Bacillus strain. The cel5 gene used as an embodiment of the present invention is the cel5 gene of 168 strains of Bacillus subtilis, and may include the nucleotide sequence of SEQ ID NO: 4. In one embodiment, the recombinant vector may include a promoter having the nucleotide sequence of SEQ ID NO: 5 in front of the Bacillus subtilis 168 cellulase gene. In addition, in one embodiment, the recombinant vector may have the nucleotide sequence of SEQ ID NO: 6.

[SEQ ID NO: 4]

[SEQ ID NO: 5]

[SEQ ID NO: 6]

In one aspect of the present invention, the step of preparing the recombinant vector can be performed by amplifying a strong promoter and a cellulase encoding gene in an expression vector by PCR, and then inserting the amplified gene.

In one aspect of the present invention, the transformed FannBacillus CAA11 strain may be prepared as a competent cell. According to one embodiment, the step of preparing the water-soluble cells comprises culturing the Fanny Bacillus CAA11 strain in LB (Luria-Bertani), 0.5 M sorbitol medium overnight at 37 ° C; Subculturing Fanny Bacillus CAA11 strain in the LB, 0.5M sorbitol medium, harvesting at OD ₆₀₀ of 0.8 and harvesting by centrifugation at 6000 rpm at 4 ° C for 10 minutes; Washing 4 times with 30 ml of cold wash buffer (0.5 M sorbitol, 0.5 M mannitol, 0.25 mM KH2PO4, 0.25 mM K2HPO4, 0.5 mM MgCl2, 10% glycerol); And resuspending in a 1/40 wash buffer volume of the culture volume and storing at -70 < 0 > C.

In another aspect of the present invention, the step of transforming may comprise an electroporation step.

In one aspect, the step of perforating electricity comprises the steps of: transferring 300-400 ng of purified plasmid DNA to 60 μl of a very coldly refrigerated cell suspension and transferring them to very cold chilled electroporation cuvettes; Electroporation under the conditions of 21 kV / cm, 200 Ω, and 25 μF (time constant 5 ms); LB, 0.5 M sorbitol and 0.38 M mannitol medium were mixed with pulsed cells and agitated at 200 rpm for 3 hours at 37 ° C .; And spreading the cell mixture.

Prepared according to one aspect of the present invention Waist CAA11 recombinant Bacillus strains can be overexpression of the cellulase (cellulase) by being transformed by a recombinant vector containing the cellulase gene. Therefore, when culturing the Bacillus Waist CAA11 recombinant strain from a cellulose medium, it is possible to increase the production of formic acid, acetic acid, ethanol, by a cellulase over-expression.

Thus, one aspect of the present invention can provide a culture method comprising culturing under a gene recombinant strain (Paenibacillus sp. CAA11-Cel) the wood-based biomass or cellulose presence of the Bacillus Waist CAA11. Specifically, it may include culturing in the presence of cellulose. In one embodiment, the recombinant strain of Fanny Bacillus CAA11 ( Paenibacillus sp . CAA11-Cel) can be applied to the culture method of Fanny Bacillus CAA11 described in this specification without limitation.

Further, one aspect is a recombinant strain of which the deposit number Bacillus Waist CAA11 of the present invention (Paenibacillus sp . CAA11-Cel). ≪ / RTI > In one aspect the method of production is genetically modified strain of the Bacillus CAA11 Companion (Paenibacillus sp . CAA11-Cel) in the presence of woody biomass or cellulose. In another aspect, the culture may be an anaerobic culture or an aerobic culture. In one embodiment, the recombinant strain of Fanny Bacillus CAA11 ( Paenibacillus sp . CAA11-Cel) can be applied without limitation to a method for producing a fermentation substance from Fanny Bacillus CAA11 described in the present specification.

In one aspect culturing said Waist Bacillus CAA11 or Companion Bacillus CAA11 genetically modified strain, the Companion Bacillus CAA11 or Companion Bacillus CAA11 cellulose recombinant strain as a carbon source, comprising: culturing in a culture medium containing the yeast extract or the like as a nitrogen source can do. In one embodiment, the cellulose may be treated with 85% phosphoric acid at 50 < 0 > C for 6 hours to remove crystals.

Production method of fermentation products from one point of view, a gene recombinant strain of the Bacillus CAA11 Companion (Paenibacillus sp . The CAA11-Cel) is the decomposition of wood-based biomass or cellulose to produce glucose, one or more of the xylose and cellobiose, and the use of the production of glucose, one or more of the xylose and cellobiose in the Companion Bacillus CAA11 The recombinant strain Paenibacillus sp . CAA11-Cel). ≪ / RTI >

In one aspect, the fermentation material may include at least one selected from the group consisting of formic acid, acetic acid, and ethanol. The Waist Bacillus gene recombinant strain (Paenibacillus sp. CAA11-Cel) of CAA11 the polysaccharide degradation is possible Companion Bacillus CAA11 (Paenibacillus sp. CAA11) for use in, because that excessive expression of the cellulase, Bio from mass of cellulose formate, acetate, ethanol, And the like.

In order to break down the polysaccharides cellulose endo cellulase, exo-cellulase, beta-glucosidase when you need multiple saccharification enzyme Days etc. But endo cellulase is cel5 of Bacillus subtilis 168 (Bacilluls subtilis 168) Bacillus subtilis 168 among them (Bacillus subtilis 168), indicating high activity.

Thus, another aspect of the invention, comprising a genetically modified strain (Paenibacillus sp CAA11-Cel.) Of the Companion Bacillus CAA11 cultured in the wood-based biomass or cellulose presence; And a step of producing bioenergy using a byproduct obtained by the culture, wherein the byproduct includes at least one selected from the group consisting of acetic acid, formic acid, and ethanol. In one embodiment the recombinant strain of Bacillus Waist CAA11 (Paenibacillus sp. CAA11-Cel) A method of producing a bio-energy from can be applied without the production of bio-energy method of Companion Bacillus CAA11 described herein limits. At this time, the recombinant strain of FANIA bacillus CAA11 overexpresses cel5, an endocellulase of Bacillus subtilis 168, so that useful products such as formic acid, acetic acid and ethanol can be produced more effectively.

In the specification, the formic acid is also referred to as formic acid. The formic acid is widely used as a preservative, and is used as a coagulant for leather production and rubber production for industrial use. In addition, technology for making low-cost fuel cells using formic acid has been developed and can be utilized for fuel cells. In the present specification, acetic acid is used in large quantities as a very important material in industrial fields such as dyeing, synthetic herb, aspirin, etc., acetic acid ester, etc., as well as vinyl acetate. In this specification, ethanol is also important as a synthesis raw material, and many studies have been made with attention as a biofuel.

Hereinafter, the present invention will be described in detail with reference to examples of the present invention. It will be apparent to those skilled in the art that the present invention has been shown by way of example only, and that the scope of the present invention is not limited by these embodiments.

[Example 1] Isolation of Cellulose Degrading Bacterium Fanny Bacillus CAA11

[Example 1-1] Strain collection

In order to isolate the strains which can directly produce the chemical raw materials by using cellulose, soil samples collected from the copper-rich tidal flats, Kwanak San, Jirisan and Indonesia, which are rich in cellulose, are spread on a flat plate containing 10 g / L of cellulose This strain was cultured overnight at 37 ° C in a constant-temperature incubator and the strain was first screened. The pure strains were re-cultured on a cellulose culture medium and secondary screening was performed by confirming whether or not the isolated strains were degraded through cellulose staining and decolorization. When the isolates were cultured, the medium was composed of the compositions shown in Tables 1 and 2 below.

Medium composition g / L K ₂ HPO ₄ 5 KH ₂ PO ₄ 3 (NH _{4) 2} SO ₄ 2 MgSO ₄ 7H ₂ O 0.4 Peptone 0.5 Beef extract 0.3 Yest extract One CaCl ₂ 2H ₂ O 0.1 트랙 요소 1 ml Cellulose 10

트랙 요소 HCl 1 ml / L ZnCl ₂ 70 mg / L MnCl2 4H2O 100 mg / L H ₃ BO ₃ 60 mg / L CoCl ₂ 6H ₂ O 200 mg / L CuCl ₂ 2H ₂ O 20 mg / L NiCl2 6H2O 20 mg / L NaMoO ₄ 2H ₂ O 40 mg / L

As a result, as shown in Figs. 1a and 1b, the strain obtained from the tidal flats of the tidal flat showed high cellulose decomposing activity. FIGS. 1A and 1B show the results of culturing bacteria on a medium containing cellulose, and FIG. 1B is a result of culturing the cells with Congolest reagent in a medium on which the bacteria were grown as shown in FIG. 1A. When Congo's Reagent is added and dyed, as shown in Fig. 1B, the Concha Red reagent adheres to the cellulose and becomes red. However, when the bacteria secrete the cellulase out of the cells and decompose the cellulose contained in the medium, the cellulose around the bacteria is degraded, so that the Congo's reagent is not stained. That is, the bacterial periphery is not stained and appears transparent. This can be confirmed in a transparent cross shape at the center of FIG. 1B, which means that the strain according to an embodiment of the present invention is a part of cellulose decomposition.

[Example 1-2] Strain analysis and identification

The genes of each strain were amplified by PCR using the colonies of the strains collected from the Dongmu tidal flat, which had the best cellulase activity. The primers used for PCR amplification were 27F (5'-AGAGTTTGATCTGCTCAG-3 ', SEQ ID NO: 1) and 1492R (5'-AAGGAGGTGATCCAGCCGCA-3', SEQ ID NO: 2) C for 1 minute, and 72 ° C for 1 minute and 30 seconds. The cycle was repeated 30 times and the reaction was continued at 72 ° C for 10 minutes. Thus requests the 16S rRNA sequencing of the resultant PCR product in Macrogen (Macrogen), a result of proceeding with the next sequence number obtained sequences, such as 3 Phylogenetic analysis based on it (Fig. 2), Waist Bacillus the strain CAA11 ( Paenibacillus sp. CAA11).

[SEQ ID NO: 3]

[Experimental Example 1] Determination of optimized culture conditions of Fanny Bacillus CAA11 strain

[Experimental Example 1-1] Determination of the composition of the culture medium

The extent of growth of the Fanny Bacillus CAA11 strain in the M9 medium, which is mainly used for culturing Bacillus strains, was measured over time.

The M9 medium is used as an example for culturing the Fanny Bacillus strain. The composition of the M9 medium is shown in Table 3 below. The trace elements in Table 3 below are the same as those in Table 2, except that FeSO ₄ was added separately. The culture of the strain was incubated at 37 DEG C with stirring at 200 rpm for 24 hours. At this time, 5 g / L of glucose was added. The growth of the strain was confirmed by measuring the absorbance (OD ₆₀₀ ). Specifically, a part of the cultured culture was diluted with distilled water and measured at 600 nm using a spectrophotometer.

Medium composition Na ₂ HPO ₄ O ₇ H ₂ O 12.8 g / L KH ₂ PO ₄ 3g / L NaCl 0.5 g / L NH ₄ Cl 1 g / L O MgSO ₄ 7H ₂ O 0.492 g / L CaCl ₂ 0.111 g / L Trace element 1ml pH 7

As a result, as shown in Fig. 3, the growth of FANIBACUSUS CAA11 strain was remarkably increased in the experimental group to which NaCl 2% (wt / v) was further added compared with the medium containing M9 medium + 5 g / L glucose + 0.1% yeast extract Increase in the number of workers.

[Experimental example 1-2] Determination of pH condition of strain culture

The following experiments were conducted to compare the growth of Fanny Bacillus CAA11 strain according to culture pH conditions.

First, Experimental Example 1-1 was inoculated to Companion Bacillus CAA11 M9 medium having the composition shown in Table 3 according to overnight, the overnight in M9 medium appropriate to the addition of glucose 5g / L, and pH 5, 6 and 7 respectively The cultured seed culture was inoculated and subcultured at 37 DEG C with stirring at 200 rpm. Absorbance values were measured at OD ₆₀₀ using a spectrophotometer (Cary 60 UV-Vis, Agilent Technologies, USA) at intervals of 10 minutes, and a growth rate graph as shown in FIG. 4 was prepared therefrom.

As a result, the growth of the strain was observed under the conditions of pH 6 and pH 7 as shown in FIGS. 4A to 4C, and it was observed that the strain was explosively grown at pH 7 in particular.

[Experimental Example 1-3] Determination of temperature condition of strain culture

The following experiments were performed to compare the growth of Fanny Bacillus CAA11 strain according to the culture temperature conditions.

First, Fanny Bacillus CAA11 was inoculated on M9 medium having the composition shown in Table 3 described in Experimental Example 1-1, followed by incubation overnight. Then, the medium was titrated to pH 7 and cultured overnight in M9 medium supplemented with glucose 5 g / seed culture was inoculated and subcultured at 30 ° C, 37 ° C, 50 ° C and 200rpm with stirring. Absorbance value was measured at OD ₆₀₀ using a spectrophotometer (Cary 60 UV-Vis, manufactured by Agilent Technologies, USA), and the growth rate graph as shown in FIGS. 5A to 5C was prepared therefrom.

As a result, as shown in Figs. 5A to 5C, it was observed that the strain was explosively grown at 30 ° C and 37 ° C, respectively, as compared with 50 ° C.

[Experimental Example 2-1] Confirmation of saccharification and efficacy of Fanny Bacillus CAA11 strain

The following experiment was carried out to confirm that Fanny Bacillus CAA11 strain can use other saccharides besides glucose.

First, colonies of Fanny Bacillus CAA11 were inoculated on a medium supplemented with 0.1% yeast extract, 2% NaCl and 5 g / L of M9 having the composition shown in Table 3 above, and then cultured overnight at 37 ° C with stirring at 200 rpm. The seed culture was subcultured to 1/100 of the medium (M9 + 0.1% yeast extract + 2% NaCl) supplemented with glucose, xylose and cellobiose as carbon sources, respectively. Thereafter, 1 mL of the bacterial solution was sampled at an appropriate time with stirring at 37 DEG C and 200 rpm. Agilent 1260 (Waldbronn, Germany), refractive index detector (RID), and Aminex HPX-87H Ion (manufactured by Nippon Kayaku Co., Ltd.) were used for the dilution of glucose, xylose and cellobiose and the content of acetic acid, Exclusion Column (300 mm x 7.8 mm, Bio-Rad, Hercules, Calif., USA).

As a result, as shown in Figs. 6A to 6C, the Fanny Bacillus CAA11 strain of the present invention was prepared by using glucose of the hexose saccharide and xylose of the pentose saccharide as a carbon source, and using acetic acid (acetic acid). In addition, it has been confirmed that the Fanny Bacillus CAA11 strain of the present invention produces acetic acid which can be used as a raw material for bioenergy even when the disaccharide cellobiose is treated with a carbon source. Thus, the Fanny Bacillus CAA11 strain of the present invention can utilize various saccharides produced in the saccharification process of biomass as a carbon source, and can carry out a fermentation process for producing a material usable as a raw material of bioenergy through fermentation process .

OK Test Example 2-2 to glycosylated and efficacy of Companion CAA11 Bacillus strain of more than two kinds of carbon source present

Experiments were carried out to confirm the saccharification and efficacy of the Fanny Bacillus CAA11 strain when two or more types of carbon sources were present under the same conditions as Experimental Example 2-1. Specifically, the type of carbon source was divided into experimental group in which glucose and cellobiose were present and experiment group in which cellobiose and xylose were present, and the remaining conditions were the same as Experimental Example 2-1.

As a result, as shown in FIG. 7A and FIG. 7B, the Fanny Bacillus CAA11 strain of the present invention produced acetic acid and lactic acid by using glucose of the monosaccharide, hexose, and cellobiose, which is a disaccharide, . 7A and 7B, it can be seen that the Fanny Bacillus CAA11 strain of the present invention produces acetic acid and lactic acid simultaneously using xylose, a monosaccharide, and cellobiose, a disaccharide, as carbon sources at the same time I could. 7A and 7B, since the Fanny Bacillus CAA11 strain of the present invention can simultaneously use glucose, which is the main reducing sugar of cellulose, and cellobiose, which is the main reducing sugar of hemicellulose, as a carbon source, biomass, specifically, The effective saccharification and fermentation process of the cellulose and hemicellulose obtained after the pretreatment process of the mass can be advanced.

[Example 3] check Companion glycosylated and to the efficacy of the Bacillus strains according to the solubility of the cellulose CAA11

10 g of crystallized cellulose Avicel PH-101 was mixed with 100 ml of 85% phosphoric acid and then treated at 50 ° C for 6 hours. After washing 4 times with sterilized distilled water, 30 ml of 2N NaOH was added, and the mixture was allowed to stand at 4 째 C overnight, followed by washing with sterilized distilled water until the pH reached about 7.0. Regenerated amorphous cellulose (RAC) was used as insoluble cellulose, and carboxymethyl cellulose (CMC) was used as soluble cellulose.

M9, 0.1% (wt / v) yeast extract, 2% (wt / v) NaCl and glucose 5 g / (Wt / v) NaCl and 5 g / L of CMC (carboxymethyl cellulose), which is a soluble cellulose, and a medium containing 0.1% (wt / v) yeast extract, 2% (wt / v) NaCl and insoluble cellulose And 5 g / L of regenerated amorphous cellulose (RAC). As a control, strains cultured in the same medium except for the absence of cellulose were used. The cells were incubated for 42 hours at 37 ° C with stirring at 200 rpm. One mL of the culture was sampled at the appropriate time to measure the extent of Fannibacillus CAA11 growth.

First, when the soluble cellulose was cultured in the medium containing CMC as the soluble cellulose, the soluble cellulose was dissolved in the medium and showed a transparent form. Therefore, the absorbance was measured at a wavelength of ₆₀₀ nm using a spectrophotometer (Cary 60 UV-Vis, Agilent Technologies, USA) The absorbance value was measured and is shown in Fig. In the case of culturing in a medium supplemented with RAC which is insoluble cellulose, since the cellulose exists in an opaque state in the culture medium, its degree of growth can not be known due to absorbance. Therefore, the growth of bacteria was confirmed by colony forming unit (CFU) for each sampling time. The degree of colony formation (CFU) was determined by diluting the sampled bacterial solution with 1/10 ³ -10 ⁸ as a culture medium, measuring the number of colonies produced after the bacteria were stained in a solid medium, and calculating the number of colonies produced per 1 mL of the bacterial solution , Which is shown in Fig. As shown in Figs. 8 and 9, Companion Bacillus CAA11 strain was confirmed that the growth regardless of the solubility whether the liquid of cellulose available, and thus can maintain an excellent glycation and to effect without being affected by the kind of the cellulose .

[Experimental Example 4] Confirmation of saccharification and efficacy of Fanny Bacillus CAA11 strain under anaerobic and aerobic conditions

The seeds cultured overnight in M9 medium supplemented with 0.1% (wt / v) yeast extract, 2% (wt / v) NaCl and 5 g / ) yeast extract, 2% (wt / v) NaCl and 5 g / L of regenerated amorphous cellulose (RAC), insoluble cellulose, were cultured in aerobic and anaerobic conditions.

The aerobic conditions were as follows: 20 mL of the medium was placed in a 100 mL flask, and the mixture was stirred at 37 ° C and 200 rpm. In the case of the anaerobic culture, inoculum was inoculated in the medium purged with argon in a serum bottle, And cultured under the same conditions (37 ° C, 200 rpm). After 1 mL of the culture was sampled at the appropriate time, the degree of growth of the bacteria was measured by the degree of colony formation (CFU), and the product was diluted 1/10 by HPLC. In this case, since RAC, which is an insoluble cellulose, is present in an opaque state in the culture medium, the degree of growth can not be known due to the absorbance. Therefore, the growth of the bacteria was confirmed by colony forming unit (CFU) at each sampling time.

For the CFU measurement, the sampled bacterial fluid was diluted to 1/10 ³ -10 ⁸ with the culture medium, and the number of colonies produced after the staining on the solid medium was measured to calculate the number of colonies generated per 1 mL of the bacterial fluid. The products acetic acid, formic acid and ethanol were analyzed by HPLC (manufactured by Agilent, product name: Agilent 1260, Waldbronn, Germany), refractive index detector (RID), Aminex HPX- 87 H Ion Exclusion Column (300 mm x 7.8 mm, Hercules, Calif., USA). The results are summarized in Figures 10a-10d.

As a result, as shown in FIGS. 10A to 10D, it can be seen that the Fanny Bacillus strain CAA11 can grow well under both aerobic conditions and anaerobic conditions, and can produce fermented products under both aerobic and anaerobic conditions.

[Example 3] Search for promoter to be used for transfection for overexpression of protein in Fanny Bacillus CAA11

Fanny Bacillus  Construction of transgenic method for CAA11

Since Fanny bacillus was not constructed as a transformed strain, various vectors such as a cloning vector usable in Bacillus, a vector for inserting a genome, and a vector capable of forming a target genetic label can be used in the Bacillus Genetic Stock Center ; BGSC).

Transformation technique for DNA introduction under various conditions resulted in establishment of DNA introduction method in CAA11. Since Fanny Bacillus CAA11 has a thick cell wall as a Gram-positive bacterium, 0.5m sorbitol was added to the buffer solution to increase the efficiency of DNA transfection. As a result, transfection was tested with plasmids of various sizes. As a result, it was confirmed that the plasmid was successfully introduced into CAA11 as shown in Table 4 below.

Plasmid pNW33N pAD123 pHCMC02 pAD43-25 pHCMC05 Negative Size (bp) 4217 5952 6866 7262 8321 colony 105 80 37 13 0 0

Preparation of vector for promoter intensity measurement

A vector for measuring the promoter strength was prepared by inserting 8 combinations of promoters into the pAD123 vector containing the GFP gene, which is a fluorescent protein in the plasmid. The promoters used in Table 5 are shown. 11A shows a production diagram of the vector (pAD123-promoter).

The PCR was carried out by one cycle of predenaturation at 98 ° C for 2 minutes followed by denaturation at 98 ° C for 10 seconds - annealing at 60 ° C for 10 seconds - elongation at 72 ° C for 10 seconds elongation) was repeated for 30 cycles. And an additional elongation at 72 ° C for 5 minutes for one more cycle.

Then, the pAD123 and promoter PCR products were digested with SmaI and BamHI restriction enzymes present at the MCS site of pAD123 plasmid (digestion), and the promoter PCR product and pAD123 were ligated.

Finally, the pAD123-promoter was transformed into E. coli DH5a, plated on an LB agar plate containing 25 μg / ml Cm, and then a recombinant plasmid was identified by inoculation with a colony.

The promoter used to measure the promoter strength

Number of Uses Promoter used sequence 1 promoter Pspac (SEQ ID NO: 7) aggccttacacagcccagtccagactattcggcactgaaattatgggtgaagtggtcaagacctcactaggcaccttaaaaatagcgcaccctgaagaagatttatttgaggtagcccttgcctacctagcttccaagaaagatatcctaacagcacaagagcggaaagatgttttgttctacatccagaacaacctctgctaaaattcctgaaaaattttgcaaaaagttgttgactttatctacaaggtgtggcataatgtgtggaattgtgagcggataacaattaagcttaaggaggtga PHpaII (SEQ ID NO: 8) gatcttctcaaaaaatactacctgtcccttgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaaaagaaaggtcttaaaggttttatggttttggtcggcactgccgacagcctcgcagagcacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgatttacat P43 (SEQ ID NO: 9) tgataggtggtatgttttcgcttgaacttttaaatacagccattgaacatacggttgatttaataactgacaaacatcaccctcttgctaaagcggccaaggacgctgccgccggggctgtttgcgtttttgccgtgatttcgtgtatcattggtttacttatttttttgccaaagctgtaatggctgaaaattcttacatttattttacatttttagaaatgggcgtgaaaaaaagcgcgcgattatgtaaaatataaagtgatagc 2 promoters Pspac P43 (SEQ ID NO: 10) aggccttacacagcccagtccagactattcggcactgaaattatgggtgaagtggtcaagacctcactaggcaccttaaaaatagcgcaccctgaagaagatttatttgaggtagcccttgcctacctagcttccaagaaagatatcctaacagcacaagagcggaaagatgttttgttctacatccagaacaacctctgctaaaattcctgaaaaattttgcaaaaagttgttgactttatctacaaggtgtggcataatgtgtggaattgtgagcggataacaattaagcttaaggaggtgaggatcctgataggtggtatgttttcgcttgaacttttaaatacagccattgaacatacggttgatttaataactgacaaacatcaccctcttgctaaagcggccaaggacgctgccgccggggctgtttgcgtttttgccgtgatttcgtgtatcattggtttacttatttttttgccaaagctgtaatggctgaaaattcttacatttattttacatttttagaaatgggcgtgaaaaaaagcgcgcgattatgtaaaatataaagtgatagc
PHpaII P43 (SEQ ID NO: 11) gatcttctcaaaaaatactacctgtcccttgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaaaagaaaggtcttaaaggttttatggttttggtcggcactgccgacagcctcgcagagcacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgatttacatggatcctgataggtggtatgttttcgcttgaacttttaaatacagccattgaacatacggttgatttaataactgacaaacatcaccctcttgctaaagcggccaaggacgctgccgccggggctgtttgcgtttttgccgtgatttcgtgtatcattggtttacttatttttttgccaaagctgtaatggctgaaaattcttacatttattttacatttttagaaatgggcgtgaaaaaaagcgcgcgattatgtaaaatataaagtgatagc
P43 P43 (SEQ ID NO: 5) tgataggtggtatgttttcgcttgaacttttaaatacagccattgaacatacggttgatttaataactgacaaacatcaccctcttgctaaagcggccaaggacgctgccgccggggctgtttgcgtttttgccgtgatttcgtgtatcattggtttacttatttttttgccaaagctgtaatggctgaaaattcttacatttattttacatttttagaaatgggcgtgaaaaaaagcgcgcgattatgtaaaatataaagtgatagcggatcctgataggtggtatgttttcgcttgaacttttaaatacagccattgaacatacggttgatttaataactgacaaacatcaccctcttgctaaagcggccaaggacgctgccgccggggctgtttgcgtttttgccgtgatttcgtgtatcattggtttacttatttttttgccaaagctgtaatggctgaaaattcttacatttattttacatttttagaaatgggcgtgaaaaaaagcgcgcgattatgtaaaatataaagtgatagc

Preparation of cellulase expression vector

A cellulase expression vector was prepared as shown in FIG. 12 using the P43 P43 promoter (SEQ ID NO: 5) among the two promoters showing strong expression of the gene through comparison of the promoter strength. The P43 promoter was inserted into E. coli and the Bacillus shuttle vector pNW33N vector, and the P43 promoter, signal peptide (nprB), and cellulase (cel5) were ligated to overlap vector pNW33N P43 vector. The promoter (P43) cellulase (cel5) and the signal peptide (nprB) were amplified by PCR using the genomic DNA of 168 strains of Bacillus subtilis as a template. The amplified PCR product was amplified by overlap PCR to obtain the P43-nprB-cel5 product.

The pNW33N vector and P43 were treated with SmaI and BamHI for ligation, transformed into E. coli DH5a, plated on an LB agar plate containing 25 μg / ml Cm, and then inoculated with pNW33N -P43 recombinant plasmid was identified.

The resulting pNW33N-P43 vector and P43-nprB-cel5 were treated with BamHI and XbaI for ligation, and transformed into E. coli DH5a as described above. Then, LB agar plates containing 25 μg / ml Cm ), And then colony was inoculated to confirm pNW33N-P43-P43 nprB cel5 recombinant plasmid (SEQ ID NO: 6).

Fanny Bacillus  Preparation of CAA11 Recombinant Strain

After transformed by introducing a recombinant vector prepared in the Companion CAA11 Bacillus strain, were cultured in a medium containing Cm 10 μg / mL Waist Bacillus CAA11 To prepare a recombinant strain.

At this time, the transformation was carried out by the following method.

First, the Fanny Bacillus CAA11 strain was cultured overnight at 37 ° C in LB (Luria-Bertani) medium containing 0.5 M sorbitol, subcultured again in the same medium, and then centrifuged at 6000 rpm at 4 ° C for 10 minutes And harvested at an OD ₆₀₀ of 0.8. And washed four times with 30 ml of cold wash buffer (0.5 M sorbitol, 0.5 M mannitol, 0.25 mM KH2PO4, 0.25 mM K2HPO4, 0.5 mM MgCl2, 10% glycerol) and redispersed in a 1/40 wash buffer volume of the culture volume resuspend) to prepare competent cells.

Then, 300 μl of the purified plasmid DNA and 60 μl of the cell suspension refrigerated very cold were mixed and transferred to electroporation cuvettes which were refrigerated very coolly. Then, the cells were cultured under conditions of 21 kV / cm, 200 Ω, 25 μF (time constant 5 ms) Lt; / RTI > 1 mL of LB, 0.5 M sorbitol and 0.38 M mannitol medium was mixed with pulsed cells and agitated at 37 ° C for 3 hours at 200 rpm. The cell mixture was mixed with 10 μg / mL of Cm Transformation was performed by spreading in the medium.

[Experimental Example 5] Comparison of Promoter Strength

The vector for the production promoter strength measurements were introduced into the Bacillus SPF35 similar Waist Bacillus CAA11. After incubation in LB containing 5 μg / ml of Cm, the completed vector was transformed into Paenibacillus sp. The intensity of the promoter was analyzed by measuring the intensity of fluorescence by introducing it into CAA11. As a result, as shown in FIG. 11B, when two promoters were inserted in a row, the fluorescence expression was the highest when one promoter was used. Based on the experimental results, two promoters were used to express the desired gene in the isolate.

[Experimental Example 6] In solid medium Comparison of Cellulose Degradability of Fanny Bacillus CAA11 Recombinant Strain

PNW33N-P43 P43-nprB cel5 vector with two promoters linked thereto was prepared (FIG. 12). In order to investigate the degree of degradation of the cellulose degradation of the recombinant strain after transformation into CAA11, an empty vector And compared with the transformed control group.

First, soluble in the liquid solution of cellulose is carboxymethyl cellulose then (CMC) are cultured for 24 hours Companion Bacillus CAA11 strain and cellulase A Companion Bacillus CAA11 strain containing the excess expression vector comprising only an empty vector in the M9 medium containing 10g / L of cellulose After dyeing with 0.1% Congo Red solution, which can analyze the degree of degradation, we discolored with 1M NaCl and compared the size of halo zone decomposed cellulose. As a result, it was confirmed that cellulose degradation ability of the cellulase overexpressing strain was increased as shown in Fig.

[Experimental Example 7] Cellulose degradation ability and production material analysis of Fanny Bacillus CAA11 recombinant strain in liquid medium

Cellulase overexpressing strains were cultured on cellulose medium and cellulase degradation was quantified and analyzed. Cellulose (RAC; regenerated amorphous cellulose) to be used for the medium was prepared as follows. 10 g of crystallized cellulose Avicel PH-101 was mixed with 100 ml of 85% phosphoric acid and then treated at 50 ° C for 6 hours. After washing 4 times with sterilized distilled water, 30 ml of 2N NaOH was added. The mixture was allowed to stand overnight at 4 ° C, and then washed with sterilized distilled water until pH 7.0.

In the M9 medium containing 5 g / L of glucose, Fanny Bacillus CAA11 was cultured overnight, inoculated into 50 ml of M9 medium containing 5 g / L of the cellulose prepared above, and then cultured under aerobic and anaerobic conditions at 37 ° C and 200 rpm .

The samples were centrifuged at 13,000 rpm for 10 min. The supernatant was analyzed for the product, and the cellulosic precipitated with pellet was used for cellulosic determination. Analysis of the product was carried out on an Aminex HPX-87 H Ion Exclusion Column (300 mm x 7.8 mm, Bio-Rad, Hercules, Calif.) Using HPLC (manufacturer: Agilent 1260, Waldbronn, Germany) USA).

In order to remove cells, cellulase was added at a concentration of 40 μg / ml. Cells were incubated at 37 ° C for 30 min. Then, 20 μl of Bug Buster reagent from Novagen was added and incubated at room temperature for 30 min. Lt; / RTI > After washing 2 times with distilled water, it was diluted 1/10. 10μl of sample, 200μl of sulfuric acid solution and 20μl of 5% phenol solution were added to a 96-well plate and reacted at room temperature for 30 minutes. Absorbance was measured at 490 nm and the amount of cellulose remaining in the culture was measured.

Cellulose degradation enhancing strain was cultured in M9 medium containing 10 g / L of RAC and 1 g / L of yeast extract under aerobic and anaerobic conditions. As a result, as shown in FIGS. 14A to 14E, 41% decomposition of cellulose, 0.11 g / L of acetic acid was produced, 62% of cellulose was decomposed in cellulose degrading ability improving strain and 0.6 g / L of acetic acid was produced. In the anaerobic condition, the control group decomposed 30% of cellulose and produced 0.14 g / L formic acid, 0.12 g / L acetic acid, 0.13 g / L ethanol and 0.06 g / L lactic acid. Cellulose degradation enhanced the celluloses by 54% and yielded 0.7 g / L formic acid, 0.74 g / L acetic acid, 0.65 g / L ethanol and 0.16 g / L lactic acid.

Fanny Bacillus CAA11 was able to decompose cellulose under both aerobic and anaerobic conditions. It was found to produce acetic acid under aerobic conditions, acetic acid, formic acid, ethanol and lactic acid under anaerobic conditions and overexpressed Cel5 to improve cellulose degradation. Fanny Bacillus CAA11 Of recombinant strains of Paenibacillus sp . CAA11-Cel), because the decomposition of the cellulose better up, it was confirmed that the above Waist Bacillus production material than the CAA11 increased by about 5-6 times the plasma that is not converted.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Microorganism Conservation Center KCCM11602P 20141106 Korea Microorganism Conservation Center KCCM11825P 20160324

<110> Korea Institute of Science and Technology <120> Paenibacillus sp. CAA11 capable of saccharification and          fermentation of cellulose and transformed strain thereof <130> 16p126ind <150> KR 10-2015-0047444 <151> 2015-04-03 <160> 11 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying strain Example 1-1 <400> 1 agagtttgat ctgctcag 18 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying strain of Example 1-1 <400> 2 aaggaggtga tccagccgca 20 <210> 3 <211> 1437 <212> DNA <213> Artificial Sequence <220> <223> Sequence of amplified strain of Example 1-1 <400> 3 ctatactgca gtcgagcgga gttatttaga agcttgcttc taaataactt agcggcggac 60 gggtgagtaa cacgtaggca acctgcctgt aagactggga taactaccgg aaacggtagc 120 taataccgga tacacaagtt cctcgcatga gggatttggg aaagacggag caatctgtca 180 cttacggatg ggcctgcggc gcattagcta gttggtgggg taacggctcca ccaaggcgac 240 gatgcgtagc cgacctgaga gggtgaacgg ccacactggg actgagacac ggcccagact 300 cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc 360 cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gccagggaag aacgcttgag 420 agagtaactg ctcttaaggt gacggtacct gagaagaaag ccccggctaa ctacgtgcca 480 gcagccgcgg taatacgtag ggggcaagcg ttgtccggaa ttattgggcg taaagcgcgc 540 gcaggcggcc atttaagtct ggtgtttaat cctggagctc aactccgggt cgcactggaa 600 actgggtggc ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt 660 agagatgtgg aggaacacca gtggcgaagg cgactctctg ggctgtaact gacgctgagg 720 cgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg 780 aatgctaggt gttaggggtt tcgataccct tggtgccgaa gttaacacat taagcattcc 840 gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacggggac ccgcacaagc 900 agtggagtat tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc 960 ctctgaccgg tacagagatg tacctttcct ttacggacaa aggaaacagg tgggtgcatg 1020 gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta 1080 actttagttg ccagcaggtc aagctgggca ctctagagtg actgccggtg acaaaccgga 1140 ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtacta 1200 caatggccgg tacaacggga agcgaaggag cgatctggag cgaatcctag aaaagccggt 1260 ctcagttcgg attgcaggct gcaactcgcc tgcatgaagt cggaattgct agtaatcgcg 1320 gatcagcatg ccgcggtgaa tacgttcccg ggtcttgtac acaccgcccg tcacaccacg 1380 agagtttaca acacccgaag tcggtgaggt aacccgcaag ggggccagcc gccgaag 1437 <210> 4 <211> 1410 <212> DNA <213> Artificial Sequence <220> <223> sequence of bacillus subtilis 168 cellulase (cel5) <400> 4 gcagggacaa aaacgccagt agccaagaat ggccagctta gcataaaagg tacacagctc 60 gttaaccgag acggtaaagc ggtacagctg aaggggatca gttcacacgg attgcaatgg 120 tatggagaat atgtcaataa agacagctta aaatggctga gagatgattg gggtatcacc 180 gtttgccgtg cagcgatgta tacggcagat ggcggttata ttgacaaccc gtccgtgaaa 240 aataaagtaa aagaagcggt tgaagcggca aaagagcttg ggatatatgt catcattgac 300 tggcatatct taaatgacgg taatccaaac caaaataaag agaaggcaaa agaattcttc 360 aaggaaatgt caagccttta cggaaacacg ccaaacgtca tttatgaaat tgcaaacgaa 420 ccaaacggtg atgtgaactg gaagcgtgat attaaaccat atgcggaaga agtgatttca 480 gttatccgca aaaatgatcc agacaacatc atcattgtcg gaaccggtac atggagccag 540 gatgtgaatg atgctgccga tgaccagcta aaagatgcaa acgttatgta cgcacttcat 600 ttttatgccg gcacacacgg ccaattttta cgggataaag caaactatgc actcagcaaa 660 ggagcaccta tttttgtgac agagtgggga acaagcgacg cgtctggcaa tggcggtgta 720 ttccttgatc aatcgaggga atggctgaaa tatctcgaca gcaagaccat tagctgggtg 780 aactggaatc tttctgataa gcaggaatca tcctcagctt taaagccggg ggcatctaaa 840 acaggcggct ggcggttgtc agatttatct gcttcaggaa cattcgttag agaaaacatt 900 ctcggcacca aagattcgac gaaggacatt cctgaaacgc catcaaaaga taaacccaca 960 caggaaaatg gtatttctgt acagtacaga gcaggggatg ggagtatgaa cagcaaccaa 1020 atccgtccgc agcttcaaat aaaaaataac ggcaatacca cggttgattt aaaagatgtc 1080 actgcccgtt actggtataa agcgaaaaac aaaggccaaa actttgactg tgactacgcg 1140 cagattggat gcggcaatgt gacacacaag tttgtgacgt tgcataaacc aaagcaaggt 1200 gcagatacct atctggaact tggatttaaa aacggaacgt tggcaccggg agcaagcaca 1260 gggaatattc agctccgtct tcacaatgat gactggagas attatgcaca aagcggcgat 1320 tattcctttt tcaaatcaaa tacgtttaaa acaacgaaaa aaatcacatt atatgatcaa 1380 ggaaaactga tttggggaac agaaccaaat 1410 <210> 5 <211> 542 <212> DNA <213> Artificial Sequence <220> <223> sequence of promoter P43 P43 <400> 5 tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60 taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 120 tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 180 aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240 gcgattatgt aaaatataaa gtgatagcgg atcctgatag gtggtatgtt ttcgcttgaa 300 cttttaaata cagccattga acatacggtt gatttaataa ctgacaaaca tcaccctctt 360 gctaaagcgg ccaaggacgc tgccgccggg gctgtttgcg tttttgccgt gatttcgtgt 420 atcattggtt tacttatttt tttgccaaag ctgtaatggc tgaaaattct tacatttatt 480 ttacattttt agaaatgggc gtgaaaaaaa gcgcgcgatt atgtaaaata taaagtgata 540 gc 542 <210> 6 <211> 6291 <212> DNA <213> Artificial Sequence <220> <223> sequence of vector pNW33N-P43-P43 nprB cel5 <400> 6 aattcgagct ctgataggtg gtatgttttc gcttgaactt ttaaatacag ccattgaaca 60 tacggttgat ttaataactg acaaacatca ccctcttgct aaagcggcca aggacgctgc 120 cgccggggct gtttgcgttt ttgccgtgat ttcgtgtatc attggtttac ttattttttt 180 gccaaagctg taatggctga aaattcttac atttatttta catttttaga aatgggcgtg 240 aaaaaaagcg cgcgattatg taaaatataa agtgatagcg gatcctgata ggtggtatgt 300 tttcgcttga acttttaaat acagccattg aacatacggt tgatttaata actgacaaac 360 atcaccctct tgctaaagcg gccaaggacg ctgccgccgg ggctgtttgc gtttttgccg 420 tgatttcgtg tatcattggt ttacttattt ttttgccaaa gctgtaatgg ctgaaaattc 480 ttacatttat tttacatttt tagaaatggg cgtgaaaaaa agcgcgcgat tatgtaaaat 540 ataaagtgat agcggtacca ttataggtaa gagaggaatg tacacatgcg caacttgacc 600 aagacatctc tattactggc cggcttatgc atagcggccc aaatggtttt tgtaacacat 660 gccccagctg cagggacaaa aacgccagta gccaagaatg gccagcttag cataaaaggt 720 acacagctcg ttaaccgaga cggtaaagcg gtacagctga aggggatcag ttcacacgga 780 ttgcaatggt atggagaata tgtcaataaa gacagcttaa aatggctgag agatgattgg 840 ggtatcaccg ttttccgtgc agcgatgtat acggcagatg gcggttatat tgacaacccg 900 tccgtgaaaa ataaagtaaa agaagcggtt gaagcggcaa aagagcttgg gatatatgtc 960 atcattgact ggcatatctt aaatgacggt aatccaaacc aaaataaaga gaaggcaaaa 1020 gaattcttca aggaaatgtc aagcctttac ggaaacacgc caaacgtcat ttatgaaatt 1080 gcaaacgaac caaacggtga tgtgaactgg aagcgtgata ttaaaccata tgcggaagaa 1140 gtgatttcag ttatccgcaa aaatgatcca gacaacatca tcattgtcgg aaccggtaca 1200 tggagccagg atgtgaatga tgctgccgat gaccagctaa aagatgcaaa cgttatgtac 1260 gcacttcatt tttatgccgg cacacacggc caatttttac gggataaagc aaactatgca 1320 ctcagcaaag gagcacctat ttttgtgaca gagtggggaa caagcgacgc gtctggcaat 1380 ggcggtgtat tccttgatca atcgagggaa tggctgaaat atctcgacag caagaccatt 1440 agctgggtga actggaatct ttctgataag caggaatcat cctcagcttt aaagccgggg 1500 gcatctaaaa caggcggctg gcggttgtca gatttatctg cttcaggaac attcgttaga 1560 gaaaacattc tcggcaccaa agattcgacg aaggacattc ctgaaacgcc atcaaaagat 1620 aaacccacac aggaaaatgg tatttctgta cagtacagag caggggatgg gagtatgaac 1680 agcaaccaaa tccgtccgca gcttcaaata aaaaataacg gcaataccac ggttgattta 1740 aaagatgtca ctgcccgtta ctggtataaa gcgaaaaaca aaggccaaaa ctttgactgt 1800 gactacgcgc agattggatg cggcaatgtg acacacaagt ttgtgacgtt gcataaacca 1860 aagcaaggtg cagataccta tctggaactt ggatttaaaa acggaacgtt ggcaccggga 1920 gcaagcacag ggaatattca gctccgtctt cacaatgatg actggagcaa ttatgcacaa 1980 agcggcgatt attccttttt caaatcaaat acgtttaaaa caacgaaaaa aatcacatta 2040 tatgatcaag gaaaactgat ttggggaaca gaaccaaatc atcatcatca tcatcattag 2100 tctagagtcg acctgcaggc atgcaagctt ggcgtaatca tggtcatagc tgtttcctgt 2160 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 2220 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 2280 tttccagtcg ggaaacctgt cgtgccagcc cttcaaactt cccaaaggcg agccctagtg 2340 acattagaaa accgactgta aaaagtacag tcggcattat ctcatattat aaaagccagt 2400 cattaggcct atctgacaat tcctgaatag agttcataaa caatcctgca tgataaccat 2460 cacaaacaga atgatgtacc tgtaaagata gcggtaaata tattgaatta cctttattaa 2520 tgaattttcc tgctgtaata atgggtagaa ggtaattact attattattg atatttaagt 2580 taaacccagt aaatgaagtc catggaataa tagaaagaga aaaagcattt tcaggtatag 2640 gtgttttggg aaacaatttc cccgaaccat tatatttctc tacatcagaa aggtataaat 2700 cataaaactc tttgaagtca ttctttacag gagtccaaat accagagaat gttttagata 2760 cccatcaaa aattgtataa agtggctcta acttatccca ataacctaac tctccgtcgc 2820 tattgtaacc agttctaaaa gctgtatttg agtttatcac ccttgtcact aagaaaataa 2880 atgcagggta aaatttatat ccttcttgtt ttatgtttcg gtataaaaca ctaatatcaa 2940 tttctgtggt tatactaaaa gtcgtttgtt ggttcaaata atgattaaat atctcttttc 3000 tcttccaatt gtctaaatca attttattaa agttcatttg atatgcctcc taaattttta 3060 tctaaagtga atttaggagg cttacttgtc tgctttcttc attagaatca atcctttttt 3120 aaaagtcaat cccgtttgtt gaactactct ttaataaaat aatttttccg ttcccaattc 3180 cacattgcaa taatagaaaa tccatcttca tcggcttttt cgtcatcatc tgtatgaatc 3240 aaatcgcctt cttctgtgtc atcaaggttt aattttttat gtatttcttt taacaaacca 3300 ccataggaga ttaacctttt acggtgtaaa ccttcctcca aatcagacaa acgtttcaaa 3360 ttcttttctt catcatcggt cataaaatcc gtatccttta caggatattt tgcagtttcg 3420 tcaattgccg attgtatatc cgatttatat ttatttttcg gtcgaatcat ttgaactttt 3480 acatttggat catagtctaa tttcattgcc tttttccaaa attgaatcca ttgtttttga 3540 ttcacgtagt tttctgtatt cttaaaataa gttggttcca cacataccaa tacatgcatg 3600 tgctgattat aagaattatc tttattattt attgtcactt ccgttgcacg cataaaacca 3660 acaagatttt tattaatttt tttatattgc atcattcggc gaaatccttg agccatatct 3720 gacaaactct tatttaattc ttcgccatca taaacatttt taactgttaa tgtgagaaac 3780 aaccaacgaa ctgttggctt ttgtttaata acttcagcaa caaccttttg tgactgaatg 3840 ccatgtttca ttgctctcct ccagttgcac attggacaaa gcctggattt acaaaaccac 3900 actcgataca actttctttc gcctgtttca cgattttgtt tatactctaa tatttcagca 3960 caatctttta ctctttcagc ctttttaaat tcaagaatat gcagaagttc aaagtaatca 4020 acattagcga ttttcttttc tctccatggt ctcacttttc cactttttgt cttgtccact 4080 aaaacccttg atttttcatc tgaataaatg ctactattag gacacataat attaaaagaa 4140 acccccatct atttagttat ttgtttggtc acttataact ttaacagatg gggtttttct 4200 gtgcaaccaa ttttaagggt tttcaatact ttaaaacaca tacataccaa cacttcaacg 4260 cacctttcag caactaaaat aaaaatgacg ttatttctat atgtatcaag aatagaaaga 4320 actcgttttt cgctacgctc aaaacgcaaa aaaagcactc attcgagtgc tttttcttat 4380 cgctccaaat catgcgattt tttcctcttt gcttttcttt gctcacgaag ttctcgatca 4440 cgctgcaaaa catcttgaag cgaaaaagta ttcttctttt cttccgatcg ctcatgctga 4500 cgcacgaaaa gccctctagg cgcataggaa caactcctaa atgcatgtga ggggttttct 4560 cgtccatgtg aacagtcgca tacgcaatat tttgtttccc atactgcatt aatgaatcgg 4620 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 4680 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 4740 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 4800 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 4860 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 4920 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 4980 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 5040 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 5100 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 5160 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 5220 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 5280 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 5340 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 5400 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 5460 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 5520 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 5580 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 5640 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 5700 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 5760 agatttatca gcaataaacc agccagcccg atatgggaaa caaaatattg cgtatgcgac 5820 tgttcacatg gacgagaaaa cccctcacat gcatttagga gttgttccta tgcgcctaga 5880 gggcttttcg tgcgtcagca tgagcgatcg gaagaaaaga agaatacttt ttcgcttcaa 5940 gaggttttgc agcgtgatcg agaacttcgt gagcaaagaa aagcaaagag gaaaaaatcg 6000 catgatttgg agcgataaga aaaagcactc gaatgagtgc tttttttgcg ttttgagcgt 6060 agcgaaaaac gagttctttc tattcttgat acatatagaa ataacgtcat ttttatttta 6120 gttgctgaaa ggtgcgttga agtgttggta tgtatgtgat tcaataattt cttttactcg 6180 ctcgttatag tcgatcggtt catcattcac caaatcataa ttttcatgtg accgttcttt 6240 atcaatatcg ggattcgttt tactttcccg ttctctctga ttgtgaaatt g 6291 <210> 7 <211> 304 <212> DNA <213> Artificial Sequence <220> <223> sequence of promoter Pspac <400> 7 aggccttaca cagcccagtc cagactattc ggcactgaaa ttatgggtga agtggtcaag 60 acctcactag gcaccttaaa aatagcgcac cctgaagaag atttatttga ggtagccctt 120 gcctacctag cttccaagaa agatatccta acagcacaag agcggaaaga tgttttgttc 180 tacatccaga acaacctctg ctaaaattcc tgaaaaattt tgcaaaaagt tgttgacttt 240 atctacaagg tgtggcataa tgtgtggaat tgtgagcgga taacaattaa gcttaaggag 300 gtga 304 <210> 8 <211> 287 <212> DNA <213> Artificial Sequence <220> <223> sequence of promoter PHpaII <400> 8 gatcttctca aaaaatacta cctgtccctt gctgattttt aaacgagcac gagagcaaaa 60 cccccctttg ctgaggtggc agagggcagg tttttttgtt tcttttttct cgtaaaaaaa 120 agaaaggtct taaaggtttt atggttttgg tcggcactgc cgacagcctc gcagagcaca 180 cactttatga atataaagta tagtgtgtta tactttactt ggaagtggtt gccggaaaga 240 gcgaaaatgc ctcacatttg tgccacctaa aaaggagcga tttacat 287 <210> 9 <211> 268 <212> DNA <213> Artificial Sequence <220> <223> sequence of promoter P43 <400> 9 tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60 taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 120 tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 180 aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240 gcgattatgt aaaatataaa gtgatagc 268 <210> 10 <211> 578 <212> DNA <213> Artificial Sequence <220> <223> sequence of promoter Pspac P43 <400> 10 aggccttaca cagcccagtc cagactattc ggcactgaaa ttatgggtga agtggtcaag 60 acctcactag gcaccttaaa aatagcgcac cctgaagaag atttatttga ggtagccctt 120 gcctacctag cttccaagaa agatatccta acagcacaag agcggaaaga tgttttgttc 180 tacatccaga acaacctctg ctaaaattcc tgaaaaattt tgcaaaaagt tgttgacttt 240 atctacaagg tgtggcataa tgtgtggaat tgtgagcgga taacaattaa gcttaaggag 300 gtgaggatcc tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat 360 acggttgatt taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc 420 gccggggctg tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg 480 ccaaagctgt aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga 540 aaaaaagcgc gcgattatgt aaaatataaa gtgatagc 578 <210> 11 <211> 561 <212> DNA <213> Artificial Sequence <220> <223> sequence of promoter PHpaII P43 <400> 11 gatcttctca aaaaatacta cctgtccctt gctgattttt aaacgagcac gagagcaaaa 60 cccccctttg ctgaggtggc agagggcagg tttttttgtt tcttttttct cgtaaaaaaa 120 agaaaggtct taaaggtttt atggttttgg tcggcactgc cgacagcctc gcagagcaca 180 cactttatga atataaagta tagtgtgtta tactttactt ggaagtggtt gccggaaaga 240 gcgaaaatgc ctcacatttg tgccacctaa aaaggagcga tttacatgga tcctgatagg 300 tggtatgttt tcgcttgaac ttttaaatac agccattgaa catacggttg atttaataac 360 tgacaaacat caccctcttg ctaaagcggc caaggacgct gccgccgggg ctgtttgcgt 420 ttttgccgtg atttcgtgta tcattggttt acttattttt ttgccaaagc tgtaatggct 480 gaaaattctt acatttattt tacattttta gaaatgggcg tgaaaaaaag cgcgcgatta 540 tgtaaaatat aaagtgatag c 561

Claims (17)

delete In Bacillus subtilis 168 (Bacillus subtilis 168) gene recombinant strain (Paenibacillus sp. CAA11-Cel) of the transformed, Companion Bacillus CAA11 (Paenibacillus sp. CAA11) by a vector comprising a cellulase gene,
The accession number of Fanny Bacillus CAA11 is KCCM11602P,
The accession number of the recombinant strain of Fanny Bacillus CAA11 is KCCM11825P,
Genetic recombinant strain of Fanny Bacillus CAA11 ( Paenibacillus sp. CAA11-Cel). delete [Claim 3] The Bacillus subtilis 168 cellulase gene according to claim 2, wherein the gene is a recombinant strain of Paenibacillus sp. CAA11-Cel of Fannibacillus CAA11 having the nucleotide sequence of SEQ ID NO: 4. 3. The recombinant strain of Fannibacillus CAA11 ( Paenibacillus sp. CAA11-Cel) according to claim 2, wherein said vector comprises a promoter having the nucleotide sequence of SEQ ID NO: 5 in front of said Bacillus subtilis 168 cellulase gene. 3. The vector according to claim 2, wherein said vector is a gene recombinant strain of Fannibacillus CAA11 ( Paenibacillus &lt; RTI ID = 0.0 &gt; sp . CAA11-Cel). A method for producing a recombinant strain of Paenibacillus sp. CAA11-Cel of Fanny Bacillus CAA11 according to any one of claims 2 and 4 to 6,
Inserting a promoter into a shuttle vector to produce an expression vector;
Adding the promoter, the signal peptide and the cellulase encoding gene to the expression vector by overlap PCR, and cloning the recombinant vector; And
Introducing the recombinant vector into a strain of FannBacillus CAA11 and transforming it;
&Lt; / RTI &gt; 8. The method of claim 7, wherein the step of transforming comprises an electroporation step. delete Bacillus subtilis 168 (Bacillus subtilis 168) transformed by the vector containing the cellulase gene of claim 2 and claim 4 to claim 6 any one of the Companion Bacillus CAA11 gene recombinant strain (Paenibacillus sp. CAA11- of wherein Cel). Claim, wherein the culture which comprises 2) and (4) to (6 incubated in any one of the recombinant strains of Bacillus Companion (Paenibacillus sp. CAA11-Cel) the wood-based biomass or cellulose in the presence of anti-CAA11. 12. The method of claim 11,
The culture method, culture method, which comprises culturing under aerobic or anaerobic conditions, the genetically modified strain (Paenibacillus sp. CAA11-Cel) of the Companion Bacillus CAA11. As a method for producing a fermentation substance,
Claim and comprising the step of culturing a 2) and (4) to (6 wherein any one of the Companion Bacillus CAA11 of gene recombinant strain (Paenibacillus sp. CAA11-Cel) of,
Wherein the fermentation material comprises at least one selected from the group consisting of formic acid, acetic acid, and ethanol. The method of claim 13, wherein the production method is a method of production of fermentation products which comprises cultured in the wood-based biomass or cellulose present a recombinant strain (Paenibacillus sp. CAA11-Cel) of the Companion Bacillus CAA11. 15. The method of claim 14 wherein the genetically modified strain of Bacillus Companion (Paenibacillus sp. CAA11-Cel) of the CAA11 and by decomposing the wood-based biomass or cellulose to produce glucose, one or more of the xylose and cellobiose,
A method of producing fermentation products, which comprises the production of a culture of glucose, xylose and gene recombinant strain of the Bacillus CAA11 Companion using at least one of cellobiose (Paenibacillus sp. CAA11-Cel) . delete Comprising the steps of: culturing under a 2) and (4) to (6 any one of the recombinant strains of Bacillus Companion (Paenibacillus sp CAA11-Cel.) The wood-based biomass or cellulose in the presence of anti-CAA11; And
And a step of producing bio-energy comprising producing bio-energy using a by-product obtained by the above-
Wherein said byproduct comprises at least one selected from the group consisting of acetic acid, formic acid and ethanol.

Images