Preparation of engineering bacteria for improving catalytic efficiency of glucuronic acid and conversion method of glucuronic acid

Abstract

The invention discloses a method for preparing engineering bacteria capable of improving the catalytic efficiency of glucuronic acid and a method for converting glucuronic acid, which comprises the following steps of: through carrier construction, mutant construction and expression strain preparation, adding inositol transport protein to an inositol oxidase expression strain, and mutating lazy acid at the 57th site of inositol oxidase into alanine, the catalytic efficiency of the new genetic engineering strain on substrate inositol within the same time is improved by 43%, the substrate can be converted into a product glucuronic acid more quickly, and the risk that inositol oxidase is low in stability and incomplete in catalysis is greatly reduced.

Description

Technical Field

[0001] The invention relates to the technical field of lactic acid fermentation, and in particular to a method for preparing an engineering bacterium for improving the catalytic efficiency of glucuronic acid and a method for converting glucuronic acid. Background Art

[0002] Glucuronic acid (Glucuronic acid) is also called glucuronic acid, which is the uronic acid formed by the oxidation of the C-6 hydroxyl group of glucose to a carboxyl group. Glucuronic acid can be combined with unconjugated bilirubin to generate conjugated bilirubin. It has high solubility and low toxicity and is excreted from the body through the bile duct. Glucuronic acid can be combined with a variety of harmful substances in the liver to play a detoxifying role. Glucuronic acid can not only be used as a liver detoxifier, but also as an intermediate of many drugs, and has a very broad application prospect. Chinese patent CN109423469B discloses a method for producing glucuronic acid and its special engineering bacteria, using inositol as a substrate, producing glucuronic acid by inositol oxidase, but due to the low stability of inositol oxidase, it is easy to inactivate, resulting in the potential risk of low catalytic efficiency in production. Summary of the invention

[0003] The first technical problem to be solved by the present invention is: in view of the deficiencies in the prior art, a method for preparing an engineering bacterium for improving the catalytic efficiency of glucuronic acid is provided, which has high catalytic efficiency.

[0004] To solve the above-mentioned first technical problem, the technical solution of the present invention is:

[0005] A method for preparing an engineering bacterium for improving the catalytic efficiency of glucuronic acid comprises the following steps:

[0006] S1. Vector construction

[0007] S1-01, Genes and Primers

[0008] Take inositol oxidase (MIOX) and inositol transporter (ioIT), wherein the nucleotide sequence of the ioIT is shown in SEQ ID NO.1, and the amino acid sequence of the ioIT is shown in SEQ ID NO.2;

[0009]

[0010]

[0011] S1-02, construction of vector pBAD / HisB-MIOX+ioIT

[0012] ① MIOX target gene acquisition: 2× high-fidelity enzyme mix 50μL, template 1μL, MIOX F 2μL, MIOX R 2μL, ddH 2O to make up 100 μL, reaction procedure: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 20 s, cooling to annealing temperature 55℃ annealing for 20 s, 72℃ extension for 1 min, a total of 30 cycles; 72℃ extension for 10 min, 4℃ insulation. Then PCR product purification;

[0013] ioIT target gene acquisition: 2× high-fidelity enzyme mix 50μL, template 1μL, ioIT F 2μL, ioIT R 2μL, ddH 2 O to make up 100 μL, reaction procedure: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 20 s, cooling to annealing temperature 55℃ annealing for 20 s, 72℃ extension for 1 min, a total of 30 cycles; 72℃ extension for 10 min, 4℃ insulation. Then PCR product purification;

[0014] MIOX+ioIT target gene acquisition: 2× high-fidelity enzyme mix 50μL, template 1μL, MIOX F 2μL, ioIT R 2μL, ddH 2 O to make up 100 μL, reaction procedure: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 20 s, cooling to annealing temperature 55℃ annealing for 20 s, 72℃ extension for 2 min, a total of 30 cycles; 72℃ extension for 10 min, 4℃ insulation. Then PCR product purification;

[0015] ②Double restriction enzyme system and reaction conditions: XhoI / EcoRI double restriction enzyme digestion was performed on plasmid pBAD / HisB and target gene MIOX+ioIT, 10*buffer 5μL, BSA 2μL, XhoI / EcoRI 2 / 2μL, DNA 20μL, ddH 2 O 19μL, a total of 50μL, 37℃ digestion for 1h, recover the target vector fragment and target gene fragment;

[0016] ③T4 DNA ligase reaction system and conditions: vector DNA 1μg, target fragment 2μg, T4 ligase 1μL, 10×T4 ligase buffer 1μL, ddH 2 O to make up to 10 μL, and connect at 16°C for 2 h;

[0017] ④Chemical transformation: Take 10 μL of the ligation product and add it to DH5α competent cells, place it on ice for 30 min, heat shock it at 42°C for 90 s, place it on ice for 2 min, add 600 μL of antibiotic-free LB liquid medium, incubate it at 37°C and 160-180 rpm for 60 min, and spread it on LB (Str 50 μg / mL) resistance plates;

[0018] ⑤ Screening of positive clones of cloning strains: After colony PCR verification, the plasmid was sent to BGI for sequencing verification. The vector with the correct clone was named: pBAD / HisB-MIOX+ioIT;

[0019] S2. Construction of mutants

[0020] S2-01. Screening of mutation sites

[0021] Through AlphaFold site prediction, MIOX predicted the key site K57, where the amino acid sequence 57th amino acid was mutated to alanine;

[0022] S2-02, Primer

[0023]

[0024] S2-03, Mutation PCR system and procedure

[0025] 5×Phusion HF buffer 2μL, dNTP (2.5mmol / L) 0.8μL, DNA (vector pB AD / HisB-MIOX+ioIT) template 0.2μL, K57A F / K57A R 0.3μL each, DMSO 0.3μL, Phu high-fidelity DNA polymerase (2U / μl) 0.1μL, ddH 2 O 6μL, total volume 10μL; pre-denaturation: 98℃ denaturation for 3min; amplification cycle: 98℃ denaturation for 10s, 55℃ annealing for 30s, 72℃ extension for 4.5min, 32 cycles; fill-in: 72℃ extension for 10min;

[0026] S2-04, DpnI digestion template system (10 μL) and reaction conditions

[0027] 10× buffer 1 μL, DNA PCR product 8 μL, DpnI 1 μL; 37°C, 1 h;

[0028] S2-05, Chemical Conversion

[0029] After the PCR product was digested with DpnI at 37°C for 1 hour, 10 μL was added to the competent E. coli DH 5α, ice bathed for 30 minutes, heat-shocked in a 42°C water bath for 90 seconds, and placed on ice for 5 minutes. Add 600 μL LB liquid culture medium and spread on the LB (Str50 μg / mL) plate until the bacterial liquid is completely absorbed. Invert the plate and culture at 37°C for 12 to 16 hours until the corresponding colonies are visible;

[0030] S2-06. Screening of positive clones of clonal strains

[0031] The extracted plasmid was sent to BGI for sequencing verification, and the correctly cloned vector was named: pBAD / HisB-MIOX / K57A+ioIT;

[0032] S3. Preparation of expression strain

[0033] Take 1 μL of the correctly sequenced vector pBAD / HisB-MIOX / K57A+ioIT and add it to the competent E. coli BW25113, place it in an ice bath for 30 minutes, heat shock it in a 42℃ water bath for 90 seconds, and let it stand on ice for 5 minutes. Add 600 μL of LB liquid culture medium and spread it on the LB (Str50μg / mL) plate until the bacterial liquid is completely absorbed. Invert the plate and culture it at 37℃ for 12 to 16 hours. The corresponding colonies can be seen and named BW25113::pBAD / HisB-MIOX / K57A+ioIT.

[0034] Among them, the gene sequence and amino acid sequence of inositol oxidase have been disclosed in Chinese patent CN109423469B;

[0035] The nucleotide sequence of inositol transporter (SEQ ID NO.1) is:

[0036]

[0037] The amino acid sequence of inositol transporter (SEQ ID NO.2) is:

[0038] MNKQGNQMSFLRTIILVSTFGGLLFGYDTGVLNGALPYMGEPDQLNLNAFTEGLVTSSLLFGAALGAVFGGRMSDFNGRRKNILFLAVIFFISTIGCTFAPNVTVMIISRFVLGIAVG GASVTVPAYLAEMSPVESRGRMVTQNELMIVSGQLLAFVFNAILGTTMGDNSHVWRFMLVIASLPALFLFFGMIRMPESPRWLVSKGRKEDALRVLKKIRDEKRAAAELQEIEFAFKKE DQLEKATFKDLSVPWVRRIVFIGLGIAIVQQITGVNSIMYYGTEILRNSGFQTEAALIGNIANGVISVLATFVGIWLLGRVGRRPMLMTGLIGTTTALLLIGIFSLVLEGSPALPYVV LSLTVTFLAFQQGAISPVTWLMLSEIFPLRLRGLGMGVTVFCLWMVNFAVSFTFPILLAAIGLSTTFFIFVGLGICSVLFVKRFLPETKGLSLEQLEENFRAYDHSGAKKDSGAEVIG.

[0039] Preferably, the LB solid culture medium is composed of 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, and 20 g / L agar powder.

[0040] Preferably, the LB liquid culture medium is composed of 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

[0041] The second technical problem to be solved by the present invention is: in view of the deficiencies in the prior art, a method for preparing an engineering bacterium with improved glucuronic acid catalytic efficiency is provided, which has high catalytic efficiency.

[0042] To solve the above second technical problem, the technical solution of the present invention is:

[0043] A method for converting glucuronic acid comprises the steps of taking the engineered bacteria for improving the catalytic efficiency of glucuronic acid, carrying out bacterial fermentation, and then adding inositol for conversion to obtain a conversion liquid.

[0044] Preferably, the bacterial fermentation control conditions are as follows: pick a single colony BW25113::pBAD / HisB-MIOX / K57A+ioIT and culture it in 4 mL LB liquid medium for 10-14 h, add the bacterial solution to 100 mL TB medium at a volume of 0.5-1.5% v / v, and culture for 2-3 h. 600 When the pH reached 0.6-0.8, arabinose was added at a final concentration of 2 g / L and induced at 16°C for 22 h.

[0045] Preferably, the transformation system is OD 600 =100 bacteria, 100mM inositol, 2mM ferrous sulfate, 2mM cysteine.

[0046] Preferably, the reaction conditions of the conversion system are 40° C. water bath shaker, 180 rpm, and reaction time of 40 min.

[0047] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0048] By adding inositol transporter to the inositol oxidase expression strain and mutating the amino acid at position 57 of inositol oxidase to alanine, the catalytic efficiency of the obtained genetically engineered strain for the substrate inositol was increased by 43% in the same time, and the substrate could be converted into the product glucuronic acid more quickly, greatly reducing the risk of incomplete catalysis due to low stability of inositol oxidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is the liquid chromatogram of the conversion liquid in Example 2 of the present invention. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with the embodiments.

[0051] Example 1

[0052] A method for preparing an engineering bacterium for improving the catalytic efficiency of glucuronic acid comprises the following steps:

[0053] S1. Vector construction

[0054] S1-01, Genes and Primers

[0055] Take inositol oxidase (MIOX) and inositol transporter (ioIT), wherein the nucleotide sequence of the ioIT is shown in SEQ ID NO.1, and the amino acid sequence of the ioIT is shown in SEQ ID NO.2;

[0056]

[0057] S1-02, construction of vector pBAD / HisB-MIOX+ioIT

[0058] ① MIOX target gene acquisition: 2× high-fidelity enzyme mix 50μL, template 1μL, MIOX F 2μL, MIOX R 2μL, ddH 2 O to make up 100 μL, reaction procedure: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 20 s, cooling to annealing temperature 55℃ annealing for 20 s, 72℃ extension for 1 min, a total of 30 cycles; 72℃ extension for 10 min, 4℃ insulation. Then PCR product purification;

[0059] ioIT target gene acquisition: 2× high-fidelity enzyme mix 50μL, template 1μL, ioIT F 2μL, ioIT R 2μL, ddH 2 O to make up 100 μL, reaction procedure: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 20 s, cooling to annealing temperature 55℃ annealing for 20 s, 72℃ extension for 1 min, a total of 30 cycles; 72℃ extension for 10 min, 4℃ insulation. Then PCR product purification;

[0060] MIOX+ioIT target gene acquisition: 2× high-fidelity enzyme mix 50μL, template 1μL, MIOX F 2μL, ioIT R 2μL, ddH 2 O to make up 100 μL, reaction procedure: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 20 s, cooling to annealing temperature 55℃ annealing for 20 s, 72℃ extension for 2 min, a total of 30 cycles; 72℃ extension for 10 min, 4℃ insulation. Then PCR product purification;

[0061] ②Double restriction enzyme system and reaction conditions: XhoI / EcoRI double restriction enzyme digestion was performed on plasmid pBAD / HisB and target gene MIOX+ioIT, 10*buffer 5μL, BSA 2μL, XhoI / EcoRI 2 / 2μL, DNA 20μL, ddH 2 O 19μL, a total of 50μL, 37℃ digestion for 1h, recover the target vector fragment and target gene fragment;

[0062] ③T4 DNA ligase reaction system and conditions: vector DNA 1μg, target fragment 2μg, T4 ligase 1μL, 10×T4 ligase buffer 1μL, ddH 2 O to make up to 10 μL, and connect at 16°C for 2 h;

[0063] ④Chemical transformation: Take 10 μL of the ligation product and add it to DH5α competent cells, place it on ice for 30 min, heat shock it at 42°C for 90 s, place it on ice for 2 min, add 600 μL of antibiotic-free LB liquid medium, incubate it at 37°C and 160-180 rpm for 60 min, and spread it on LB (Str 50 μg / mL) resistance plates;

[0064] ⑤ Screening of positive clones of cloning strains: After colony PCR verification, the plasmid was sent to BGI for sequencing verification. The vector with the correct clone was named: pBAD / HisB-MIOX+ioIT;

[0065] S2. Construction of mutants

[0066] S2-01. Screening of mutation sites

[0067] Through AlphaFold site prediction, MIOX predicted the key site K57, where the amino acid sequence 57th amino acid was mutated to alanine;

[0068] S2-02, Primer

[0069]

[0070] S2-03, Mutation PCR system and procedure

[0071] 5×Phusion HF buffer 2μL, dNTP (2.5mmol / L) 0.8μL, DNA (vector pB AD / HisB-MIOX+ioIT) template 0.2μL, K57A F / K57A R 0.3μL each, DMSO 0.3μL, Phu high-fidelity DNA polymerase (2U / μl) 0.1μL, ddH 2 O 6μL, total volume 10μL; pre-denaturation: 98℃ denaturation for 3min; amplification cycle: 98℃ denaturation for 10s, 55℃ annealing for 30s, 72℃ extension for 4.5min, 32 cycles; fill-in: 72℃ extension for 10min;

[0072] S2-04, DpnI digestion template system (10 μL) and reaction conditions

[0073] 10× buffer 1 μL, DNA PCR product 8 μL, DpnI 1 μL; 37°C, 1 h;

[0074] S2-05, Chemical Conversion

[0075] After the PCR product was digested with DpnI at 37°C for 1 hour, 10 μL was added to the competent E. coli DH 5α, ice bathed for 30 minutes, heat-shocked in a 42°C water bath for 90 seconds, and placed on ice for 5 minutes. Add 600 μL LB liquid culture medium and spread on the LB (Str50 μg / mL) plate until the bacterial liquid is completely absorbed. Invert the plate and culture at 37°C for 12 to 16 hours until the corresponding colonies are visible;

[0076] S2-06. Screening of positive clones of clonal strains

[0077] The extracted plasmid was sent to BGI for sequencing verification, and the correctly cloned vector was named: pBAD / HisB-MIOX / K57A+ioIT;

[0078] S3. Preparation of expression strain

[0079] Take 1 μL of the correctly sequenced vector pBAD / HisB-MIOX / K57A+ioIT and add it to the competent E. coli BW25113, place it in an ice bath for 30 minutes, heat shock it in a 42℃ water bath for 90 seconds, and let it stand on ice for 5 minutes. Add 600 μL of LB liquid culture medium and spread it on the LB (Str50μg / mL) plate until the bacterial liquid is completely absorbed. Invert the plate and culture it at 37℃ for 12 to 16 hours. The corresponding colonies can be seen and named BW25113::pBAD / HisB-MIOX / K57A+ioIT.

[0080] The LB solid culture medium is composed of 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, and 20 g / L agar powder.

[0081] The LB liquid culture medium is composed of 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

[0082] Example 2

[0083] A method for converting glucuronic acid comprises the steps of taking the engineered bacteria for improving the catalytic efficiency of glucuronic acid, carrying out bacterial fermentation, and then adding inositol for conversion to obtain a conversion liquid.

[0084] The bacterial fermentation control conditions are as follows: pick a single colony BW25113::pBAD / HisB-MIOX / K57A+ioIT and culture it in 4 mL LB liquid medium for 10-14 h, add the bacterial solution to 100 mL TB medium at a volume of 0.5-1.5% v / v, and culture it for 2-3 h. 600 When the pH reached 0.6-0.8, arabinose was added at a final concentration of 2 g / L and induced at 16°C for 22 h.

[0085] Transformation system to OD 600=100 bacteria, 100mM inositol, 2mM ferrous sulfate, 2mM cysteine.

[0086] The reaction conditions of the conversion system were 40°C water bath shaker, 180 rpm, and reaction time of 40 min.

[0087] Chromatographic detection showed that the content of glucuronic acid in the conversion solution was 4.5 g / L, and the conversion rate was 25%.

[0088] Comparative Example

[0089] The bacterial fermentation control conditions are as follows: pick a single colony BW25113::pBAD / HisB-MIOX and culture it in 4 mL LB liquid medium for 10-14 h, add the bacterial solution to 100 mL TB medium at a volume of 0.5-1.5% v / v, and culture it for 2-3 h. 600 When the pH reached 0.6-0.8, arabinose was added at a final concentration of 2 g / L and induced at 16°C for 22 h.

[0090] Transformation system to OD 600 =100 bacteria, 100mM inositol, 2mM ferrous sulfate, 2mM cysteine.

[0091] The reaction conditions of the conversion system were 40°C water bath shaker, 180 rpm, and reaction time of 40 min.

[0092] Chromatographic detection showed that the content of glucuronic acid in the conversion solution was 3.15 g / L, and the conversion rate was 17.5%.

[0093] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it 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 also fall within the scope limited by the appended claims of the application.

Claims

1. A method for preparing an engineered bacterium for improving the catalytic efficiency of glucuronic acid, characterized in that The following steps are involved: S1. Vector construction S1-01, Genes and Primers Take inositol oxidase (MIOX) and inositol transporter (ioIT), wherein the nucleotide sequence of the ioIT is as shown in SEQ ID NO.1, and the amino acid sequence of the ioIT is as shown in SEQ ID NO.2; S1-02, construction of vector pBAD / HisB-MIOX+ioIT ① Obtaining MIOX target gene: 2× high-fidelity enzyme mix 50μL, template 1μL, MIOX F 2μL, MIOX R 2μL, ddH2O 100μL, reaction procedure: 98℃ pre-denaturation for 2min; 98℃ denaturation for 20s, cooling to annealing temperature 55℃ annealing for 20s, 72℃ extension for 1min, a total of 30 cycles; 72℃ extension for 10min, 4℃ insulation. Then PCR product purification; ioIT target gene acquisition: 2× high-fidelity enzyme mix 50μL, template 1μL, ioIT F 2μL, ioIT R 2μL, ddH2O fill 100μL, reaction procedure: 98℃ pre-denaturation 2min; 98℃ denaturation 20s, cooling to annealing temperature 55℃ annealing 20s, 72℃ extension 1min, a total of 30 cycles; 72℃ extension 10min, 4℃ insulation. Then PCR product purification; MIOX+ioIT target gene acquisition: 2× high-fidelity enzyme mix 50μL, template 1μL, MIOX F 2μL, ioIT R 2μL, ddH2O fill 100μL, reaction procedure: 98℃ pre-denaturation 2min; 98℃ denaturation 20s, cooling to annealing temperature 55℃ annealing 20s, 72℃ extension 2min, a total of 30 cycles; 72℃ extension 10min, 4℃ insulation. Then PCR product purification; ②Double restriction enzyme system and reaction conditions: Double restriction enzyme digestion of plasmid pBAD / HisB and target gene MIOX+ioIT with XhoI / EcoRI, 10*buffer 5μL, BSA 2μL, XhoI / EcoRI 2 / 2μL, DNA 20μL, ddH2O 19μL, a total of 50μL, 37℃ restriction enzyme digestion for 1h, and recovery of target vector fragment and target gene fragment; ③T4 DNA ligase reaction system and conditions: vector DNA 1μg, target fragment 2μg, T4 ligase 1μL, 10×T4 ligase buffer 1μL, ddH2O 10μL, 16℃ ligation for 2h; ④Chemical transformation: Take 10 μL of the ligation product and add it to DH5α competent cells, place it on ice for 30 min, heat shock it at 42°C for 90 s, place it on ice for 2 min, add 600 μL of antibiotic-free LB liquid medium, incubate it at 37°C and 160-180 rpm for 60 min, and spread it on LB (Str 50 μg / mL) resistance plates; ⑤ Screening of positive clones of cloning strains: After colony PCR verification, the plasmid was sent to BGI for sequencing verification. The vector with the correct clone was named: pBAD / HisB-MIOX+ioIT; S2. Construction of mutants S2-01. Screening of mutation sites Through AlphaFold site prediction, MIOX predicted the key site K57, where the amino acid sequence 57th amino acid was mutated to alanine; S2-02, Primer S2-03, Mutation PCR system and procedure 5×Phusion HF buffer 2μL, dNTP (2.5mmol / L) 0.8μL, DNA (vector pB AD / HisB-MIOX+ioIT) template 0.2μL, K57A F / K57A R 0.3μL each, DMSO 0.3μL, Phu high-fidelity DNA polymerase (2U / μl) 0.1μL, ddH2O 6μL, total volume 10μL; pre-denaturation: 98℃ denaturation 3min; amplification cycle: 98℃ denaturation 10s, 55℃ annealing 30s, 72℃ extension 4.5min, 32 cycles; fill-in: 72℃ extension 10min; S2-04, DpnI digestion template system (10 μL) and reaction conditions 10× buffer 1 μL, DNA PCR product 8 μL, DpnI 1 μL; 37°C, 1 h; S2-05, Chemical Conversion After the PCR product was digested with DpnI at 37°C for 1 hour, 10 μL was added to the competent E. coli DH 5α, ice bathed for 30 minutes, heat-shocked in a 42°C water bath for 90 seconds, and placed on ice for 5 minutes. Add 600 μL LB liquid culture medium and spread on the LB (Str50 μg / mL) plate until the bacterial liquid is completely absorbed. Invert the plate and culture at 37°C for 12 to 16 hours until the corresponding colonies are visible; S2-06. Screening of positive clones of clonal strains The extracted plasmid was sent to BGI for sequencing verification, and the correctly cloned vector was named: pBAD / HisB-MIOX / K57A+ioIT; S3. Preparation of expression strain Take 1 μL of the correctly sequenced vector pBAD / HisB-MIOX / K57A+ioIT and add it to the competent E. coli BW25113, place it in an ice bath for 30 minutes, heat shock it in a 42℃ water bath for 90 seconds, and let it stand on ice for 5 minutes. Add 600 μL of LB liquid culture medium and spread it on the LB (Str50μg / mL) plate until the bacterial liquid is completely absorbed. Invert the plate and culture it at 37℃ for 12 to 16 hours. The corresponding colonies can be seen and named BW25113::pBAD / HisB-MIOX / K57A+ioIT.

2. The method for preparing an engineered bacterium for improving the catalytic efficiency of glucuronic acid according to claim 1, characterized in that: The LB solid culture medium is composed of 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, and 20 g / L agar powder.

3. The method for preparing an engineered bacterium for improving the catalytic efficiency of glucuronic acid according to claim 1, characterized in that: The LB liquid culture medium is composed of 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

4. A method for converting glucuronic acid, characterized in that: The engineered bacteria according to any one of claims 1 to 3 are fermented, and then inositol is added for conversion to obtain a conversion solution.

5. A method for converting glucuronic acid, characterized in that: The bacterial fermentation control conditions are as follows: pick a single colony BW25113::pBAD / HisB-MIOX / K57A+ioIT and culture it in 4 mL LB liquid medium for 10-14 h, add the bacterial solution to 100 mL TB medium at a volume of 0.5-1.5% v / v, and culture it for 2-3 h. 600 When the pH reached 0.6-0.8, arabinose was added at a final concentration of 2 g / L and induced at 16°C for 22 h.

6. A method for converting glucuronic acid, characterized in that: Transformation system to OD 600 =100 bacteria, 100mM inositol, 2mM ferrous sulfate, 2mM cysteine.

7. A method for converting glucuronic acid, characterized in that: The reaction conditions of the conversion system were 40°C water bath shaker, 180 rpm, and reaction time of 40 min.

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