Recombinant bacteria for expressing BASP gene and CGCBP gene and application of recombinant bacteria in preparation of cellobiose

Abstract

The invention discloses a recombinant bacterium for expressing a BASP gene and a CGCBP gene and application of the recombinant bacterium in preparation of cellobiose. The present invention provides a recombinant microorganism having a BASP gene and a CGCBP gene. The invention also provides a method for preparing the recombinant microorganism for producing cellobiose, which comprises the following steps: introducing the BASP gene and the CGCBP gene into host bacteria to obtain the recombinant microorganism with the BASP gene and the CGCBP gene, namely the recombinant microorganism for producing cellobiose. The invention also provides a method for preparing cellobiose, which comprises the following step of: preparing cellobiose by taking cane sugar and glucose as raw materials and the recombinant microorganism as a whole-cell catalyst. The method can generate huge economic benefits for cellobiose production, and has great popularization and application values.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a recombinant bacterium expressing a BASP gene and a CGCBP gene and an application thereof in preparing cellobiose. Background Art

[0002] Cellobiose is a reducing disaccharide, which is formed by the condensation of two molecules of β-D-glucose through a β (1→4) glycosidic bond. The repeating unit of its chemical composition is glucose. Cellobiose is both the basic structural unit of cellulose and the main product of cellulose hydrolysis. In the synthesis of fungal cellulases, cellobiose can induce or inhibit the synthesis of cellulases and significantly inhibit the hydrolytic activity of exocellulases. Cellobiose has attracted more and more attention and has a wide range of applications in the food, health care products, feed and other industries. In the prior art, the main applications of cellobiose are as follows: (1) The sweetness of cellobiose is about 30% of that of sucrose, and it is considered to be a new type of "zero-calorie" sweetener; (2) As a soluble dietary fiber, cellobiose has biological functions similar to prebiotics and supports the growth of beneficial microorganisms; (3) As a functional oligosaccharide, cellobiose has health functions similar to oligofructose (FOS); (4) As an animal feed additive, cellobiose can enhance animal health and reduce the use of antibiotics.

[0003] At present, the production method of cellobiose is mainly through chemical or high-temperature acid hydrolysis and bio-enzyme hydrolysis of cellulose raw materials. However, the extraction rate of cellobiose is low, the process of refining the crude product is complicated, and a large amount of acid pollutants are generated, causing environmental pollution.

[0004] In summary, there is an urgent need in the art to develop a new method for efficiently producing cellobiose. Summary of the invention

[0005] The technical problem solved by the present invention is how to efficiently synthesize cellobiose. Therefore, the present invention provides an engineered bacterium for preparing cellobiose.

[0006] The invention discloses a recombinant bacterium expressing a BASP gene and a CGCBP gene and an application of the recombinant bacterium in preparing cellobiose.

[0007] The present invention provides a recombinant microorganism having a BASP gene and a CGCBP gene. The present invention also provides a method for preparing a recombinant microorganism for producing cellobiose, comprising the following steps: introducing BASP gene and CGCBP gene into the microorganism to obtain a recombinant microorganism having BASP gene and CGCBP gene, that is, the recombinant microorganism for producing cellobiose.

[0008] The present invention also provides the use of the recombinant microorganism in preparing cellobiose.

[0009] In the application, the recombinant microorganism serves as a whole-cell catalyst.

[0010] In the application, cellobiose is prepared using sucrose and glucose as raw materials.

[0011] The recombinant microorganism may be the recombinant microorganism after induction culture.

[0012] The present invention also provides a method for preparing cellobiose, comprising the following steps: using sucrose and glucose as raw materials and the recombinant microorganism as a whole-cell catalyst to prepare cellobiose.

[0013] The recombinant microorganism may be the recombinant microorganism after induction culture.

[0014] Specifically, the method for preparing cellobiose further comprises the following steps: after the preparation is completed, adding saccharomyces cerevisiae into the system and culturing to remove fructose (by-product).

[0015] Specifically, the Saccharomyces cerevisiae may be Saccharomyces cerevisiae SC001.

[0016] The present invention also provides the use of biological material I in preparing recombinant microorganisms for producing cellobiose; The biological material I includes BASP gene-related biological materials and CGCBP gene-related biological materials; the BASP gene-related biological materials are BASP genes, expression cassettes with BASP genes or recombinant vectors with BASP genes; the CGCBP gene-related biological materials are CGCBP genes, expression cassettes with CGCBP genes or recombinant vectors with CGCBP genes.

[0017] The biological material I also includes microorganisms (microorganisms as receptor materials).

[0018] In the application, cellobiose is prepared using sucrose and glucose as raw materials.

[0019] The present invention also provides the use of biological material II in preparing recombinant microorganisms for producing cellobiose; The biological material II includes double-gene related biological materials; the double genes are BASP gene and CGCBP gene; the double-gene related biological materials are the double genes, expression boxes with the double genes or recombinant vectors with the double genes.

[0020] The biological material II also includes microorganisms (microorganisms as receptor materials).

[0021] In the application, cellobiose is prepared using sucrose and glucose as raw materials.

[0022] The present invention also provides the use of biomaterial III in the preparation of cellobiose; The biomaterial III includes BASP gene-related biomaterials and CGCBP gene-related biomaterials; the BASP gene-related biomaterials are BASP genes, expression cassettes with BASP genes, recombinant vectors with BASP genes or recombinant microorganisms with BASP genes; the CGCBP gene-related biomaterials are CGCBP genes, expression cassettes with CGCBP genes, recombinant vectors with CGCBP genes or recombinant microorganisms with CGCBP genes.

[0023] The biological material III also includes microorganisms (microorganisms as receptor materials).

[0024] The recombinant microorganism may be the recombinant microorganism after induction culture.

[0025] In the application, cellobiose is prepared using sucrose and glucose as raw materials.

[0026] The present invention also provides the use of biomaterial IV in the preparation of cellobiose; The biological material IV includes double-gene related biological materials; the double genes are BASP gene and CGCBP gene; the double-gene related biological materials are the double genes, expression cassettes with the double genes, recombinant vectors with the double genes or recombinant microorganisms with the double genes.

[0027] The biological material IV also includes microorganisms (microorganisms as receptor materials).

[0028] The recombinant microorganism may be the recombinant microorganism after induction culture.

[0029] In the application, cellobiose is prepared using sucrose and glucose as raw materials.

[0030] Any of the above BASP genes encodes BASP protein.

[0031] Specifically, the function of BASP protein is: sucrose phosphorylase.

[0032] Specifically, the BASP protein is derived from Bifidobacterium adolescentis ( Bifidobacterium adolescentis ).

[0033] The BASP protein is the protein shown in SEQ ID NO:1.

[0034] The BASP protein is as follows (a1) or (a2) or (a3) ​​or (a4): (a1) the protein shown in SEQ ID NO: 1; (a2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (a1); (a3) a protein obtained by substituting and / or deleting and / or adding one or more amino acid residues in (a1) and having the same function as (a1); (a4) A protein that is 98% or more identical to (a1) and has the same function as (a1).

[0035] Any of the above CGCBP genes encodes CGCBP protein.

[0036] Specifically, the function of CGCBP protein is: cellobiose phosphorylase.

[0037] Specifically, the CGCBP protein is derived from Cellulomonas gibbsii ( Cellulomonas gilvus ).

[0038] The CGCBP protein is the protein shown in SEQ ID NO:3.

[0039] The CGCBP protein is as follows (b1) or (b2) or (b3) or (b4): (b1) the protein shown in SEQ ID NO: 3; (b2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (b1); (b3) a protein obtained by substituting and / or deleting and / or adding one or more amino acid residues in (b1) and having the same function as (b1); (b4) A protein that is 98% or more identical to (b1) and has the same function as (b1).

[0040] Specifically, the BASP gene is derived from Bifidobacterium adolescentis ( Bifidobacterium adolescentis ).

[0041] Specifically, the BASP gene is a DNA molecule whose coding region is shown in SEQ ID NO:2.

[0042] Specifically, the BASP gene is as follows (c1) or (c2) or (c3): (c1) a DNA molecule having a coding region as shown in SEQ ID NO: 2; (c2) a DNA molecule having more than 90% identity with (c1) and encoding the protein; (c3) A DNA molecule that hybridizes to the nucleotide sequence defined in (c1) under stringent conditions and encodes the protein.

[0043] Specifically, the CGCBP gene is derived from Cellulomonas gibbsii ( Cellulomonas gilvus ).

[0044] Specifically, the CGCBP gene is a DNA molecule whose coding region is shown in SEQ ID NO:4.

[0045] Specifically, the CGCBP gene is as follows (d1) or (d2) or (d3): (d1) a DNA molecule having a coding region as shown in SEQ ID NO: 4; (d2) a DNA molecule having more than 90% identity with (d1) and encoding the protein; (d3) A DNA molecule that hybridizes to the nucleotide sequence defined in (d1) under stringent conditions and encodes the protein.

[0046] Specifically, the recombinant vector having the BASP gene may be a recombinant plasmid obtained by inserting the BASP gene into an expression vector. Specifically, the expression vector may be a vector pBAD / HisB.

[0047] Specifically, the recombinant microorganism having the BASP gene may be a recombinant microorganism obtained by introducing the BASP gene into a starting microorganism. Specifically, the recombinant microorganism having the BASP gene may be a recombinant microorganism obtained by introducing a recombinant vector having the BASP gene into a starting microorganism. Specifically, the microorganism may be a bacterium. Specifically, the microorganism may be Escherichia coli. Specifically, the microorganism may be Escherichia coli BW25113.

[0048] Specifically, the recombinant vector having the CGCBP gene may be a recombinant plasmid obtained by inserting the CGCBP gene into an expression vector. Specifically, the expression vector may be a vector pBAD / HisB. Specifically, the recombinant microorganism having the CGCBP gene may be a recombinant microorganism obtained by introducing the CGCBP gene into a starting microorganism. Specifically, the recombinant microorganism having the CGCBP gene may be a recombinant microorganism obtained by introducing a recombinant vector having the CGCBP gene into a starting microorganism. Specifically, the microorganism may be a bacterium. Specifically, the microorganism may be Escherichia coli. Specifically, the microorganism may be Escherichia coli BW25113.

[0049] Specifically, the recombinant vector with the double genes may be a recombinant plasmid obtained by inserting the BASP gene and the CGCBP gene into an expression vector. Specifically, the expression vector may be a vector pBAD / HisB. Specifically, the recombinant vector with the double genes may be: using the vector pBAD / HisB as a starting plasmid, replacing the CGCBP gene shown in SEQ ID NO: 4 with Xho I and HopeI enzyme cuts the small fragment between the recognition sequences and replaces it with the BASP gene shown in SEQ ID NO: 2 Hope I and Psst I enzyme cuts the small fragment between the recognition sequences to obtain a recombinant plasmid. Specifically, the recombinant vector with the double gene can be: using the vector pBAD / HisB as the starting plasmid, replacing the BASP gene shown in SEQ ID NO: 2 Xho I and Hope I enzyme cuts the small fragment between the recognition sequences and replaces it with the CGCBP gene shown in SEQ ID NO: 4 Hope I and Psst I enzyme cuts the small fragment between the recognition sequences to obtain the recombinant plasmid.

[0050] Specifically, the recombinant microorganism with the double gene may be a recombinant microorganism obtained by introducing the BASP gene and the CGCBP gene into a starting microorganism. Specifically, the recombinant microorganism with the double gene may be a recombinant microorganism obtained by introducing a recombinant vector with the BASP gene and the CGCBP gene into a starting microorganism. Specifically, the microorganism may be a bacterium. Specifically, the microorganism may be Escherichia coli. Specifically, the microorganism may be Escherichia coli BW25113.

[0051] Specifically, the microorganism is a bacterium, and the microorganism is a recombinant bacterium.

[0052] Specifically, the microorganism is Escherichia coli, and the microorganism is recombinant Escherichia coli.

[0053] Specifically, the Escherichia coli is Escherichia coli BW25113.

[0054] Specifically, the recombinant microorganism after induced culture is obtained by induced culture of the recombinant microorganism.

[0055] Exemplarily, the induction culture is: L-arabinose induction culture. Exemplarily, the induction culture is: using L-arabinose induction culture at a concentration of 0.1-0.3g / 100mL. Exemplarily, the induction culture is: using L-arabinose induction culture at a concentration of 0.2g / 100mL. Exemplarily, the temperature of the induction culture is: 25-40°C. Exemplarily, the temperature of the induction culture is: 30°C. Exemplarily, the time of the induction culture is: 10-16 hours. Exemplarily, the time of the induction culture is: 12-16 hours. Exemplarily, the time of the induction culture is: 12 hours. Exemplarily, the induction culture is shaking culture. Exemplarily, the rotation speed of the shaking culture is 100-300rpm. Exemplarily, the rotation speed of the shaking culture is 200rpm. Exemplarily, the conditions of the induction culture are shaking culture at 30°C and 200rpm for 12 hours.

[0056] Exemplarily, the method for preparing the recombinant microorganism after induced culture comprises the following steps: ① The recombinant microorganism was inoculated into a liquid culture medium and cultured until OD 600nm The value is 0.6~0.8; ②After completing step ①, add L-arabinose to the culture system for induction culture.

[0057] ③ After completing step ②, collect the bacterial precipitate, which is the recombinant microorganism after induced culture.

[0058] In the step ①, the liquid culture medium may be liquid LB culture medium.

[0059] In the step ①, the liquid culture medium may be a liquid LB culture medium containing ampicillin.

[0060] In the step ①, the liquid culture medium may be a liquid LB culture medium containing 50 μg / mL ampicillin.

[0061] In step ①, the culture conditions are: 37° C., 220 rpm shaking culture.

[0062] In the step ②, the concentration of L-arabinose in the system is 0.1-0.3 g / 100 mL.

[0063] In step ②, the concentration of L-arabinose in the system is 0.2 g / 100 mL.

[0064] In step ②, the culture time is 10-16 hours.

[0065] In step ②, the culture time is: 12-16 hours.

[0066] In the step ②, the culture time is: 12 hours.

[0067] In step ②, the culture temperature is 25-40°C.

[0068] In step ②, the culture temperature is 30°C.

[0069] Exemplarily, the induction culture is a shaking culture. Exemplarily, the rotation speed of the shaking culture is 100-300 rpm. Exemplarily, the rotation speed of the shaking culture is 200 rpm.

[0070] In step ②, the culture conditions are: 30° C., 200 rpm shaking culture for 12 hours.

[0071] In the step ③, the bacterial precipitate is collected by centrifugation.

[0072] Exemplarily, the centrifugation parameters are: 6000 rpm for 15 min.

[0073] Specifically, in the method for preparing cellobiose, the reaction temperature is 30°C-60°C.

[0074] Specifically, in the method for preparing cellobiose, the reaction temperature is 37°C.

[0075] Specifically, in the method for preparing cellobiose, the reaction time is 12-24 hours.

[0076] Specifically, in the method for preparing cellobiose, the reaction time is 12 hours.

[0077] Specifically, in the method for preparing cellobiose, the reaction pH is 6.0-8.0.

[0078] Specifically, in the method for preparing cellobiose, the reaction pH is 6.0.

[0079] Specifically, in the method for preparing cellobiose, the contents of sucrose and glucose in the reaction system are both 0.5M-1000M.

[0080] In the method for preparing cellobiose, the content of the recombinant microorganism in the reaction system is 20-40 g (wet weight) / L.

[0081] In the method for preparing cellobiose, the content of the recombinant microorganism in the reaction system is 30 g (wet weight) / L.

[0082] In the method for preparing cellobiose, the reaction system is composed of the recombinant microorganism, sucrose, glucose monohydrate and phosphate buffer. In the reaction system, the cell content of the recombinant microorganism is 30 g (wet weight) / L, the sucrose content is 342 g / L, and the glucose monohydrate content is 198 g / L.

[0083] In the method for preparing cellobiose, shaking culture is used. Exemplarily, the rotation speed of the shaking culture is 100-300 rpm. Exemplarily, the rotation speed of the shaking culture is 200 rpm.

[0084] In the method for preparing cellobiose, the reaction conditions are: 37° C., 200 rpm shaking reaction for 12 hours.

[0085] The invention will generate huge economic benefits for the production of cellobiose and has great promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Schematic diagram of the pathway by which BASP and CGCBP convert sucrose and glucose into cellobiose.

[0087] Figure 2 HPLC spectra of glucose-1-phosphate standard, cellobiose standard, phosphate standard, glucose standard and fructose standard.

[0088] Figure 3 This is the time curve of the production of cellobiose by the engineered bacteria CB02. DETAILED DESCRIPTION

[0089] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0090] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples are all repeated three times, and the results are averaged. Unless otherwise specified, the phosphate buffer in the examples is pH 6.0, 50mM phosphate buffer. The vector pBAD / HisB is provided by the pBAD / His kit, which is a product of Invitrogen, with the product catalog number V43001. Escherichia coli BW25113: NBRP E.coli strain, with the product catalog number ME9062. Sucrose (CAS number: 57-50-1; molecular formula: C 12 H 22 O 11 ; Molecular weight: 342.3): Sinopharm Chemical Reagent Co., Ltd., product catalog number 10021463. Glucose monohydrate (CAS number: 14431-43-7; molecular formula: C6H 12 O6·H2O; molecular weight: 198.17): Sinopharm Chemical Reagent Co., Ltd., product catalog number 10010518. Cellobiose (CAS number: 528-50-7; molecular formula: C 12 H 22 O 11 ; Molecular weight: 342.3): Aladdin Company, catalog number C111867. α-D-Glucose-1-phosphate disodium salt (CAS number: 56401-20-8; EINECS number: 260-154-1; molecular formula: C6H 11 Na2O9P: Merck (Sigma-Aldrich). Saccharomyces cerevisiae SC001: Saccharomyces cerevisiae with CGMCC number 2.3973 ( Saccharomyces cerevisiae ). Preparation method of SC001 seed solution of Saccharomyces cerevisiae: inoculate SC001 of Saccharomyces cerevisiae into YPD medium, and culture at 30℃ and 220rpm with shaking until the bacterial concentration reaches 20 cfu / ml. YPD medium (pH 6.0): contains 10g / L yeast powder, 20g / L peptone and 20g / L glucose, and the balance is water.

[0091] The BASP gene is shown in SEQ ID NO:2, and encodes the protein shown in SEQ ID NO:1.

[0092] The CGCBP gene is shown in SEQ ID NO:4, and encodes the protein shown in SEQ ID NO:3.

[0093] Example 1. Construction of recombinant bacteria 1. Construction of recombinant plasmid pCBPSP Using vector pBAD / HisB as the starting plasmid, the CGCBP gene shown in SEQ ID NO: 4 was replaced Xho I and Hope I enzyme cuts the small fragment between the recognition sequences and replaces it with the BASP gene shown in SEQ ID NO: 2 Hope I and Psst I enzyme cut the small fragment between the recognition sequences to obtain the recombinant plasmid pCBPSP.

[0094] The sequence of relevant elements in this recombinant plasmid is: Xho I-CGCBP gene Hope I-BASP gene- Psst I.

[0095] The recombinant plasmid pCBPSP was verified by sequencing.

[0096] 2. Construction of recombinant plasmid pSPCBP Using vector pBAD / HisB as the starting plasmid, the BASP gene shown in SEQ ID NO: 2 was replaced Xho I and Hope I enzyme cuts the small fragment between the recognition sequences and replaces it with the CGCBP gene shown in SEQ ID NO: 4 Hope I and Psst I enzyme cut the small fragment between the recognition sequences to obtain the recombinant plasmid pSPCBP.

[0097] The sequence of relevant elements in this recombinant plasmid is: Xho I-BASP gene- Hope I-CGCBP gene Psst I.

[0098] The recombinant plasmid pSPCBP was verified by sequencing.

[0099] 3. Construction of engineered bacteria The recombinant plasmid pCBPSP was introduced into Escherichia coli BW25113 to obtain the recombinant bacteria, which was named engineered bacteria CB01.

[0100] The recombinant plasmid pSPCBP was introduced into Escherichia coli BW25113 to obtain a recombinant bacterium, which was named engineered bacterium CB02.

[0101] Example 2: Synthesis of cellobiose from sucrose and glucose using engineered bacteria The engineered bacteria are the engineered bacteria CB01 or the engineered bacteria CB02 prepared in Example 1.

[0102] 1. Take a single clone of the engineered bacteria and inoculate it into a liquid LB medium containing 50 μg / mL ampicillin. Cultivate at 37°C and 220 rpm with shaking until the OD 600nm =0.6~0.8.

[0103] 2. After completing step 1, add L-arabinose to the culture system and make its concentration in the system 0.2 g / 100 mL, and culture at 30°C and 200 rpm for 12 hours.

[0104] 3. After completing step 2, take the entire culture system, centrifuge it at 4°C and 6000 rpm for 15 min, and collect the bacterial precipitate.

[0105] 4. Prepare the initial reaction system, and then shake the reaction at 37°C and 200 rpm for 12 hours. During the reaction, take samples every 1-2 hours.

[0106] The initial reaction system consists of the bacterial precipitate obtained in step 3, sucrose, glucose monohydrate and phosphate buffer. In the initial reaction system, the bacterial content is 30 g (wet weight) / L, the sucrose content is 342 g / L, and the glucose monohydrate content is 198 g / L.

[0107] 5. Take the sample, centrifuge at 6000g for 10 minutes, and collect the supernatant. Dilute the supernatant to 20 times the volume with distilled water, then filter with a filter membrane with a pore size of 0.22µm and collect the filtrate. The filtrate is tested by HPLC.

[0108] The relevant parameters of HPLC detection are as follows: HPLC chromatograph: Agilent 1260 high performance liquid chromatograph; Chromatographic column: Aminex HPX-87H (300 mm × 7.8 mm); Mobile phase: 5mmol / L sulfuric acid aqueous solution; Mobile phase flow rate: 0.3 mL / min; Column temperature: 40°C; Detector:RID.

[0109] Glucose standards, glucose-1-phosphate standards, cellobiose standards, fructose standards, and phosphate standards were prepared according to the same HPLC method and parameters. The chromatograms are shown in Figure 2The retention time of glucose-1-phosphate is 12.593 min, the retention time of cellobiose is 14.112 min, the retention time of phosphate is 15.816 min, the retention time of glucose is 17.620 min, and the retention time of fructose is 19.363 min. For HPLC under the same condition parameters, the peaks in the chromatogram with a retention time of ±0.1 min from the standard peak can be identified as the peaks of the same substance.

[0110] Each standard product was used in accordance with the same HPLC method and parameters to prepare a standard curve of standard product content and peak area, and then the content of each target substance in the system at the time of sampling was calculated based on the standard curve. The results of the engineered bacteria CB02 are shown in Figure 3 . In addition to the target product cellobiose, the reaction system also contains a large amount of by-product fructose. When the engineered bacteria CB02 is used, the cellobiose content in step 4 is 240.0 g / L (conversion rate is 70%) after 12 hours of reaction. When the engineered bacteria CB01 is used, the cellobiose content in step 4 is 188.5 g / L (conversion rate is 55%) after 12 hours of reaction.

[0111] 6. Add 0.5L of the reaction product solution from step 4 and 100mL of Saccharomyces cerevisiae SC001 seed solution to the reaction vessel (1L fermenter) and ferment for 12-24 hours. During the fermentation process, the dissolved oxygen was controlled at 30% by adjusting the speed (500rpm-1200rpm) and the pH was controlled at 6.0 by adding 20% ​​NaOH aqueous solution.

[0112] During the fermentation process, samples were taken at intervals of 1-2 hours to detect the fructose content and cellobiose content (the method is the same as step 5). After 24 hours of fermentation, no fructose remained in the fermentation system. After 24 hours of fermentation, the cellobiose content in the fermentation system and the corresponding conversion rate are shown in Table 1.

[0113] Table 1

[0114] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Recombinant microorganisms with BASP gene and CGCBP gene; The BASP gene encodes the protein shown in SEQ ID NO: 1; The CGCBP gene encodes the protein shown in SEQ ID NO:

3.

2. The recombinant microorganism according to claim 1, characterized in that: The BASP gene is derived from Bifidobacterium adolescentis; The CGCBP gene is derived from Cellulomonas gibbsii.

3. The recombinant microorganism according to claim 1, characterized in that: The coding region of the BASP gene is shown in SEQ ID NO: 2; The coding region of the CGCBP gene is shown in SEQ ID NO:

4.

4. A method for preparing a recombinant microorganism for producing cellobiose, comprising the following steps: introducing a BASP gene and a CGCBP gene into a microorganism to obtain a recombinant microorganism having the BASP gene and the CGCBP gene, that is, a recombinant microorganism for producing cellobiose; the BASP gene is the BASP gene described in claim 1, 2 or 3; the CGCBP gene is the CGCBP gene described in claim 1, 2 or 3.

5. Use of the recombinant microorganism according to any one of claims 1 to 3 in the preparation of cellobiose.

6. A method for preparing cellobiose, comprising the following steps: using sucrose and glucose as raw materials and the recombinant microorganism according to any one of claims 1 to 3 as a whole-cell catalyst to prepare cellobiose.

7. Application of biomaterial I in the preparation of recombinant microorganisms for producing cellobiose; The biological material I includes BASP gene-related biological materials and CGCBP gene-related biological materials; the BASP gene-related biological materials are BASP genes, expression cassettes with BASP genes or recombinant vectors with BASP genes; the CGCBP gene-related biological materials are CGCBP genes, expression cassettes with CGCBP genes or recombinant vectors with CGCBP genes; The BASP gene is the BASP gene described in claim 1, 2 or 3; the CGCBP gene is the CGCBP gene described in claim 1, 2 or 3.

8. Application of biomaterial II in the preparation of recombinant microorganisms for producing cellobiose; The biological material II includes a double-gene related biological material; the double gene is the BASP gene and the CGCBP gene; the double-gene related biological material is the double gene, an expression cassette having the double gene, or a recombinant vector having the double gene; The BASP gene is the BASP gene described in claim 1, 2 or 3; the CGCBP gene is the CGCBP gene described in claim 1, 2 or 3.

9. Application of biomaterial III in the preparation of cellobiose; The biological material III includes BASP gene-related biological materials and CGCBP gene-related biological materials; the BASP gene-related biological materials are BASP genes, expression cassettes with BASP genes, recombinant vectors with BASP genes, or recombinant microorganisms with BASP genes; the CGCBP gene-related biological materials are CGCBP genes, expression cassettes with CGCBP genes, recombinant vectors with CGCBP genes, or recombinant microorganisms with CGCBP genes; The BASP gene is the BASP gene described in claim 1, 2 or 3; the CGCBP gene is the CGCBP gene described in claim 1, 2 or 3.

10. Application of biomaterial IV in the preparation of cellobiose; The biological material IV includes a double-gene related biological material; the double gene is a BASP gene and a CGCBP gene; the double-gene related biological material is the double gene, an expression cassette having the double gene, a recombinant vector having the double gene, or a recombinant microorganism having the double gene; The BASP gene is the BASP gene described in claim 1, 2 or 3; the CGCBP gene is the CGCBP gene described in claim 1, 2 or 3.

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