Foal bacillus for producing bacterial cellulose by efficiently utilizing cellobiose as well as construction method and application of foal bacillus

Abstract

The invention discloses foal bacillus capable of efficiently utilizing cellobiose to produce bacterial cellulose and a construction method and application thereof, the foal bacillus capable of efficiently utilizing cellobiose to produce bacterial cellulose (Komagaeibacter xylinus) GX071 (CGMCC No.26247) or the foal bacillus capable of efficiently utilizing cellobiose to produce bacterial cellulose (Komagaeibacter xylinus) GX073 (CGMCC No.26248) is fermented, the utilization rate of GX071 cellobiose is increased by 19%, the yield of bacterial cellulose is increased by 24%, and the yield of bacterial cellulose is increased by 24%. The utilization rate of cellobiose of GX073 is increased by 31%, and the yield of bacterial cellulose is increased by 38%. Lignocellulose resources such as forestry and agricultural residues are effectively utilized, green biological manufacturing is achieved, the production cost is effectively reduced, and the requirement for large-scale industrial production of bacterial cellulose is met.

Description

Technical Field

[0001] The invention relates to the field of bacterial cellulose production, in particular to a bacterial strain for efficiently utilizing cellobiose to produce bacterial cellulose and its application. Background Art

[0002] Bacterial cellulose (BC) is a nanopolymer synthesized by microbial fermentation. It is composed of glucose and has extremely high purity and biodegradability. Functionalized bacterial cellulose has broad application prospects in biomedicine, food, papermaking and other fields.

[0003] Reducing the production cost of bacterial cellulose, especially the cost of culture medium, is the basis for realizing large-scale industrial production of bacterial cellulose.

[0004] Lignocellulose is the most abundant green renewable resource. Effective utilization of lignocellulose resources such as agricultural and forestry waste is a key way to reduce the production cost of bio-based products and green biomanufacturing. The bacterial cellulose production strain Gluconacetobacter xylinus (G xylinus for short) can produce bacterial cellulose using glucose in lignocellulose hydrolysate. However, glucose has a significant carbon metabolism inhibitory effect on other carbon sources in the hydrolysate, such as xylose and arabinose, which affects the co-fermentation rate of the composite carbon source.

[0005] Traditional wild-type bacterial cellulose production strains have a weak ability to utilize cellobiose. Therefore, there is an urgent need for a strain that can efficiently utilize cellobiose to produce bacterial cellulose, so as to improve the fermentation efficiency of lignocellulose hydrolysate, effectively reduce the production cost of bacterial cellulose, and meet the needs of large-scale industrial production of bacterial cellulose. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a bacillus xylostella which can efficiently utilize cellobiose to produce bacterial cellulose.

[0007] The second object of the present invention is to provide a method for constructing Bacillus xylostella which can efficiently utilize cellobiose to produce bacterial cellulose.

[0008] The third object of the present invention is to provide a use of Bacillus xylostella which can efficiently utilize cellobiose to produce bacterial cellulose.

[0009] The fourth object of the present invention is to provide another strain of Bacillus xylem that efficiently utilizes cellobiose to produce bacterial cellulose.

[0010] The fifth object of the present invention is to provide another method for constructing Bacillus xylem that efficiently utilizes cellobiose to produce bacterial cellulose.

[0011] The sixth object of the present invention is to provide another use of Bacillus xylostella which efficiently utilizes cellobiose to produce bacterial cellulose in efficiently utilizing cellobiose to produce bacterial cellulose.

[0012] The technical solution of the present invention is summarized as follows:

[0013] Komagataeibacter xylinus GX071, CGMCC No.26247, which efficiently utilizes cellobiose to produce bacterial cellulose.

[0014] The method for constructing the above-mentioned Xylinobacterium is to introduce a recombinant plasmid pBLy94A into Gluconacetobacter xylinus CGMCC No. 2955, wherein the recombinant plasmid pBLy94A contains the LacY gene and the cep94A gene;

[0015] The nucleotide sequence of the recombinant plasmid pBLy94A is shown in SEQ ID No. 1;

[0016] The nucleotide sequence of the LacY gene is shown in SEQ ID No.2;

[0017] The nucleotide sequence of the cep94A gene is shown in SEQ ID No.3.

[0018] The use of the above-mentioned bacillus in efficiently utilizing cellobiose to produce bacterial cellulose.

[0019] The second type of bacterial cellulose produced by efficiently utilizing cellobiose is Komagataeibacterxylinus GX073, CGMCC No.26248.

[0020] The second method for constructing Bacillus xylinus is to introduce a recombinant plasmid pBLy3CA into Gluconacetobacter xylinus CGMCC No. 2955, wherein the recombinant plasmid pBLy3CA contains the LacY gene and the Bg13c gene;

[0021] The nucleotide sequence of the recombinant plasmid pBLy3CA is shown in SEQ ID No.4;

[0022] The nucleotide sequence of the LacY gene is shown in SEQ ID No.2;

[0023] The nucleotide sequence of the Bg13c gene is shown in SEQ ID No.5.

[0024] The use of the second type of Bacillus mentioned above in efficiently utilizing cellobiose to produce bacterial cellulose.

[0025] Advantages of the present invention:

[0026] The invention introduces LacY gene and cep94A gene or LacY gene and Bg13c gene into xylinobacter, and obtains recombinant Komagataeibacter xylinus GX071, CGMCC No. 26247 and recombinant Komagataeibacter xylinus GX073, CGMCC No. 26248 respectively. The two recombinant bacteria are fermented respectively, and the utilization rate of cellobiose is improved. The utilization rate of cellobiose of GX071 is improved by 19%, and the bacterial cellulose yield is improved by 24%. The utilization rate of cellobiose of GX073 is improved by 31%, and the bacterial cellulose yield is improved by 38%. The wood cellulose resources such as agricultural and forestry wastes are effectively utilized, and green biological manufacturing is carried out, the production cost of bacterial cellulose is effectively reduced, and the needs of large-scale industrial production of bacterial cellulose are met. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The effects of 2955 (abbreviation of CGMCC No. 2955), GX071, and GX073 on cellobiose utilization and bacterial cellulose production.

[0028] Figure 2 The microscopic network structure of bacterial cellulose membranes produced by 2955, GX071, and GX073 in CM medium;

[0029] (A) Microscopic network structure of bacterial cellulose film produced by 2955 in CM medium;

[0030] (B) Microscopic network structure of bacterial cellulose film produced by GX071 in CM medium;

[0031] (C) Microstructure of bacterial cellulose film produced by GX073 in CM medium. DETAILED DESCRIPTION

[0032] Gluconacetobacter xylinus was deposited on March 18, 2009 at the General Microbiology Center of China Culture Collection Administration, located at Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No.2955.

[0033] Komagataeibacter xylinus GX071 was deposited on December 26, 2022 at the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 26247.

[0034] Komagataeibacter xylinus GX073 was deposited on December 26, 2022 at the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 26248.

[0035] The bacterium with the deposit number of CGMCC No. 2955 is called Gluconacetobacter xylinus. The bacterium is used as the starting bacterium. Two recombinant bacteria are constructed in the present invention. The recombinant Gluconacetobacter xylinus is Komagataeibacter xylinus.

[0036] The present invention will be further described below by means of specific examples.

[0037] Example 1

[0038] Construction of recombinant strains:

[0039] 1. Using plasmid pBLy (as shown in SEQ ID No.6, modified from commercial plasmid pBla (SEQ ID No.12)) as template, the target gene LacY (SEQ ID No.2) was amplified using upstream primer PtlacyF (SEQ ID No.7) and downstream primer PtlacyR (SEQ ID No.8);

[0040] Using plasmid pBcep94A (shown in SEQ ID No. 9, modified from commercial plasmid pBla (SEQ ID No. 12)) as a template, upstream primer pBceF (SEQ ID No. 10) and downstream primer pBceR (SEQ ID No. 11) were used to amplify the target gene cep94A (SEQ ID No. 3).

[0041] The DNA polymerase used to amplify the target gene was Max Super-Fidelity DNA Polymerase; use agarose gel electrophoresis to check the fragment size, and purify the correct band using Tiangen Biochemical Technology Co., Ltd. agarose gel recovery kit. The reaction system is: Max Super-Fidelity DNA Polymerase, 1μL; dNTPMix (10mM), 1μL; 2×Phanta Max Buffer, 25μL; upstream primer (10μM), 2μL; downstream primer (10μM), 2μL; template DNA, 0.5μL; ddH2O, 18.5μL. Reaction conditions: 98℃, 3min; 95℃, 15s; 53℃, 15s; 72℃, 3min; 32 cycles; 72℃, 5min.

[0042] 2. Treat the vector pBla (SEQ ID No. 12) with XbaI and BglⅡ restriction endonucleases at 37°C for 2 h, perform agarose gel electrophoresis after enzyme digestion, and purify using the agarose gel recovery kit of Tiangen Biochemical Technology Co., Ltd. to obtain a linearized vector.

[0043] 3. The target gene LacY, cep94A and linearized vector were connected using the ClonExpressUltra One Step Cloning Kit. Prepare the reaction system in an ice water bath, use a pipette to mix the components in the solution thoroughly, centrifuge briefly and react in a 37°C metal bath for 30 minutes. After the reaction, immediately put the reaction tube into the ice water mixture to cool for 5 minutes, and then transform E. coli (E. coli DH5α). The reaction system is: 5xCE MultiS Buffer, 4μL; Exnase MultiS, 2μL; linearized vector, 1μL; insert amplification product LacY, cep94A fragment, 1.5μL each; dd H2O, 10μL.

[0044] 4. Place the competent cells of E. coli (E. coli DH5α) on ice for 5 minutes, add 5 μL of the product obtained in step 3 to the competent cells, mix well and place on ice for 30 minutes. Heat shock at 42°C for 90 seconds, immediately place on ice for 3 minutes, add 900 μL of preheated LB liquid culture medium, and shake culture at 37°C for 1.5 hours. Centrifuge at 6000r / min for 3 minutes, discard an appropriate amount of supernatant, resuspend the remaining supernatant and bacterial pellet, apply to LB solid culture plate containing 100 μg / mL kanamycin, and culture overnight at 37°C.

[0045] 5. Pick a single colony as a template for PCR reaction. The reaction system is: 2x GoTaq Green PCR mastermix, 5 μL; upstream primer ZCCF (SEQ ID No. 13) 0.25 μmol; downstream primer ZCCR (SEQ ID No. 14) 0.25 μmol; sterile water 5 μL.

[0046] The verified positive transformants were inoculated into LB liquid culture medium and cultured at 37°C and 200 r / min for 16 h. The plasmids were extracted and sequenced to obtain the recombinant plasmid pBLy94A (SEQ ID No. 1). The recombinant plasmid pBLy94A contained the LacY gene (SEQ ID No. 2) and the cep94A gene (SEQ ID No. 3).

[0047] 6. Using the recombinant plasmid pBLy94A as a template, the upstream primer pBlyF (SEQ ID No. 15) and the downstream primer pBlyR (SEQ ID No. 16) were used to amplify the target gene LacY (SEQ ID No. 2);

[0048] Using the E. coli DH5α genome as a template, the target gene Bgl3C (SEQ ID No. 5) was amplified using the upstream primer bgF (SEQ ID No. 17) and the downstream primer bgR (SEQ ID No. 18).

[0049] The DNA polymerase used to amplify the target gene was Max Super-Fidelity DNA Polymerase; use agarose gel electrophoresis to check the fragment size, and purify the correct band using Tiangen Biochemical Technology Co., Ltd. agarose gel recovery kit. The reaction system is: Max Super-Fidelity DNA Polymerase, 1μL; dNTPMix (10mM), 1μL; 2×Phanta Max Buffer, 25μL; upstream primer (10μM), 2μL; downstream primer (10μM), 2μL; template DNA, 0.5μL; ddH2O, 18.5μL. Reaction conditions: 98℃, 3min; 95℃, 15s; 53℃, 15s; 72℃, 3min; 32 cycles; 72℃, 5min.

[0050] 7. Treat the vector pBla (SEQ ID No. 12) with XbaI and BglⅡ restriction endonucleases at 37°C for 2 h, perform agarose gel electrophoresis after enzyme digestion, and purify using the agarose gel recovery kit of Tiangen Biochemical Technology Co., Ltd. to obtain a linearized vector.

[0051] 8. The target genes LacY, Bgl3C and the linearized vector were connected using the ClonExpressUltra One Step Cloning Kit. Prepare the reaction system in an ice-water bath, use a pipette to mix the components in the solution thoroughly, centrifuge briefly and react in a 37°C metal bath for 30 minutes. After the reaction, immediately put the reaction tube into the ice-water mixture to cool for 5 minutes, and then transform E. coli (E. coli DH5α). The reaction system is: 5xCE MultiS Buffer, 4μL; Exnase MultiS, 2μL; linearized vector, 1μL; insert amplification products LacY, Bgl3C fragments, 1.5μL each; dd H2O, 10μL.

[0052] 9. Place the competent cells of E. coli (E. coli DH5α) on ice for 5 minutes, add 5 μL of the product obtained in step 8 to the competent cells, mix well and place on ice for 30 minutes. Heat shock at 42°C for 90 seconds, immediately place on ice for 3 minutes, add 900 μL of preheated LB liquid culture medium, and shake culture at 37°C for 1.5 hours. Centrifuge at 6000r / min for 3 minutes, discard an appropriate amount of supernatant, resuspend the remaining supernatant and bacterial pellet, apply to LB solid culture plate containing 100 μg / mL kanamycin, and culture overnight at 37°C.

[0053] 10. Pick a single colony as a template for PCR reaction. The reaction system is: 2x GoTaq Green PCR mastermix, 5 μL; upstream primer ZCCF (SEQ ID No. 13) 0.25 μmol; downstream primer LaBgR (SEQ ID No. 19) 0.25 μmol; sterile water 5 μL.

[0054] The verified positive transformants were inoculated into LB liquid culture medium and cultured at 37°C and 200 r / min for 16 h. The plasmids were extracted and sequenced to obtain the recombinant plasmid pBLy3CA (SEQ ID No. 4). The recombinant plasmid pBLy3CA contained the LacY gene (SEQ ID No. 2) and the Bg13c gene (SEQ ID No. 5).

[0055] 11. Pick a single colony of Gluconacetobacter xylinus (CGMCC No. 2955) from a culture dish with an inoculating loop, transfer it to a test tube containing 5 mL of liquid culture medium with glucose as the carbon source (HS culture medium for short, with a formula of 10 g / L peptone, 7.5 g / L yeast extract powder, 10 g / L disodium hydrogen phosphate, 25 g / L glucose, pH=5.0, and the balance is water; 2% agar is added to form HS solid culture medium), culture at 30°C and 180 r / min for 24 h, and then draw 1.5 mL of bacterial solution from the test tube with an inoculum size of 3% (v / v) and transfer it to a 250 mL shake flask containing 50 mL HS culture medium, and continue to culture with shaking for 3 h.

[0056] 12. Add 100 μL of cellulase solution (Taijin) to the bacterial solution in step 11, shake and culture until OD600 is 0.6-0.8, and place at 4°C;

[0057] 13. Take 35 mL of the bacterial solution from step 12 into a 50 mL pre-cooled sterile centrifuge tube, centrifuge at 6000 r / min, 4°C for 6 min, and discard the supernatant.

[0058] 14. Add 5 mL of 1 mM HEPES, mix thoroughly, centrifuge at 6000 r / min, 4°C for 6 min, discard the supernatant, and repeat twice.

[0059] 15. Add 5 ml of 15% glycerol, pipette repeatedly to resuspend the bacterial pellet, and obtain G. xylinus (CGMCC No. 2955) competent cells.

[0060] 16. Take 100 μL of the product obtained in step 15 and place it in two pre-cooled sterile 1.5 mL EP tubes. Place the electroporation cup on ice and inject 100 μL of G. xylinus (CGMCC No. 2955) competent cells. Add 5 μL of pBLy94A and 5 μL of pBLy3CA plasmids respectively, and electroporate after ice bathing for 20 minutes. The electroporation conditions are: 2500 V, 200 Ω, 25 μF.

[0061] 17. Under a sterile environment, repeatedly pipette and aspirate the competent cells with 1 mL of HS medium, aspirate the bacterial solution into a 1.5 mL EP tube, and culture at 30°C and 180 rpm for 12 hours.

[0062] 18.6000r / min, centrifuge for 3min, and discard the supernatant. Add 100μL HS medium to resuspend the cells, spread on HS solid medium containing 100μg / mL kanamycin, and pick the transformants after culturing for 2-3d, and perform colony PCR verification and plasmid sequencing verification. Komagataeibacter xylinus GX071 (referred to as GX071) and Komagataeibacterxylinus GX073 (referred to as GX073) were obtained for efficient use of cellobiose to produce bacterial cellulose and were preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, and survived.

[0063] Example 2

[0064] Determination of bacterial cellulose production

[0065] 1. Determination of bacterial cellulose production under static fermentation conditions in glucose medium

[0066] 1) Use an inoculation loop to pick single colonies of strains CGMCC No.2955, GX071 and GX073 from the culture medium and place them in 5 mL of HS culture medium. Shake and culture at 180 rpm and 30°C for 72 h.

[0067] 2) Pipette the culture solution from step 1) into 30 mL HS medium until the initial OD 600 =0.1, and cultured in a 30°C incubator for 9 days.

[0068] 3) On the third, sixth and ninth days, 3 mL of the fermentation broth was taken and centrifuged at 12000 r / min for 10 min to remove bacterial cellulose and bacterial precipitate. After dilution, the glucose content was determined using an SBA-40E biosensor (commercial product).

[0069] 4) After nine days of cultivation, the bacterial cellulose membrane in the shake flask was taken out and repeatedly washed with distilled water to remove the fermentation liquid and bacterial residues on the surface of the bacterial cellulose membrane.

[0070] 5) Soak the bacterial cellulose membrane in 0.1 M NaOH aqueous solution, and soak it repeatedly until the membrane turns milky white.

[0071] 6) Continue soaking in distilled water until it becomes neutral.

[0072] 7) The bacterial cellulose film was made into a dry sheet of constant weight using a sheet machine at 95°C.

[0073] There were little differences between GX071, GX073 and the control strain CGMCC No.2955 in bacterial cellulose film production. The bacterial cellulose film production of the control strain CGMCC No.2955 was 5.6 g / L, and the bacterial cellulose film production of GX071 and GX073 was 5.4 g / L, proving that the introduction of the cellobiose metabolic pathway did not have a negative impact on the growth of the bacteria.

[0074] 2. Determination of carbon source consumption and bacterial cellulose production under cellobiose medium conditions

[0075] 1) Use an inoculation loop to pick single colonies of the control strain CGMCC No. 2955, the constructed strains GX071 and GX073 from the culture medium and place them in 5 mL of cellobiose liquid medium (CM medium), and culture them in a shaker at 180 r / min and 30° C. for 96 h.

[0076] The formula of CM medium is as follows: cellobiose 10 g / L, yeast extract powder 7.5 g / L, peptone 10 g / L, disodium hydrogen phosphate 10 g / L, pH=5.0, and the balance is water.

[0077] 2) Pipette the culture solution obtained in step 1) into 30 mL of CM medium until the initial OD 600 =0.1, and cultured in a 30°C incubator for 9 days.

[0078] 3) On the third, sixth and ninth days, 3 mL of fermentation broth was taken and centrifuged at 12000 r / min for 10 min to remove bacterial cellulose and bacterial precipitate. After dilution, the cellobiose content was determined by HPLC (differential). The supernatant fermentation broth was filtered using a 0.22 μm water filter membrane, with 5 mM H2SO4 passing through a 0.22 μm water membrane as the mobile phase and Biorad Aminex HPX-87H as the chromatographic column. The detection conditions were set as follows: ultrasonic degassing of the mobile phase for 40 min, injection volume of 10 μL, detection time of 20 min, flow rate of 0.6 mL / min, and column temperature of 65 °C.

[0079] 4) After nine days of culture, the bacterial cellulose membrane in the shake flask was taken out and repeatedly washed with distilled water to remove the fermentation liquid and bacterial residues on the surface of the cellulose membrane.

[0080] 5) Soak the cellulose membrane in 0.1 M NaOH aqueous solution, and soak it repeatedly until the membrane turns milky white.

[0081] 6) Continue soaking in distilled water until it becomes neutral.

[0082] 7) The bacterial cellulose film was made into a dry sheet of constant weight using a sheet machine at 95°C.

[0083] from Figure 1 It can be seen that the construction of the cellobiose metabolic pathway effectively increased the yield of bacterial cellulose (BC) membrane. Among them, GX073 performed more prominently. Compared with CGMCC No.2955.

[0084] GX073 showed a 31% increase in cellobiose utilization and a 38% increase in bacterial cellulose production.

[0085] GX071 showed a 19% increase in cellobiose utilization and a 24% increase in bacterial cellulose production.

[0086] 8) The purified bacterial cellulose membrane was placed in liquid nitrogen for 20 minutes and then transferred to a vacuum freeze dryer until the sample temperature was room temperature. A thin layer was torn off from the dried sample and placed on the sample tray. After gold plating, it was placed in the scanning electron microscope sample chamber for observation and photography. A field emission scanning electron microscope was used. The scanning electron microscope parameters were HV = 1.00 kW, spot = 3.2. The scanning electron microscope results are shown in Figure 1. Figure 2 As shown (where A is 2955, B is GX071, and C is GX073), the fiber diameter and network structure of the bacterial cellulose membrane produced by the engineered strain introduced into the cellobiose metabolic pathway did not change significantly.

Claims

1. Komagataeibacter xylinus GX071, CGMCC No.26247, which efficiently utilizes cellobiose to produce bacterial cellulose.

2. The method for constructing the bacillus xylem according to claim 1, characterized in that Introducing a recombinant plasmid pBLy94A into Gluconacetobacter xylinus CGMCC No.2955, wherein the recombinant plasmid pBLy94A contains a LacY gene and a cep94A gene; The nucleotide sequence of the recombinant plasmid pBLy94A is shown in SEQ ID No. 1; The nucleotide sequence of the LacY gene is shown in SEQ ID No.2; The nucleotide sequence of the cep94A gene is shown in SEQ ID No.

3.

3. Use of the bacillus according to claim 1 in efficiently utilizing cellobiose to produce bacterial cellulose.

4. Komagataeibacter xylinus GX073, CGMCC No.26248, which efficiently utilizes cellobiose to produce bacterial cellulose.

5. The method for constructing the Xylocoporus spp. according to claim 4, characterized in that Introducing a recombinant plasmid pBLy3CA into Gluconacetobacter xylinus CGMCC No.2955, wherein the recombinant plasmid pBLy3CA contains the LacY gene and the Bg13c gene; The nucleotide sequence of the recombinant plasmid pBLy3CA is shown in SEQ ID No.4; The nucleotide sequence of the LacY gene is shown in SEQ ID No.2; The nucleotide sequence of the Bg13c gene is shown in SEQ ID No.

5.

6. Use of the bacillus according to claim 4 in efficiently utilizing cellobiose to produce bacterial cellulose.

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