Recombinant strain for producing glucuronic acid and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a recombinant strain for producing glucuronic acid and application of the recombinant strain. The invention provides a recombinant strain for producing glucuronic acid, and the recombinant strain does not express uronic acid isomerase and overexpresses inositol oxidase. The technical scheme of the invention has the following advantages: (1) uronic acid isomerase uxaC from escherichia coli K12 is knocked out, so that glucuronic acid accumulation is increased; and (2) successfully integrating the target gene of the inositol oxidase from the mouse into the escherichia coli K12MG1655. And (3) when the engineering strain is used for catalyzing inositol, the content of glucuronic acid in a system after the reaction is finished is 52.50 g / L, and the conversion rate is as high as 97.23%.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a recombinant bacterial strain that produces glucuronic acid and its uses. Background Technology

[0002] D-glucuronic acid is a uronic acid formed by oxidizing the hydroxyl group at the C-6 position of glucose to a carboxyl group. Its molecular formula is C6H2O. 10 O7 has a molecular weight of 194.14. Glucuronic acid is a white needle-like crystal or powder with a melting point of 155℃-157℃.

[0003] Glucuronic acid can be converted into glucuronide, which is a valuable drug for treating hepatitis, cirrhosis, and connective tissue diseases. my country is a major producer of glucuronide. Existing methods include polysaccharide hydrolysis (obtaining glucuronic acid by hydrolyzing polysaccharides containing uronic acid) and chemical oxidation. However, the chemical oxidation process suffers from high energy consumption, poor selectivity, low product yield, and severe environmental pollution. Therefore, there is an urgent need to develop a new, efficient method for producing glucuronic acid. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defects in the prior art, thereby providing a recombinant strain for producing glucuronic acid and its uses.

[0005] A recombinant strain producing glucuronic acid, wherein the recombinant does not express the uronic acid isomerase uxaC and overexpresses inositol oxidase. The uronic acid isomerase uxaC (GenBank: AKK15070.2); the amino acid sequence of inositol oxidase is shown in SEQ ID NO.4.

[0006] Preferably, the recombinant strain originates from Escherichia coli; the Escherichia coli is E. coli K12MG1655.

[0007] Preferably, the recombinant strain overexpresses an inositol transporter mutant, said mutant being any one of the following (A)-(C):

[0008] (A) Its amino acid sequence is shown in SEQ ID NO.2; the mutation site is G347A;

[0009] (B) Proteins that have 95% or 97% or more of the same amino acid sequence as defined in (A) and have the same function;

[0010] (C) A fusion protein obtained by attaching a tag to the end of the protein defined in (A) or (B).

[0011] Preferably, the coding gene for the inositol transporter mutant is shown in SEQ ID NO.1. Positions 1039-1041 are mutated from the original GGA to GCC.

[0012] Preferably, the promoter controlling the overexpression of the gene encoding inositol oxidase is the pTrc promoter; the gene sequence of the pTrc promoter is shown in positions 1-30 of SEQ ID NO.3.

[0013] In one embodiment, the method for overexpressing a mutant inositol transporter in a recombinant strain includes: inserting pTrc-iolT into the ybgC pseudogene site in the E. coli K12MG1655 genome. G347A The fragment, as shown in SEQ ID NO.3, has the pTrc promoter in positions 1-30 and the iolT promoter in positions 31-1449. G347A The gene sequence has a terminator at positions 1450-1539.

[0014] pTrc-iolT was inserted into the ybgC pseudogene locus in the E. coli K12 MG1655 genome. G347A The fragment method includes the following steps: ybgC::pTrc-iolT G347A Gene integration fragments, pREDCas9 plasmids, and plasmids containing DNA fragments with the ybgC target sequence were introduced into the starting strain;

[0015] ybgC::pTrc-iolT G347A The gene integration fragment is pTrc-iolT G347A The 5' and 3' ends of the fragment (SEQ ID NO.3) are connected to the upstream and downstream homologous arms of the ybgC gene, respectively, which are used for homologous recombination at the ybgC pseudogene site.

[0016] The preparation method of the upstream homologous arm of the ybgC gene includes the following steps: using the E. coli K12 MG1655 genome as a template, the upstream homologous arm of the ybgC gene is amplified by PCR using primers ybgC-US (SEQ ID NO.39) and ybgC-UA (SEQ ID NO.40); using the E. coli K12 MG1655 genome as a template, the downstream homologous arm of the ybgC gene is amplified by PCR using primers ybgC-DS (SEQ ID NO.41) and ybgC-DA (SEQ ID NO.42). The PCR system used in both cases is as follows: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer (10 uM) 2 μL; downstream primer (10 uM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL. The PCR program described above is as follows: pre-denaturation at 95℃ for 30 seconds, denaturation at 95℃ for 15 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 60 seconds, repeating the three steps of denaturation, annealing, and extension 30 times. Final extension at 72℃ for 5 minutes.

[0017] The method for preparing a DNA fragment containing the ybgC target sequence includes the following steps: using F8 (SEQ ID NO.45) and R8 (SEQ ID NO.46) as primers and the E. coli K12 MG1655 genome as a template, a DNA fragment containing the ybgC target sequence is constructed by PCR. PCR system: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; F6 (10 uM) 2 μL; R6 (10 uM) 2 μL; template 1; DNA polymerase 1 μL; ddH2O 18 μL. PCR program: pre-denaturation 95℃ for 30 s, denaturation 95℃ for 15 s, annealing 60℃ for 45 s, extension 72℃ for 60 s, (denaturation, annealing, extension) cycles 30 times. Final extension 72℃ for 5 min.

[0018] Preferred methods for not expressing uronic acid isomerase include: knocking out the uronic acid isomerase gene uxaC in the genome of the recombinant strain;

[0019] Knocking out uxaC and inserting the pTrc-mioX gene fragment at the uxaC gene site involves the following steps: a plasmid containing a DNA fragment of the uxaC target sequence and a pREDCas9 plasmid, and the uxaC::pTrc-mioX gene integration fragment are transferred into the starting strain.

[0020] The uxaC::pTrc-mioX gene integration fragment is the ptrc-miox fragment with its 5' and 3' ends connected to the upstream and downstream homologous arms of uxaC, respectively; these homologous arms are used for homologous recombination at the uxaC site.

[0021] The upstream homologous arm of uxaC was obtained by PCR amplification of uxaC-US (SEQ ID NO.6) and uxaC-UA (SEQ ID NO.7) using the E. coli K12 MG1655 genome (Genbank number U00096) as a template.

[0022] The downstream homologous arm of uxaC was obtained by PCR amplification using the E. coli K12 MG1655 genome as a template and primers uxaC-DS (SEQ ID NO. 8) and uxaC-DA (SEQ ID NO. 9).

[0023] DNA fragments containing the uxaC target sequence were used with primers F2 (SEQ ID NO.13) and R2 (SEQ ID NO.14), and the E. coli K12 MG1655 genome was used as a template. The PCR system was as follows: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; F2 (10 uM) 2 μL; R2 (10 uM) 2 μL; template 1; DNA polymerase 1 μL; ddH2O 18 μL. The PCR program was: pre-denaturation 95℃ for 30 s, denaturation 95℃ for 15 s, annealing 60℃ for 45 s, extension 72℃ for 60 s, (denaturation, annealing, extension) cycles 30 times. The final extension was 72℃ for 5 min. The PCR system for obtaining the upstream and downstream homologous arms was as follows: 2×Buffer 25μL; dNTPMix (10mMeach) 1μL; upstream primer (10uM) 2μL; downstream primer (10uM) 2μL; template 1μL; DNA polymerase 1μL; ddH2O 18μL.

[0024] The PCR program for obtaining the upstream and downstream homologous arms was as follows: pre-denaturation at 95℃ for 30 s, denaturation at 95℃ for 15 s, annealing at 55℃ for 15 s, extension at 72℃ for 60 s, repeating the three steps of denaturation, annealing, and extension for 30 cycles. The final extension was performed at 72℃ for 5 min.

[0025] Methods for overexpressing inositol oxidase include inserting the encoding gene for inositol oxidase into one or more of the following sites: uxaC, yagT, ybdH, yiaM, ygiF, and ybgC.

[0026] In one embodiment, a pTrc-mioX gene fragment is inserted into any one or more of the following sites in the E. coli K12 MG1655 genome: the uxaC gene site, the yagT pseudogene site, the ybdH pseudogene site, the yiaM pseudogene site, the ygiF pseudogene site, and the ybgC pseudogene site. The pTrc-mioX gene fragment sequence is shown in SEQ ID NO.12, where positions 1-30 are the promoter, positions 31-924 are the mioX gene sequence, and positions 925-1011 are the terminator.

[0027] Methods for inserting the pTrc-mioX gene fragment into the yagT pseudogene locus include:

[0028] The 5' and 3' ends of the pTrc-mioX gene fragment are connected to the upstream and downstream homologous arms of the yagT gene, respectively, to obtain sequence A; these homologous arms are used to induce homologous recombination at the yagT gene site.

[0029] The sequence A, pREDCas9 plasmid, and plasmid containing the DNA fragment of the yagT target sequence were introduced into the starting strain.

[0030] The method for obtaining the upstream homologous arm of the yagT gene includes the following steps: using the E. coli K12 MG1655 genome as a template, the upstream homologous arm of the yagT gene is obtained by PCR amplification using primers yagT-US (SEQ ID NO.15) and yagT-UA (SEQ ID NO.16);

[0031] The method for obtaining the downstream homologous arm of the yagT gene includes the following steps: using the E. coli K12 MG1655 genome as a template, the downstream homologous arm of the yagT gene is obtained by PCR amplification using primers yagT-DS (SEQ ID NO.17) and yagT-DA (SEQ ID NO.18);

[0032] The method for preparing a DNA fragment containing the yagT target sequence includes the following steps: using F3 (SEQ ID NO.19) and R3 (SEQ ID NO.20) as primers and the E. coli K12 MG1655 genome as a template, a DNA fragment containing the yagT target sequence is constructed by PCR. PCR system: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer (10 uM) 2 μL; downstream primer (10 uM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL. PCR program: pre-denaturation 95℃ for 30 s, denaturation 95℃ for 15 s, annealing 60℃ for 45 s, extension 72℃ for 60 s, (denaturation, annealing, extension) cycles 30 times. Final extension 72℃ for 5 min.

[0033] Methods for inserting the pTrc-mioX gene fragment into the ybdH pseudogene locus include:

[0034] Sequence B, pREDCas9 plasmid, and plasmid containing a DNA fragment of the ybdH target sequence were introduced into the starting strain.

[0035] Sequence B was obtained by linking the upstream and downstream homologous arms of the ybdH gene to the 5' and 3' ends of the pTrc-mioX gene fragment, respectively;

[0036] The method for obtaining the upstream and downstream homologous arms of the ybdH gene includes the following steps:

[0037] Using the E. coli K12 MG1655 genome as a template, the upstream homologous arm of the ybdH gene was amplified by PCR using primers ybdH-US (SEQ ID NO.21) and ybdH-UA (SEQ ID NO.22). Similarly, the downstream homologous arm of the ybdH gene was amplified by PCR using primers ybdH-DS (SEQ ID NO.23) and ybdH-DA (SEQ ID NO.24). The PCR system consisted of: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer (10 μM) 2 μL; downstream primer (10 μM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL. The PCR program described above is as follows: pre-denaturation at 95℃ for 30 seconds, denaturation at 95℃ for 15 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 60 seconds, repeating the three steps of denaturation, annealing, and extension 30 times. Final extension at 72℃ for 5 minutes.

[0038] The method for preparing a DNA fragment containing the ybdH target sequence includes the following steps: using F4 (SEQ ID NO.25) and R4 (SEQ ID NO.26) as primers and the E. coli K12 MG1655 genome as a template, a DNA fragment containing the ybdH target sequence is constructed by PCR. PCR system: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer F4 (10 uM) 2 μL; downstream primer R4 (10 uM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL. PCR program: pre-denaturation 95℃ for 30 s, denaturation 95℃ for 15 s, annealing 60℃ for 45 s, extension 72℃ for 60 s, (denaturation, annealing, extension) cycles 30 times. Final extension 72℃ for 5 min.

[0039] Methods for inserting the pTrc-mioX gene fragment into the yiaM pseudogene locus include:

[0040] Sequence C, pREDCas9 plasmid, and plasmid containing a DNA fragment of the yiaM target sequence were introduced into the starting strain.

[0041] Sequence C is obtained by linking the upstream and downstream homologous arms of the yiaM gene to the 5' and 3' ends of the pTrc-mioX gene fragment, respectively;

[0042] The preparation method of the upstream and downstream homologous arms of the yiaM gene includes the following steps: using the E. coli K12 MG1655 genome as a template, the upstream homologous arm of the yiaM gene is amplified by PCR using primers yiaM-US (SEQ ID NO.27) and yiaM-UA (SEQ ID NO.28); using the E. coli K12 MG1655 genome as a template, the downstream homologous arm of the yiaM gene is amplified by PCR using primers yiaM-DS (SEQ ID NO.29) and yiaM-DA (SEQ ID NO.30). The PCR system for both methods is as follows: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer (10 uM) 2 μL; downstream primer (10 uM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL.

[0043] The PCR program described above is as follows: pre-denaturation at 95℃ for 30 seconds, denaturation at 95℃ for 15 seconds, annealing at 60℃ for 15 seconds, extension at 72℃ for 60 seconds, repeating the three steps of denaturation, annealing, and extension 30 times. Final extension at 72℃ for 5 minutes.

[0044] The method for preparing a DNA fragment containing the yiaM target sequence includes the following steps: using F5 (SEQ ID NO.31) and R5 (SEQ ID NO.32) as primers and the E. coli K12 MG1655 genome as a template, a DNA fragment containing the yiaM target sequence is constructed by PCR. PCR system: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer (10 uM) 2 μL; downstream primer (10 uM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL. PCR program: pre-denaturation 95℃ for 30 s, denaturation 95℃ for 15 s, annealing 60℃ for 45 s, extension 72℃ for 60 s, (denaturation, annealing, extension) cycles 30 times. Final extension 72℃ for 5 min.

[0045] Methods for inserting the pTrc-mioX gene fragment into the ygiF pseudogene locus include:

[0046] Sequence D, pREDCas9 plasmid, and plasmid containing a DNA fragment of the ygiF target sequence were introduced into the starting strain;

[0047] Sequence D was obtained by linking the upstream and downstream homologous arms of the ygiF gene to the 5' and 3' ends of the pTrc-mioX gene fragment, respectively;

[0048] The preparation method of the upstream and downstream homologous arms of the ygiF gene includes the following steps: using the E. coli K12 MG1655 genome as a template, the upstream homologous arm of the ygiF gene was amplified by PCR using primers ygiF-US (SEQ ID NO.33) and ygiF-UA (SEQ ID NO.34); the downstream homologous arm of the ygiF gene was amplified by PCR using primers ygiF-DS (SEQ ID NO.35) and ygiF-DA (SEQ ID NO.36). The PCR system consisted of: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer (10 uM) 2 μL; downstream primer (10 uM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL. The PCR program described above is as follows: pre-denaturation at 95℃ for 30 seconds, denaturation at 95℃ for 15 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 60 seconds, repeating the three steps of denaturation, annealing, and extension 30 times. Final extension at 72℃ for 5 minutes.

[0049] The preparation of the DNA fragment containing the ygiF target sequence included the following steps: using F6 (SEQ ID NO.37) and R6 (SEQ ID NO.38) as primers and the E. coli K12 MG1655 genome as a template, a DNA fragment containing the ygiF target sequence was constructed by PCR. The PCR system consisted of: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; F6 (10 uM) 2 μL; R6 (10 uM) 2 μL; template 1; DNA polymerase 1 μL; ddH2O 18 μL. The PCR program was: pre-denaturation at 95℃ for 30 s, denaturation at 95℃ for 15 s, annealing at 60℃ for 45 s, extension at 72℃ for 60 s, repeated 30 times (denaturation, annealing, and extension). The final extension was at 72℃ for 5 min.

[0050] Preferably, the promoter controlling the gene encoding the inositol transporter mutant is the pTrc promoter; the gene sequence of the pTrc promoter is shown in positions 1-30 of SEQ ID NO.3.

[0051] Preferably, the nucleotide sequence of the inositol oxidase encoding gene is shown in SEQ ID NO.5.

[0052] A method for producing glucuronic acid using inositol as a raw material and under the action of a recombinant bacterial strain.

[0053] Preferably, in the method, the reaction temperature is 30℃-40℃ and the reaction pH is 8.0-9.0.

[0054] The application of the above-mentioned recombinant strains in the preparation of glucuronic acid or glucuronolactone.

[0055] The technical solution of this invention has the following advantages:

[0056] (1) Knockout of uxaC, a glucuronic acid isomerase derived from Escherichia coli K12, led to an increase in glucuronic acid accumulation.

[0057] (2) The target gene of inositol oxidase and the mutant gene of inositol transport protein derived from mice were successfully integrated into Escherichia coli K12MG1655.

[0058] (3) When the engineered strain was used to catalyze inositol, the glucuronic acid content in the system after the reaction was 52.50 g / L, and the conversion rate was as high as 97.23%. Detailed Implementation

[0059] E. coli K12 MG1655 was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number: B98184.

[0060] pREDCas9 plasmid is from Baosai Plasmid and Strawberry Resource Company; Product No.: 71541.

[0061] Example 1: Controlling mioX gene overexpression at the uxaC gene site using the pTrc promoter

[0062] 1. Using the E. coli K12 MG1655 genome (Genbank ID U00096) as a template, the upstream homologous arm of the uxaC gene was amplified and purified by PCR using primers uxaC-US (SEQ ID NO. 6) and uxaC-UA (SEQ ID NO. 7). Using the E. coli K12 MG1655 genome as a template, the downstream homologous arm of the uxaC gene was amplified and purified by PCR using primers uxaC-DS (SEQ ID NO. 8) and uxaC-DA (SEQ ID NO. 9). uxaC-UA was designed to contain an upstream homologous sequence of the promoter, and uxaC-DS was designed to contain a downstream homologous sequence of the terminator.

[0063] The PCR system described above consisted of: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer (10 uM) 2 μL; downstream primer (10 uM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL.

[0064] The PCR program described above is as follows: pre-denaturation at 95℃ for 30 seconds, denaturation at 95℃ for 15 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 60 seconds, repeating the three steps of denaturation, annealing, and extension 30 times. Final extension at 72℃ for 5 minutes.

[0065] 2. Obtaining the pTrc-mioX gene fragment (SEQ ID NO.12): Using the artificially synthesized mioX gene as a template, and uxaC-F (SEQ ID NO.10) and uxaC-R (SEQ ID NO.11) as primers, the pTrc-mioX gene fragment was amplified by PCR. The PCR system consisted of: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer (10 uM) 2 μL; downstream primer (10 uM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL. The PCR program was: pre-denaturation 95℃ for 30 s, denaturation 95℃ for 15 s, annealing 57℃ for 15 s, extension 72℃ for 60 s, repeating the three steps (denaturation, annealing, and extension) 35 times. The final extension was 72℃ for 5 min. After purification, the fragment was obtained.

[0066] The pTrc promoter is designed in the sense primer uxaC-F for the mioX gene; the pTrc terminator is designed in the antisense primer uxaC-R for the mioX gene.

[0067] pTrc-mioX gene fragment (sequence shown in SEQ ID NO.12): where positions 1-30 are the promoter, positions 31-924 are the mioX gene sequence, and positions 925-1011 are the terminator.

[0068] 3. Using the upstream and downstream homologous arms of uxaC and the pTrc-mioX gene fragment as templates, the integrated uxaC::pTrc-mioX gene fragment was obtained by overlap PCR. The overlap PCR amplification system consisted of: 10 μL of 5×Phusion HF buffer, 4 μL of dNTPs (2.5 mmol / L), 1 μL of template, 1 μL of each primer, 0.5 μL of Phu high-fidelity DNA polymerase (2 U / μL), and 39.5 μL of ddH2O; the molar ratio of the upstream, downstream, and pTrc-mioX components in the template was 1:1:1.

[0069] Overlap PCR amplification program: pre-denaturation 95℃ for 30s, denaturation 95℃ for 15s, annealing 55℃ for 60s, extension 72℃ for 60s, (denaturation, annealing, and extension steps) 30 cycles. Final extension 72℃ for 5min.

[0070] Primers: uxaC-DS, uxaC-DA, uxaC-F, uxaC-R, uxaC-US, uxaC-UA.

[0071] 4. Primers F2 (SEQ ID NO.13) and R2 (SEQ ID NO.14) were designed. Using the E. coli K12 MG1655 genome as a template, a DNA fragment containing the uxaC target sequence was constructed by PCR. PCR system: 2×Buffer 25μL; dNTPMix (10mMeach) 1μL; F2 (10uM) 2μL; R2 (10uM) 2μL; template 1; DNA polymerase 1μL; ddH2O 18μL.

[0072] PCR program: pre-denaturation 95℃ for 30s, denaturation 95℃ for 15s, annealing 60℃ for 45s, extension 72℃ for 60s, (denaturation, annealing, and extension steps) 30 cycles. Final extension 72℃ for 5min.

[0073] The DNA fragment containing the uxaC target sequence was digested with restriction endonucleases BamHI and HindIII. The pGRB vector was also digested with restriction endonucleases BamHI and HindIII to obtain a linearized pGRB vector. The digested DNA fragment containing the uxaC target sequence was recombined with the linearized pGRB vector and transformed into E. coli DH5α competent cells. Positive transformants were selected, and the plasmid pGRB-uxaC was extracted from them.

[0074] 5. The pREDCas9 plasmid was transformed into E. coli K12 MG1655 strain.

[0075] The obtained uxaC::pTrc-mioX gene integration fragment and pGRB-uxaC plasmid were transformed into E. coli K12 MG1655 strain containing pREDCas9 plasmid via electroporation. After selecting positive transformants, the two plasmids used for gene editing were eliminated, resulting in strain E. coli K12 MG1655-1. Compared with the wild-type strain, strain E. coli K12 MG1655-1 had the uronic acid isomerase gene uxaC knocked out from the original strain's genome, and the pTrc-mioX gene fragment was inserted at the uxaC site. The function of uronic acid isomerase uxaC (GenBank: AKK15070.2) is to induce glucuronic acid isomerization.

[0076] Example 2: Controlling mioX gene overexpression at the yagT pseudogene site using the pTrc promoter

[0077] 1. Using the E. coli K12 MG1655 genome as a template, the upstream homologous arm of the yagT gene was amplified and purified by PCR using primers yagT-US (SEQ ID NO.15) and yagT-UA (SEQ ID NO.16). Using the E. coli K12 MG1655 genome as a template, the downstream homologous arm of the yagT gene was amplified and purified by PCR using primers yagT-DS (SEQ ID NO.17) and yagT-DA (SEQ ID NO.18).

[0078] The PCR system described above consisted of: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer (10 uM) 2 μL; downstream primer (10 uM) 2 μL; template 1; DNA polymerase 1 μL; ddH2O 18 μL.

[0079] The PCR program described above is as follows: pre-denaturation at 95℃ for 30 seconds, denaturation at 95℃ for 15 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 60 seconds, repeated 30 times (denaturation, annealing, and extension steps). Final extension is performed at 72℃ for 5 minutes. The yagT-UA design contains an upstream homologous sequence to the promoter, while the yagT-DS design contains a downstream homologous sequence to the terminator.

[0080] 2. Obtaining the pTrc-mioX gene fragment (sequence shown in SEQ ID NO.12): Refer to step 2 of Example 1.

[0081] 3. Using the upstream and downstream homologous arms of yagT and the pTrc-mioX gene fragment as templates, the integrated yagT::pTrc-mioX gene fragment was obtained by overlap PCR.

[0082] Overlap PCR amplification system: 10 μL 5×Phusion HF buffer, 4 μL dNTP (2.5 mmol / L), 1 μL template, 1 μL primers, 0.5 μL Phu high-fidelity DNA polymerase (2 U / μL), 39.5 μL ddH2O; the molar ratio of upstream homologous arm, downstream homologous arm, and pTrc-mioX in the template is 1:1:1;

[0083] Overlap PCR amplification program: pre-denaturation 95℃ for 30s, denaturation 95℃ for 15s, annealing 56℃ for 60s, extension 72℃ for 60s, (denaturation, annealing, and extension steps) 30 cycles. Final extension 72℃ for 5min.

[0084] Primers: yagT-US, yagT-UA, uxaC-F, uxaC-R, yagT-DS, yagT-DA.

[0085] 4. Primers F3 (SEQ ID NO.19) and R3 (SEQ ID NO.20) were designed, and a DNA fragment containing the yagT target sequence was constructed by PCR using the E.coli K12 MG1655 genome as a template.

[0086] PCR system: 2×Buffer 25μL; dNTPMix (10mMeach) 1μL; upstream primer (10uM) 2μL; downstream primer (10uM) 2μL; template 1μL; DNA polymerase 1μL; ddH2O 18μL. PCR program: pre-denaturation 95℃ for 30s, denaturation 95℃ for 15s, annealing 60℃ for 45s, extension 72℃ for 60s, (denaturation, annealing, extension) cycles 30 times. Final extension 72℃ for 5min. After purification, a DNA fragment containing the yagT target sequence is obtained.

[0087] The DNA fragment containing the yagT target sequence was digested with restriction endonucleases BamHI and HindIII, and the pGRB vector was also digested with restriction endonucleases BamHI and HindIII to obtain a linearized pGRB vector. The digested DNA fragment containing the yagT target sequence was recombined with the linearized pGRB vector and transformed into E. coli DH5α competent cells. Positive transformants were obtained through screening, and the plasmid pGRB-yagT was extracted from them.

[0088] 5. Transform the pREDCas9 plasmid into E. coli K12 MG1655-1.

[0089] The obtained yagT::pTrc-mioX gene integration fragment and pGRB-yagT plasmid were transformed into E. coli K12 MG1655-1 strain containing pREDCas9 plasmid via electroporation. After selecting positive transformants, the two plasmids used for gene editing were eliminated, resulting in strain E. coli K12 MG1655-2. Compared with E. coli K12 MG1655-1, strain E. coli K12 MG1655-2 has the pTrc-mioX gene fragment inserted at the pseudogene site yagT (sequence shown in SEQ ID NO. 12).

[0090] Example 3: Controlling mioX gene overexpression at the ybdH pseudogene locus using the pTrc promoter

[0091] 1. Using the E. coli K12 MG1655 genome as a template, the upstream homologous arm of the ybdH gene was amplified and purified by PCR using primers ybdH-US (SEQ ID NO.21) and ybdH-UA (SEQ ID NO.22). Similarly, the downstream homologous arm of the ybdH gene was amplified and purified by PCR using primers ybdH-DS (SEQ ID NO.23) and ybdH-DA (SEQ ID NO.24). Specifically, ybdH-UA was designed to contain an upstream homologous sequence of the promoter, and ybdH-DS was designed to contain a downstream homologous sequence of the terminator.

[0092] The PCR system described above consisted of: 2×Buffer 25 μL; dNTP MiX (10 mMeach) 1 μL; upstream primer (10 μM) 2 μL; downstream primer (10 μM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL. The PCR program described above consisted of: pre-denaturation at 95℃ for 30 s, denaturation at 95℃ for 15 s, annealing at 55℃ for 15 s, extension at 72℃ for 60 s, repeated 30 times (denaturation, annealing, and extension steps). The final extension was performed at 72℃ for 5 min.

[0093] 2. Obtaining the pTrc-mioX gene fragment: Refer to step 2 of Example 1.

[0094] 3. Using the upstream and downstream homologous arms of ybdH and the pTrc-mioX gene fragment as templates, the integrated ybdH::pTrc-mioX gene fragment was obtained by overlap PCR. The overlap PCR amplification system consisted of: 10 μL of 5×Phusion HF buffer, 4 μL of dNTPs (2.5 mmol / L), 1 μL of template, 1 μL of each primer, 0.5 μL of Phu high-fidelity DNA polymerase (2 U / μL), and 39.5 μL of ddH2O; the molar ratio of the upstream, downstream, and pTrc-mioX components in the template was 1:1:1.

[0095] Overlap PCR amplification program: pre-denaturation 95℃ for 30s, denaturation 95℃ for 15s, annealing 55℃ for 60s, extension 72℃ for 60s, (denaturation, annealing, and extension steps) 30 cycles. Final extension 72℃ for 5min.

[0096] Primers: ybdH-US, ybdH-UA, uxaC-F, uxaC-R, ybdH-DS, ybdH-DA.

[0097] 4. Primers F4 (SEQ ID NO.25) and R4 (SEQ ID NO.26) were designed. Using the E. coli K12 MG1655 genome as a template, a DNA fragment containing the ybdH target sequence was constructed by PCR. PCR system: 2×Buffer 25μL; dNTPMix (10mMeach) 1μL; upstream primer (10uM) 2μL; downstream primer (10uM) 2μL; template 1μL; DNA polymerase 1μL; ddH2O 18μL. PCR program: pre-denaturation 95℃ for 30s, denaturation 95℃ for 15s, annealing 60℃ for 45s, extension 72℃ for 60s, (denaturation, annealing, extension) cycles 30 times. Final extension 72℃ for 5min.

[0098] The DNA fragment containing the ybdH target sequence was digested with restriction endonucleases BamHⅠ and HindⅢ. The pGRB vector was also digested with restriction endonucleases BamHⅠ and HindⅢ to obtain a linearized pGRB vector. The digested DNA fragment containing the ybdH target sequence was recombined with the linearized pGRB vector and transformed into E. coli DH5α competent cells. Positive transformants were selected to obtain the plasmid pGRB-ybdH.

[0099] 5. Transform the pREDCas9 plasmid into strain E.coli K12 MG1655-2.

[0100] The obtained ybdH::pTrc-mioX gene integration fragment and pGRB-ybdH plasmid were transformed into E. coli K12 MG1655-2 strain containing pREDCas9 plasmid via electroporation. After screening for positive transformants, the two plasmids used for gene editing were eliminated, finally obtaining strain E. coli K12 MG1655-3. Compared with strain E. coli K12 MG1655-2, strain E. coli K12 MG1655-3 has the inositol oxidase gene sequence pTrc-mioX (sequence shown in SEQ ID NO.12) inserted at the pseudogene site ybdH.

[0101] Example 4: Controlling mioX gene overexpression at the yiaM pseudogene site using the pTrc promoter

[0102] 1. Using the E. coli K12 MG1655 genome as a template, the upstream homologous arm of the yiaM gene was amplified and purified by PCR using primers yiaM-US (SEQ ID NO.27) and yiaM-UA (SEQ ID NO.28). Similarly, the downstream homologous arm of the yiaM gene was amplified and purified by PCR using primers yiaM-DS (SEQ ID NO.29) and yiaM-DA (SEQ ID NO.30). Specifically, yiaM-UA was designed to contain an upstream homologous sequence of the promoter, and yiaM-DS was designed to contain a downstream homologous sequence of the terminator.

[0103] The PCR system described above consisted of: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer (10 uM) 2 μL; downstream primer (10 uM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL.

[0104] The PCR program described above is as follows: pre-denaturation at 95℃ for 30 seconds, denaturation at 95℃ for 15 seconds, annealing at 60℃ for 15 seconds, extension at 72℃ for 60 seconds, repeating the three steps of denaturation, annealing, and extension 30 times. Final extension at 72℃ for 5 minutes.

[0105] 2. Obtaining the pTrc-mioX gene fragment: Refer to step 2 of Example 1.

[0106] 3. Using the upstream and downstream homologous arms of yiaM and the pTrc-mioX gene fragment as templates, the integrated yiaM::pTrc-mioX gene fragment was obtained by overlap PCR.

[0107] Overlap PCR amplification system: 10 μL 5×Phusion HF buffer, 4 μL dNTP (2.5 mmol / L), 1 μL template, 1 μL primers, 0.5 μL Phu high-fidelity DNA polymerase (2 U / μL), 39.5 μL ddH2O; the molar ratio of upstream homologous arm, downstream homologous arm, and pTrc-mioX in the template is 1:1:1;

[0108] Overlap PCR amplification program: pre-denaturation 95℃ for 30s, denaturation 95℃ for 15s, annealing 55℃ for 60s, extension 72℃ for 60s, (denaturation, annealing, and extension steps) 30 cycles. Final extension 72℃ for 5min.

[0109] Primers: yiaM-US, yiaM-UA, uxaC-F, uxaC-R, yiaM-DS, yiaM-DA.

[0110] 4. Primers F5 (SEQ ID NO.31) and R5 (SEQ ID NO.32) were designed, and a DNA fragment containing the yiaM target sequence was constructed by PCR using the E.coli K12 MG1655 genome as a template.

[0111] PCR system: 2×Buffer 25μL; dNTPMix (10mMeach) 1μL; upstream primer (10uM) 2μL; downstream primer (10uM) 2μL; template 1μL; DNA polymerase 1μL; ddH2O 18μL. PCR program: Pre-denaturation 95℃ for 30s, denaturation 95℃ for 15s, annealing 60℃ for 45s, extension 72℃ for 60s, (denaturation, annealing, extension) cycles 30 times. Final extension 72℃ for 5min. After purification, a DNA fragment containing the yiaM target sequence is obtained.

[0112] The DNA fragment containing the yiaM target sequence was digested with restriction endonucleases BamHI and HindIII. The pGRB vector was then digested with BamHI and HindIII to obtain a linearized pGRB vector. The digested DNA fragment containing the ybdH target sequence was recombined with the linearized pGRB vector and transformed into E. coli DH5α competent cells. Positive transformants were selected, from which the plasmid pGRB-yiaM was obtained.

[0113] 5. Transform the pREDCas9 plasmid into strain E.coli K12 MG1655-3.

[0114] The obtained yiaM::pTrc-mioX gene integration fragment and pGRB-yiaM plasmid were transformed into E. coli K12 MG1655-3 strain containing pREDCas9 plasmid via electroporation. After selecting positive transformants, the two plasmids used for gene editing were eliminated, finally obtaining strain E. coli K12 MG1655-4. The difference between strain E. coli K12 MG1655-4 and strain E. coli K12 MG1655-3 is that strain E. coli K12 MG1655-3 uses the pTrc promoter to control the overexpression of the miox gene at the yiaM pseudogene site.

[0115] Example 5: Controlling mioX gene overexpression at the ygiF pseudogene site using the pTrc promoter

[0116] 1. Using the E. coli K12 MG1655 genome as a template, the upstream homologous arm of the ygiF gene was amplified and purified by PCR using primers ygiF-US (SEQ ID NO.33) and ygiF-UA (SEQ ID NO.34). The downstream homologous arm of the ygiF gene was amplified and purified by PCR using primers ygiF-DS (SEQ ID NO.35) and ygiF-DA (SEQ ID NO.36). Specifically, ygiF-UA was designed to contain an upstream homologous sequence of the promoter, and ygiF-DS was designed to contain a downstream homologous sequence of the terminator.

[0117] The PCR system described above consisted of: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer (10 uM) 2 μL; downstream primer (10 uM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL.

[0118] The PCR program described above is as follows: pre-denaturation at 95℃ for 30 seconds, denaturation at 95℃ for 15 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 60 seconds, repeating the three steps of denaturation, annealing, and extension 30 times. Final extension at 72℃ for 5 minutes.

[0119] 2. Obtaining the pTrc-mioX gene fragment (sequence shown in SEQ ID NO.12): Refer to step 2 of Example 1.

[0120] 3. Using the upstream and downstream homologous arms of ygiF and the pTrc-mioX gene fragment as templates, the integrated ygiF::pTrc-mioX gene fragment was obtained by overlap PCR. The overlap PCR amplification system consisted of: 10 μL of 5×Phusion HF buffer, 4 μL of dNTPs (2.5 mmol / L), 1 μL of template, 1 μL of each primer, 0.5 μL of Phu high-fidelity DNA polymerase (2 U / μL), and 39.5 μL of ddH2O; the molar ratio of the upstream, downstream, and pTrc-mioX components in the template was 1:1:1.

[0121] Overlap PCR amplification program: pre-denaturation 95℃ for 30s, denaturation 95℃ for 15s, annealing 57℃ for 60s, extension 72℃ for 60s, (denaturation, annealing, and extension steps) 30 cycles. Final extension 72℃ for 5min.

[0122] Primers: ygiF-US, ygiF-UA, uxaC-F, uxaC-R, ygiF-DS, ygiF-DA

[0123] 4. Primers F6 (SEQ ID NO.37) and R6 (SEQ ID NO.38) were designed, and a DNA fragment containing the ygiF target sequence was constructed by PCR using the E.coli K12 MG1655 genome as a template.

[0124] PCR system: 2×Buffer 25μL; dNTPMix (10mMeach) 1μL; F6 (10uM) 2μL; R6 (10uM) 2μL; template 1; DNA polymerase 1μL; ddH2O 18μL.

[0125] PCR program: pre-denaturation 95℃ for 30s, denaturation 95℃ for 15s, annealing 60℃ for 45s, extension 72℃ for 60s, (denaturation, annealing, and extension steps) 30 cycles. Final extension 72℃ for 5min. After purification, a DNA fragment containing the ygiF target sequence is obtained.

[0126] The DNA fragment containing the ygiF target sequence was digested with restriction endonucleases BamHI and HindIII. The pGRB vector was also digested with BamHI and HindIII to obtain a linearized pGRB vector. The digested DNA fragment containing the ygiF target sequence was recombined with the linearized pGRB vector and transformed into E. coli DH5α competent cells. Positive transformants were selected, from which the plasmid pGRB-ygiF was obtained.

[0127] 5. The pREDCas9 plasmid was transformed into E. coli K12 MG1655-4 strain.

[0128] The obtained ygiF::pTrc-mioX gene integration fragment and pGRB-ygiF plasmid were transformed into E. coli K12 MG1655-4 strain containing pREDCas9 plasmid via electroporation. After screening for positive transformants, the two plasmids used for gene editing were eliminated, finally obtaining strain E. coli K12 MG1655-5. The difference between strain E. coli K12 MG1655-5 and strain E. coli K12 MG1655-4 is that strain E. coli K12 MG1655-4 inserted the inositol oxidase gene sequence pTrc-mioX (sequence shown in SEQ ID NO.12) at the ygiF pseudogene site.

[0129] Example 6: Controlling the inositol transporter mutant iolT at the ybgC pseudogene site using the pTrc promoter. G347A Gene overexpression

[0130] 1. Using the E. coli K12 MG1655 genome as a template, the upstream homologous arm of the ybgC gene was amplified and purified by PCR using primers ybgC-US (SEQ ID NO.39) and ybgC-UA (SEQ ID NO.40). Similarly, the downstream homologous arm of the ybgC gene was amplified and purified by PCR using primers ybgC-DS (SEQ ID NO.41) and ybgC-DA (SEQ ID NO.42). Specifically, ybgC-UA was designed to contain an upstream homologous sequence of the promoter, and ybgC-DS was designed to contain a downstream homologous sequence of the terminator.

[0131] The PCR system described above consisted of: 2×Buffer 25 μL; dNTPMix (10 mMeach) 1 μL; upstream primer (10 uM) 2 μL; downstream primer (10 uM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL.

[0132] The PCR program described above is as follows: pre-denaturation at 95℃ for 30 seconds, denaturation at 95℃ for 15 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 60 seconds, repeating the three steps of denaturation, annealing, and extension 30 times. Final extension at 72℃ for 5 minutes.

[0133] 2. pTrc-iolT G347A The fragment (SEQ ID NO.3) was obtained using primers F7 (SEQ ID NO.43) and R7 (SEQ ID NO.44). The iolT fragment carrying the mutation site was artificially synthesized. G347A Using the gene as a template and F7 and R7 as primers, pTrc-iolT was obtained by PCR amplification. G347A Gene fragment. The PCR system consisted of: 2×Buffer 25 μL; dNTP Mix (10 mMeach) 1 μL; upstream primer (10 μM) 2 μL; downstream primer (10 μM) 2 μL; template 1 μL; DNA polymerase 1 μL; ddH2O 18 μL. The PCR program was: pre-denaturation 95℃ for 30 s, denaturation 95℃ for 15 s, annealing 57℃ for 15 s, extension 72℃ for 60 s, repeated 35 times (denaturation, annealing, and extension). Final extension was 72℃ for 5 min. The pTrc promoter was designed in iolT... G347A The gene's sense strand primer F7; the pTrc terminator is designed in iolT G347A In the antisense primer R7 of the gene.

[0134] pTrc-iolT G347A Fragment (SEQ ID NO.3): Bits 1-30 are the pTrc promoter, bits 31-1449 are the iolT promoter. G347A The gene sequence has a terminator at positions 1450-1539.

[0135] A synthetically produced, unmutated pTrc-iolT gene fragment (SEQ ID NO.47) was used as a control. The pTrc-iolT gene fragment (SEQ ID NO.47) consists of a promoter at positions 1-30, an iolT gene sequence at positions 31-1449, and a terminator at positions 1450-1539.

[0136] 3. Based on the upstream and downstream homologous arms of ybgC and pTrc-iolT mentioned above... G347AUsing the gene fragment as a template, ybgC::pTrc-iolT was obtained by overlap PCR. G347A Gene integration fragment. Overlap PCR amplification system: 10 μL 5×Phusion HF buffer, 4 μL dNTP (2.5 mmol / L), 1 μL template, 1 μL primers, 0.5 μL Phu high-fidelity DNA polymerase (2 U / μL), 39.5 μL ddH2O; the molar ratio of upstream homologous arm, downstream homologous arm, and pTrc-mioX in the template was 1:1:1.

[0137] Overlap PCR amplification program: pre-denaturation 95℃ for 30s, denaturation 95℃ for 15s, annealing 55℃ for 60s, extension 72℃ for 60s, (denaturation, annealing, and extension steps) 30 cycles. Final extension 72℃ for 5min.

[0138] Primers: ybgC-US, ybgC-UA, F7, R7, ybgC-DS, ybgC-DA

[0139] pTrc-iolT G347A By replacing pTrc-iolT with other conditions unchanged, the ybgC::pTrc-iolT gene integration fragment was obtained as a control.

[0140] 4. Primers F8 (SEQ ID NO.45) and R8 (SEQ ID NO.46) were designed, and a DNA fragment containing the ybgC target sequence was constructed by PCR using the E.coli K12 MG1655 genome as a template.

[0141] PCR system: 2×Buffer 25μL; dNTPMix (10mMeach) 1μL; F6 (10uM) 2μL; R6 (10uM) 2μL; template 1; DNA polymerase 1μL; ddH2O 18μL.

[0142] PCR program: pre-denaturation 95℃ for 30s, denaturation 95℃ for 15s, annealing 60℃ for 45s, extension 72℃ for 60s, (denaturation, annealing, and extension steps) 30 cycles. Final extension 72℃ for 5min. After purification, a DNA fragment containing the ybgC target sequence is obtained.

[0143] The DNA fragment containing the ybgC target sequence was digested with restriction endonucleases BamHI and HindIII. The pGRB vector was also digested with BamHI and HindIII to obtain a linearized pGRB vector. The digested DNA fragment containing the ybgC target sequence was recombined with the linearized pGRB vector and transformed into E. coli DH5α competent cells. Positive transformants were selected, and the plasmid pGRB-ybgC was extracted.

[0144] 5. The pREDCas9 plasmid was transformed into E. coli K12 MG1655-5 strain.

[0145] The obtained ybgC::pTrc-iolT G347A The gene integration fragment and the pGRB-ybgC plasmid were transformed into *E. coli* K12 MG1655-5 strain containing the pREDCas9 plasmid via electroporation. After selecting positive transformants, the two plasmids used for gene editing were eliminated, resulting in strain *E. ginoC*. The difference between strain *E. ginoC* and strain *E. coli* K12 MG1655-5 is that *E. ginoC* inserts pTrc-iolT at the ybgC pseudogene site. G347A Fragment (SEQ ID NO.3).

[0146] Replace pTrc-iolT with the pTrc-iolT gene integration fragment G347A The gene integration fragment was used as a control, with other conditions remaining unchanged, to obtain E. ginoC-iolT. The only difference between E. ginoC-iolT and E. ginoC is that E. ginoC-iolT inserts pTrc-iolT (SEQ ID NO.47) at the ybgC pseudogene site.

[0147] Example 7: Application of E. ginoC in the preparation of glucuronic acid

[0148] (1) Take a single colony of E. ginoC and inoculate it into LB liquid medium. Incubate at 37°C with shaking at 120-220 rpm until OD. 600 The culture was incubated at 0.6-0.8, then shaken at 30℃ and 200 rpm for 12-16 hours. The culture was then centrifuged at 4℃ and 8000 rpm for 15 minutes, and the bacterial pellet was collected. E. ginoC-iolT was used as a control.

[0149] (2) Using inositol as a substrate, the cells obtained from E. ginoC culture were used for catalytic reaction and the conversion rate was calculated. The specific reaction system was as follows: 0.2 mM phosphate buffer pH 8.0, cell concentration 20 g (wet weight) / L, inositol 35 g / L, 1% Triton-X1000 (volume percentage). The reaction was carried out at 37℃ for 12 h.

[0150] After the reaction was complete, the solution was diluted 10-fold with distilled water, centrifuged at 12000 rpm for 10 min, filtered through a 0.22 μm filter, and detected by HPLC. The results are shown in the table below.

[0151] Table 1

[0152]

[0153] Example 8: Preparation of glucuronolactone

[0154] (1) Centrifuge the reaction solution obtained in step (2) of Example 7 at 8000 rpm for 30 min, collect the supernatant, filter the supernatant through an ultrafiltration membrane and collect the ultrafiltration membrane clear liquid; pass the ultrafiltration membrane clear liquid into a cation exchange resin for desalination and collect the desalination liquid.

[0155] (2) The desalting solution is concentrated under vacuum to obtain a concentrated solution. Phosphoric acid solution is added, with the amount of phosphoric acid solution added being 20% ​​(or 5-35%) of the volume of the concentrated solution. The mass fraction of the phosphoric acid solution is 83-98%. The lactone reaction is carried out to obtain a reaction solution.

[0156] (3) Vacuum distillation was performed on the above reaction solution, and then the crude glucuronolactone was obtained by cooling and crystallization.

[0157] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A recombinant bacterial strain that produces glucuronic acid, characterized in that, The recombinant strain does not express uronic acid isomerase and overexpresses inositol oxidase.

2. The recombinant strain according to claim 1, characterized in that, The recombinant strain originated from Escherichia coli; the Escherichia coli was E. coli K12 MG1655.

3. The recombinant strain according to claim 1 or 2, characterized in that, The recombinant strain overexpresses an inositol transporter mutant, wherein the mutant is any one of (A)-(C) below: (A) Its amino acid sequence is shown in SEQ ID NO.2; (B) Proteins that have 95% or 97% or more of the same amino acid sequence as defined in (A) and have the same function; (C) A fusion protein obtained by attaching a tag to the end of the protein defined in (A) or (B).

4. The recombinant strain according to claim 3, characterized in that, The encoding gene of the inositol transporter mutant is shown in SEQ ID NO.

1.

5. The recombinant strain according to any one of claims 1-4, characterized in that, The promoter controlling the overexpression of the gene encoding inositol oxidase is the pTrc promoter; the gene sequence of the pTrc promoter is shown in positions 1-30 of SEQ ID NO.

3.

6. The recombinant strain according to any one of claims 1-5, characterized in that, Methods to prevent the expression of uronic acid isomerase include: knocking out the uronic acid isomerase gene uxaC in the genome of recombinant strains; Methods for overexpressing inositol oxidase include inserting the coding gene for inositol oxidase into one or more of the following sites: uxaC site, yagT pseudogene site, ybdH pseudogene site, yiaM pseudogene site, ygiF pseudogene site, and ybgC pseudogene site.

7. The recombinant strain according to claim 6, characterized in that, The promoter controlling the gene encoding the inositol transporter mutant is the pTrc promoter; the gene sequence of the pTrc promoter is shown in positions 1-30 of SEQ ID NO.

3.

8. The recombinant strain according to claim 6, characterized in that, The nucleotide sequence of the gene encoding inositol oxidase is shown in SEQ ID NO.

5.

9. A method for producing glucuronic acid, characterized in that, Using inositol as a raw material, glucuronic acid is produced under the action of the recombinant strain described in any one of claims 1-8.

10. The use of the recombinant strain according to any one of claims 1-8 in the preparation of glucuronic acid or glucuronolactone.

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