Engineering bacterium co-culture fermentation preparation method of BC-HA component gradient composite membrane

Abstract

The invention discloses a method for regulating and controlling the component proportion of a bacterial cellulose / hyaluronic acid composite membrane on the basis of genetic engineering strain combination mixed fermentation, and belongs to the technical field of biological materials. The bacterial strain combination comprises at least two genetically modified gluconacetobacter xylinus engineering bacterial strains, and the engineering bacterial strains comprise a wild type bacterial strain for expressing empty plasmids, a bacterial strain for expressing HA recombinant plasmids, and a mutant bacterial strain for knocking out bcsH / Y / Z genes and expressing the HA recombinant plasmids. The composite membrane is obtained by regulating and controlling the inoculation ratio (1: 0.1-10) of different strains and carrying out static culture and post-treatment in an HS culture medium containing chloramphenicol. According to the method, regulation and control of the mass ratio (0-7.5%) of BC to HA in the composite membrane are innovatively realized; when the proportion of an HA expression strain is increased, the content of HA is adjustable between 0% and 1.3%, and the proportion of HA can be increased to 3% by adopting a bcs gene knockout strain combination. The obtained composite film has tunable physicochemical properties, and has important application value in the field of biomedical materials.

Description

1.1 Technical Field The present invention belongs to the field of biomaterial technology, and specifically relates to a method for preparing a composite film of bacterial cellulose (BC) and hyaluronic acid (HA) by combining several genetically engineered strains and then fermenting them together. Komagataeibacter xylinus ) strain combination to achieve the regulation of component ratio and nanostructure in BC-HA composite membrane, and expand its application potential in the field of biomedical materials. 1.2 Background Technology Gluconobacter xylonicum ( Komagataeibacter xylinus ) is the main BC production strain. Bacterial cellulose (BC) is a natural polymer material synthesized by microorganisms with high purity, high mechanical strength and biocompatibility, but its single component has limitations in functional applications (such as moisturizing and promoting repair). Hyaluronic acid (HA) is a natural polysaccharide with good moisturizing and biological activity, but its mechanical strength is poor. In the existing technology, the composite of BC and HA is mostly achieved through physical blending or chemical cross-linking, which has problems such as complex process, uneven component distribution, and weak interface bonding.

[0003] Direct synthesis of BC-HA composite membranes with specific component ratios using genetically engineered strains can fundamentally address these issues. However, existing strains are typically only capable of synthesizing either BC or HA alone and lack effective control over the ratio of composite membrane components. The present invention, by constructing a multi-genetically engineered strain combination and combining the synergistic effects of different strains, achieves in situ synthesis of BC and HA and controllable ratios. 1.3 Summary of the invention The present invention aims to solve the problem in the prior art that Gluconobacter xylonic acid acetobacter ( Komagataeibacter xylinus ) The engineered strain is unable to ferment and synthesize bacterial cellulose (BC) and hyaluronic acid (HA) while coordinating the ratio of the two. The specific technical solution is as follows: The first aspect: construction of genetically engineered bacteria To achieve the above object, the present invention provides five genetically engineered bacteria, characterized in that: based on Gluconacetobacter xylonicum ( K. xylinus ) production strains, and knocking out cellulose synthase-related genes (such as bcsH 、 bcsX 、 bcsY 、 bcsZ ) in an engineered strain that overexpresses genes related to the hyaluronic acid synthesis pathway, enabling strains with different BC production and structures to have the ability to simultaneously synthesize BC and HA.

[0005] The above-mentioned engineered strains for knocking out cellulose synthase-related genes specifically include: 1. Delete bcsH Genetically engineered strains K. xylinusΔbcsH ; 2. Delete bcsY Genetically engineered strains K. xylinusΔbcsX ; 3. Delete bcsY Genetically engineered strains K. xylinusΔbcsY ; 3. Delete bcsY Genetically engineered strains K. xylinusΔbcsY ; pSEVA331 and its recombinant plasmid HA were introduced into K. xylinus CGMCC 2955 or its gene-edited strains were screened for positive clones to obtain genetically engineered bacteria; The above-mentioned genetically engineered bacteria specifically include: 1. Strains expressing the pSEVA331 empty plasmid (SEQ ID NO. 1) in wild-type strains K. xylinus / pSEVA331; 2. Strains expressing pSEVA331-HA plasmid in wild-type strains K. xylinus / HA; 3. Delete bcsH The strain expressing pSEVA331-HA plasmid in the genetically engineered strain K. xylinusΔbcsH / HA; 4. Delete bcsX The strain expressing pSEVA331-HA plasmid in the genetically engineered strain K. xylinusΔbcsY / HA; 5. Delete bcsY The strain expressing pSEVA331-HA plasmid in the genetically engineered strain K. xylinusΔbcsY / HA; 6. Delete bcsZ The strain expressing pSEVA331-HA plasmid in the genetically engineered strain K. xylinusΔbcsZ / HA.

[0006] The second aspect: preparation method of BC-HA composite membrane The present invention also provides a method for preparing a BC-HA composite membrane using the genetically engineered bacteria, which specifically comprises: Fermentation culture: Seed culture: The strain was inoculated into HS solid medium (formula: 25 g / L glucose, 10 g / L Na2HPO4, 7.5 g / L yeast powder, 10 g / L peptone) containing 340 μg / mL chloramphenicol and cultured at 30°C for 3 days. Expansion culture: Pick a single colony into liquid HS medium (containing 340 μg / mL chloramphenicol and 4‰ cellulase), shake and culture at 30℃ and 180 rpm until OD 600 0.8-1.0; Static fermentation: After collecting the cells and washing them twice with 10 mM HEPES buffer, the initial OD 600 =0.05 was inoculated into HS culture medium and cultured at 30℃ for 3 days to form BC-HA composite membrane.

[0007] Post-processing: Alkali treatment: Immerse the membrane product in 0.1 M NaOH solution, changing the solution every 12 hours until the membrane turns milky white, and remove residual cells and culture medium; Water washing: Repeatedly rinse with deionized water until the pH is 7-8 to obtain a pure BC-HA composite membrane.

[0008] The third aspect: Regulation of HA content in BC-HA composite membrane The present invention provides a method for regulating BC-HA composite membrane by one-step fermentation, which specifically comprises: Fermentation culture: Seed culture: Genetically engineered strains with different HA contents in the BC-HA composite membrane were inoculated into HS solid medium (formula: 25 g / L glucose, 10 g / L Na2HPO4, 7.5 g / L yeast powder, 10 g / L peptone) containing 340 μg / mL chloramphenicol and cultured at 30°C for 3 days. Expansion culture: Pick a single colony into liquid HS medium (containing 340 μg / mL chloramphenicol and 4‰ cellulase), shake and culture at 30℃ and 180 rpm until OD 600 0.8-1.0; Static fermentation: After collecting the cells and washing them twice with 10 mM HEPES buffer, K. xylinus / pSEVA331 and strains with different BC and HA production were mixed in different proportions and the initial OD 600 =0.05 was inoculated into HS culture medium and cultured at 30℃ for 3 days to form BC-HA composite membrane.

[0009] Alkali treatment: Immerse the membrane product in 0.1 M NaOH solution, changing the solution every 12 hours until the membrane turns milky white, and remove residual cells and culture medium; Water washing: Soak with deionized water, changing the deionized water every 12 hours until the pH reaches 7-8 to obtain a pure BC-HA composite membrane.

[0010] Technological advantages In situ synthesis: Directly co-express the synthesis pathway of BC and HA through genetically engineered bacteria to avoid the interface defects of physical blending process and improve the uniformity of composite membrane.

[0011] One-step production: Only a single static fermentation is required to obtain the BC-HA composite membrane, which significantly simplifies the traditional multi-step composite process and reduces production costs.

[0012] Scalability: Based on the gene editing background of the starting strain (such as knocking out genes related to cellulose synthesis), the fiber structure (diameter 30-100 nm) and mechanical properties of the composite membrane can be further regulated.

[0013] Application Prospects The BC-HA composite film of the present invention has both high mechanical strength and biological activity, and can be widely used in the fields of medical dressings, tissue engineering scaffolds, and functional cosmetic carriers. 1.4 Description of the Figures Figure 1 HA production of engineered strains; Figure 2 pH and growth curves of engineered strains; The figure shows the pH change curves and growth curves of different engineered strains, where (a) K. xylinus / pSEVA331 (b) K. xylinus / HA (c) K. xylinusΔbcsH / HA (d) K. xylinusΔbcsX / HA (e) K. xylinusΔbcsY / HA (f) K. xylinusΔbcsZ / HA Figure 3 BC-HA composite film production after 3 days of shaking fermentation; Figure 4 The proportion of HA components in the BC-HA composite membrane fermented by mixed fermentation of engineered strains. 1.5 Specific Implementation Methods Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0016] 1.61. Preparation of Gluconacetobacter xylinum competent cells (1) Take the strain stored at -80°C, wait for the stored bacterial solution to thaw in an ice bath, then draw three lines on an HS medium agar plate and culture in a 30°C incubator for 24-48 hours.

[0017] (2) After the colonies have grown, pick a single colony and inoculate it into liquid HS medium. Add 1‰ (v / v) cellulase and culture at 30°C and 180 rpm in a shaking incubator for 24-48 h.

[0018] (3) Transfer the fermentation broth to 200 mL of fresh HS medium at a 1% (v / v) inoculum volume and add 1‰ (v / v) cellulase. Incubate the culture in a shaking incubator at 30°C and 180 rpm for 10-18 h.

[0019] (4) When the OD600 of the fermentation liquid is 0.5-0.8, place it in an ice bath for 20 min (all the following operations are performed in an ice bath).

[0020] (5) Transfer the fermentation broth to a 50 mL centrifuge tube and centrifuge (4000 rpm, 4°C, 5 min) to collect the cells and discard the supernatant.

[0021] (6) Resuspend the cells in 30 mL of HEPES (10 mM) buffer, collect the cells by centrifugation (4000 rpm, 4°C, 5 min), and discard the supernatant (repeat twice).

[0022] (7) Resuspend the cells in 1 mL of 15% glycerol and aliquot into 1.5 mL EP tubes (100 μL / tube) and store at -80°C.

[0023] 1.72. Electrotransformation of Gluconacetobacter xylinum (1) Remove the competent cells from the -80°C freezer and immediately place them in an ice bath.

[0024] (2) After the competent cells have thawed, add 3-4 μL of plasmid or DNA fragment (total mass of approximately 0.8-1 μg of DNA) and mix gently.

[0025] (3) Transfer the mixture of DNA and competent cells to a sterile electroporation cup and let it stand in an ice bath for 20 minutes.

[0026] (4) Take out the electroporation cup from the ice box and wipe off the water stains on the surface. Place the electroporation cup into the electroporator with a voltage of 2.5 kV and a pulse time of 5 ms.

[0027] (5) Remove the electroporation cup, resuspend the bacteria from the electroporation cup with the recovery solution of the corresponding strain, and transfer them to a 1.5 mL sterile EP tube.

[0028] (6) Resuscitate the cells (culture in a shaker at 30°C, 180 rpm for 4 h; for E. coli, culture in a shaker at 37°C, 220 rpm for 1 h).

[0029] (7) Collect the bacteria by centrifugation (8,000 rpm, 2 min), discard the supernatant (retain 100 μL of supernatant), and spread the resuspended bacterial solution on an agar plate with the corresponding resistance and culture medium (culture in a 30°C incubator).

[0030] (8) After the colonies grow, pick a single colony, perform colony PCR, and screen for positive clones.

[0031] 1.84. Fermentation of Gluconacetobacter xylinum (1) Mark three areas on HS agar medium containing 340 μg / L chloramphenicol and culture in a 30°C incubator until single colonies grow.

[0032] (2) Pick a single colony and inoculate it into 75 ml of HS seed medium containing 0.2% cellulase (v / v) and 340 μg / L chloramphenicol. Ferment in a shaking incubator at 30°C and 180 rpm for 72 h.

[0033] (3) Collect the cells by centrifugation, wash twice with 10 mM HEPES buffer, resuspend the cells, and inoculate into the fermentation medium at an initial OD = 0.05. For static fermentation, incubate in a 30°C incubator for 7 days in 75 ml of HS medium containing 340 μg / L chloramphenicol. For dynamic fermentation, incubate in a shaker at 30°C, 180 rpm for 7 days in 75 ml of HS medium containing 0.2% cellulase (v / v) and 340 μg / L chloramphenicol. Inoculate three fermentation bottles for each strain as a parallel experiment.

[0034] 1.95. Purification and recovery of cellulose membranes: (1) After static fermentation, the membrane was taken out and soaked in 0.1 mol / L NaOH solution. The NaOH solution was changed every 24 hours to remove the cells and culture medium in the cellulose membrane until the cellulose membrane turned completely milky white.

[0035] (2) In order to remove the residual NaOH in the cellulose membrane, the cellulose membrane was soaked in deionized water, changing it every 24 h until the pH of the water reached between 7 and 8 after soaking the cellulose membrane for 24 h.

[0036] 1.106. Determination of HA concentration: (1) Prepare HA standard solution at concentrations of 0, 20, 40, 60, 80, 120, and 160 mg / L as the standard solution for the standard curve.

[0037] (2) Add 500 μL of CTAB solution (2.5%) to 500 μL of HA solution and standard solution, respectively. Mix thoroughly and add 200 μL to each well of a 96-well plate. Measure the absorbance of the samples at 400 nm using a microplate reader. Draw a standard curve based on the absorbance of the standard solution and calculate the concentration of the HA solution based on the standard curve.

[0038] 1.11 The sources of biological materials used in each example are as follows: (1) pSEVA331 plasmid (laboratory collection) (2) pSEVA331-HA (laboratory collection) (3) K. xylinus CGMCC 2955 (China General Microbiological Culture Collection Center) (4) K. xylinusΔbcsH (Laboratory collection); (4) K. xylinusΔbcsX (Laboratory collection); (5) K. xylinusΔbcsY (Laboratory collection); (6, K. xylinusΔbcsZ (Laboratory collection); 1.12 Example 1: Construction of engineered strains This patent provides an engineered strain for preparing composite membranes. On the one hand, a strain carrying the pSEVA331 empty plasmid is provided. K. xylinus The wild-type strain CGMCC 2955 can grow under chloramphenicol resistance conditions, making it easy to co-culture with strains carrying the plasmid for HA synthesis (pSEVA331-HA). This strain produces BC but does not synthesize HA. By increasing the inoculation ratio of this strain during the mixed fermentation process, the BC ratio in the BC-HA composite film can be increased. On the other hand, this study provides four strains with different BC synthesis abilities carrying the pSEVA331-HA plasmid ( K. xylinus / HA, K. xylinusΔbcsH / HA, K. xylinusΔbcsX / HA 、K. xylinusΔbcsY / HA and K. xylinusΔbcsZ / HA), these strains.

[0039] First, the plasmid of HA synthesis pathway (pSEVA331-HA) was introduced into K. xylinus CGMCC 2955, K. xylinusΔbcsH 、 K. xylinusΔbcsX 、 K. xylinusΔ bcsY and K. xylinusΔbcsZ The specific process is as follows: 1. Preparation K. xylinus CGMCC 2955, K. xylinusΔbcsH 、 K. xylinusΔbcsX、K. xylinusΔbcsY and K. xylinusΔbcsZ Competent cells of these five bacterial strains.

[0040] 2. Introduce pSEVA331 into the cell line by electroporation K. xylinus CGMCC 2955; pSEVA331-HA was introduced into K. xylinus CGMCC 2955, K. xylinusΔbcsH 、 K. xylinusΔbcsY and K. xylinusΔ bcsZ Competent cells.

[0041] 3. Plate the competent cells after electroporation on solid medium containing 340 μg / L HS. After single colonies grow, screen for positive clones using colony PCR.

[0042] Next, the growth characteristics of the strain during the fermentation process were observed by testing the growth curve of the strain under shaking culture conditions ( Figure 1 ) to ensure that the newly constructed strains can have acceptable growth performance under fermentation conditions.

[0043] 1.13 Example 2: Characterization of the ability of engineered strains to synthesize HA In order to prepare BC-HA gradient composite membranes, the ability of different strains to synthesize HA was tested. The specific testing process is as follows: (1) Fermentation synthesis of HA: a) Colony activation: Take out the control strain from the -80°C freezer K. xylinus / pSEVA331 and experimental strains ( K. xylinus / HA, K. xylinusΔbcsH / HA, K. xylinusΔbcsX / HA 、K. xylinusΔbcsY / HA and K. xylinusΔbcsZ / HA), streak three areas on HS agar medium containing 340 μg / L chloramphenicol, and culture in a 30°C incubator until single colonies grow.

[0044] b) Seed culture: Pick a single colony of each of the five strains above and inoculate it into 75 ml of HS seed medium containing 0.2% cellulase (v / v) and 340 μg / L chloramphenicol. Ferment at 30°C, 180 rpm in a shaker until the OD value reaches approximately 0.8-1.0.

[0045] c) Fermentation: Cells were harvested by centrifugation, washed twice with 10 mM HEPES buffer, resuspended, and inoculated into fermentation medium at an initial OD of 0.05. Fermentation was carried out at 30°C, 180 rpm, and shaken for 7 days in 75 ml of HS medium supplemented with 0.2% cellulase (v / v) and 340 μg / L chloramphenicol. Three replicates were inoculated for each strain. After three days of fermentation, the fermentation broth was centrifuged and the supernatant collected.

[0046] 2) Isolation and purification of HA.

[0047] a) Add an equal volume of 25% (v / v) CTAB solution to 5 ml of supernatant and incubate at 37°C, 180 rpm on a shaker for 48 h to allow the polysaccharides in the supernatant to form complexes with CTAB.

[0048] b) Freeze and thaw the incubated solution once at -20°C. Centrifuge at 12,000 rpm and 16°C for 30 minutes, then remove the supernatant and collect the precipitate.

[0049] c) Resuspend the pellet in 5 ml of NaCl solution (1 mol / L NaOH) and incubate at 37°C for 48 hours. This process allows the acidic polysaccharide (HA) to dissolve and the neutral polysaccharide to precipitate, thereby separating the HA from undegraded BC. Collect the supernatant by centrifugation.

[0050] d) Add twice the volume of anhydrous ethanol to the supernatant and incubate at -20°C for 24 h. Collect the precipitate.

[0051] e) Dissolve the precipitate in 1 ml of deionized water, add twice the volume of anhydrous ethanol, and incubate at -20°C for 24 h. Collect the precipitate and repeat this process to completely remove the CTAB and inorganic salts from the solution.

[0052] f) Dissolve the precipitate in 1 ml of deionized water to obtain HA solution.

[0053] 3) Test HA concentration a) Add 500 μL of CTAB solution (2.5%) to 500 μL of HA solution and mix thoroughly.

[0054] b) Add 200 μL to each well of a 96-well plate in triplicate. Measure the absorbance of the samples at 400 nm using a microplate reader. Calculate the HA concentration of the solution based on the standard curve and subtract the HA concentration of the control strain from the HA concentration of the experimental strain. Figure 2 ).

[0055] The test results show that the HA production of different engineered strains is significantly different, so the proportion of HA in the BC-HA composite membrane can be controlled by adjusting the strain combination.

[0056] 1.14 Example 3: Preparation of BC-HA Gradient Composite Membrane In order to prepare the BC-HA gradient component composite membrane, different strains were mixed and fermented to test the effect of the inoculation ratio on the BC-HA ratio. The specific test process is as follows: (1) Fermentation synthesis of HA: a) Colony activation: Take out the control strain from the -80°C freezer K. xylinus / pSEVA331 and experimental strains ( K. xylinus / HA, K. xylinusΔbcsH / HA, K. xylinusΔbcsX / HA 、K. xylinusΔbcsY / HA and K. xylinusΔbcsZ / HA), streak three areas on HS agar medium containing 340 μg / L chloramphenicol, and culture in a 30°C incubator until single colonies grow.

[0057] b) Seed culture: Pick a single colony of each of the five strains above and inoculate it into 75 ml of HS seed medium containing 0.2% cellulase (v / v) and 340 μg / L chloramphenicol. Ferment at 30°C, 180 rpm in a shaker until the OD value reaches approximately 0.8-1.0.

[0058] c) Fermentation: Collect the cells by centrifugation, wash twice with 10 mM HEPES buffer, and dilute the resuspended cells to an OD of 1. Group the five strains according to Table 1 and mix them in the appropriate proportions. Inoculate the cells into the fermentation medium at an initial OD of 0.05. Cultivate the cells in a 30°C incubator for 3 days in 75 ml of HS medium supplemented with 340 μg / L chloramphenicol. Inoculate three fermentation flasks for each strain as replicates.

[0059] Table 1 Inoculation ratios for fermentation preparation of BC-HA gradient composite membranes

[0060] After the fermentation is completed, the membrane in the culture medium is removed and purified to obtain the BC-HA composite membrane. The BC-HA composite membrane is freeze-dried and then weighed to calculate the dry weight of the BC-HA composite membrane ( Figure 3 ). The results show that the composite membrane yield increases with K. xylinusThe yield of BC-HA composite membrane was above 1.5 g / L after 3 days of fermentation.

[0061] 1.15 Example 4: Characterization of the BC-HA Ratio in BC-HA Gradient Composite Membranes The BC-HA composite membrane obtained in Example 3 was immersed in water. The cellulose membrane was homogenized using a homogenizer, and then 0.2% (v / v) cellulase was added thereto. The mixture was stirred until the cellulose was completely degraded.

[0062] HA was extracted from the solution according to the steps in Example 2 and freeze-dried to obtain HA.

[0063] Add 500 μL of CTAB solution (2.5%) to 500 μL of HA solution, mix thoroughly, and add 200 μL to each well. Add three replicates of each sample to a 96-well plate. Measure the absorbance of the sample at 400 nm using a microplate reader. Calculate the concentration of the HA solution based on the standard curve, and further calculate the proportion of HA in the composite membrane ( Figure 4 The results showed that the proportion of HA components in the BC-HA composite membrane can be regulated by the combination of different engineered strains and the inoculation ratio. K. xylinus / HA, K. xylinusΔbcsH / HA, K. xylinusΔbcsY / HA, K. xylinusΔbcsZ / HA inoculation ratio increased 10 times, and HA content increased 3-5 times.

Claims

1. An engineered bacterial strain combination for bacterial cellulose (BC) and hyaluronic acid (HA) composite membrane, characterized in that Contains the following Gluconacetobacter xylonicum ( Komagataeibacter xylinus ) At least two of the genetically engineered strains: (a) Wild-type gene background strain expressing empty plasmid K. xylinus / pSEVA331; (b) Wild-type gene background strain expressing recombinant plasmid K. xylinus / HA; (c) Knockout bH strains expressing recombinant plasmids K. xylinusΔbcsH / HA; (d) Knockout bcsX strains expressing recombinant plasmids K. xylinusΔbcsX / HA; (e) Knockout bqY strains expressing recombinant plasmids K. xylinusΔbcsY / HA; (f) Knockout bZ strains expressing recombinant plasmids K. xylinusΔbcsZ / HA.

2. A method for preparing a BC / HA composite membrane, characterized in that The method of applying the strain combination according to claim 1 comprises the following steps: a) Inoculate at least two engineered strains at a viable cell count ratio of 1:0.1-10 into HS medium containing 340 μg / mL chloramphenicol; b) incubate at 30°C for 3 days to form a membrane-like product; c) The membrane product was treated with 0.1 M NaOH solution for 12 hours and then treated with deionized water to neutrality to obtain a BC / HA composite membrane.

3. The preparation method according to claim 2, wherein The strain combination is selected from any one of the following: a) K. xylinus / pSEVA331+ K. xylinus / HA: BC / HA mass ratio ranges from 0 to 1.29%; b) K. xylinus / pSEVA331 / pSEVA331 + K. xylinusΔbcsH / HA: The mass proportion of HA in BC / HA ranges from 0 to 2.02%; c) K. xylinus / pSEVA331 / pSEVA331 + K. xylinusΔbcsX / HA: The mass ratio of HA in BC / HA ranged from 0 to 2.31%; d) K. xylinus / pSEVA331 / pSEVA331 + K. xylinusΔbcsY / HA: the mass of HA in the BC / HA mass ratio ranged from 0 to 2.76%; e) K. xylinus / pSEVA331 / pSEVA331 + K. xylinusΔbcsZ / HA: The mass ratio of HA in BC / HA ranges from 0 to 3.35%.

4. The preparation method according to claim 3, characterized in that The BC / HA ratio is regulated by the strain combination type and inoculation ratio: a) When K. xylinus / HA inoculation ratio increased 10-fold, HA content increased 3 to 5 times; b) When K. xylinusΔbcsH / When the HA inoculation ratio increases 10-fold, the BC content increases 3 to 5 times; c) When K. xylinusΔbcsY / HA inoculation ratio increased 10-fold, BC content increased 3 to 4 times; d) When K. xylinusΔbcsZ When the / HA inoculation ratio increased 10 times, the BC content increased 3 to 5 times.

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