Glucose dehydrogenase, recombinant raoultella ornithinolytica and application thereof

Abstract

This invention provides a glucose dehydrogenase, a recombinant ornithine-degrading Raoulbacterium, and their applications, belonging to the field of biotechnology. Specifically, it provides a glucose dehydrogenase PQQ-GDH, the amino acid sequence of which is SEQ ID NO.2; and a recombinant ornithine-degrading Raoulbacterium, which overexpresses glucose dehydrogenase PQQ-GDH, thereby reducing the production of the intermediate product 5-hydroxymethyl-2-furanoic acid (HMFCA) and increasing the yield of 2,5-furandicarboxylic acid (FDCA).

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a glucose dehydrogenase, recombinant ornithine-degrading Raoulbacterium, and their applications. Background Technology

[0002] 2,5-Furfurandicarboxylic acid (FDCA) is an important bio-based platform compound and a key monomer in the synthesis of polyethylene 2,5-furandicarboxylate (PEF). PEF is a renewable polyester material that outperforms traditional polyethylene terephthalate (PET) in terms of gas barrier properties and biodegradability, showing broad application prospects. In addition, FDCA can also be used to produce other high-performance polymers, plasticizers, coatings, and fragrances. With increasing global focus on sustainable development and environmental protection, the market demand for FDCA as a bio-based chemical is growing rapidly.

[0003] However, in the current process of synthesizing FDCA using whole-cell catalysis of 5-hydroxymethylfurfural (HMF), there is a common problem of accumulation of the intermediate product 5-hydroxymethyl-2-furanoic acid (HMFCA), which leads to low FDCA yield and limits the large-scale industrial application of FDCA. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a glucose dehydrogenase, recombinant ornithine-degrading Raoulbacterium, and their applications.

[0005] The technical solution of the present invention is as follows:

[0006] A glucose dehydrogenase PQQ-GDH, the amino acid sequence of which is SEQ ID NO.2.

[0007] A gene encoding the glucose dehydrogenase PQQ-GDH.

[0008] Preferably, the nucleotide sequence of the encoding gene is SEQ ID NO.1.

[0009] A recombinant expression vector comprising the above-mentioned coding gene SEQ ID NO.1.

[0010] A recombinant ornithine-releasing Raoulbacterium, comprising the above-mentioned recombinant expression vector.

[0011] Preferably, the host bacterium of the recombinant ornithine-releasing Raoulbacterium is ornithine-releasing Raoulbacterium ROBF60-DERBS.

[0012] The above-mentioned method for constructing recombinant ornithine-lysin-free Raoulbacterium includes the following steps:

[0013] The recombinant plasmid pACYCDuet-1-hmfH-PQQ-GDH containing the target gene SEQ ID NO.1 was electroporated into ROBF60-DERBS competent cells of *Rauvolus ornithine-lysinus*, and positive clones were screened to obtain recombinant *Rauvolus ornithine-lysinus*.

[0014] The preferred method for constructing the recombinant plasmid pACYCDuet-1-hmfH-PQQ-GDH includes the following steps:

[0015] Using plasmid pACYC-trc-hmfH as a template, the linear pACYC-trc-hmfH plasmid fragment was amplified by PCR using pACYCDuet-1-F and pACYCDuet-1-R as primers. Using the genome of ROBF60-DERBS of Ornithine-eating Raouli as a template, the target fragment PQQ-GDH was amplified by PCR using PQQ-GDH-F and PQQ-GDH-R as primers. The target fragment PQQ-GDH was then ligated into the linear pACYC-trc-hmfH plasmid fragment using a one-step cloning kit to obtain the recombinant plasmid pACYCDuet-1-hmfH-PQQ-GDH.

[0016] The plasmid pACYC-trc-hmfH was extracted from ornithine-lysin-ROBF60-DERBS.

[0017] The primer sequence for pACYCDuet-1-F is SEQ ID NO.3, the primer sequence for pACYCDuet-1-R is SEQ ID NO.4, the primer sequence for PQQ-GDH-F is SEQ ID NO.5, and the primer sequence for PQQ-GDH-R is SEQ ID NO.6.

[0018] The application of the above-mentioned glucose dehydrogenase PQQ-GDH, recombinant expression vector, or recombinant ornithine-degrading Raoultella in the preparation of 2,5-furandicarboxylic acid.

[0019] The above-mentioned method for preparing 2,5-furandicarboxylic acid from recombinant ornithine-releasing Raoultella includes the following steps:

[0020] Using a recombinant ornithine-releasing Raoultella resting cell suspension, the OD of the suspension was... 600 To prepare 2,5-furandicarboxylic acid, 5-hydroxymethylfurfural was added to the bacterial suspension to a final concentration of 150-400 mM, and CaCO3 was added at a concentration of 30-80 g / L. The mixture was then converted at 25-37℃ and 150-220 rpm for 50-150 h.

[0021] The beneficial effects of this invention include at least the following:

[0022] This invention is the first to identify PQQ-dependent membrane-bound glucose dehydrogenase (PQQ-GDH), a key enzyme catalyzing the further oxidation of HMFCA in the FDCA synthesis pathway. By overexpressing it in the engineered bacterium ROBF60-DERBS, the generation of the intermediate product HMFCA is reduced, thereby increasing the yield of FDCA. The recombinant ROBF60-DERBS prepared by this invention provides a novel microbial platform with great potential for the biotechnology and chemical industries. Attached Figure Description

[0023] Figure 1 This is a process route diagram for the biocatalytic synthesis of FDCA from HMF.

[0024] Figure 2 This is an agarose gel electrophoresis image.

[0025] In the figure: A is an agarose gel electrophoresis image of the linear pACYC-trc-hmfH plasmid fragment; B is an agarose gel electrophoresis image of the gene fragment PQQ-GDH.

[0026] Figure 3 A graph showing the whole-cell catalytic process of recombinant ornithine-resolving Raoulbacterium tumefaciens;

[0027] In the diagram: HMF is 5-hydroxymethylfurfural, HMFCA is 5-hydroxymethyl-2-furanic acid, DFF is 2,5-dicarboxyfuran, FFCA is 5-formyl-2-furanic acid, and FDCA is 2,5-furanic acid.

[0028] Figure 4 A whole-cell catalytic process curve of ROBF60-DERBS, a bacterium that degrades ornithine;

[0029] In the diagram: HMF is 5-hydroxymethylfurfural, HMFCA is 5-hydroxymethyl-2-furanic acid, DFF is 2,5-dicarboxyfuran, FFCA is 5-formyl-2-furanic acid, and FDCA is 2,5-furanic acid. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods involved in the embodiments are carried out in accordance with the prior art; unless otherwise specified, the drugs and reagents involved in the embodiments are all commercially available products.

[0031] ROBF60-DERBS, a strain of ornithine-lysinic Rauvolf, has been described in existing technical literature, “Combinatorial synthetic pathway fine-tuning and comparative transcriptomics for metabolic engineering of…” Raoultella ornithinolytica The ornithine-solubilizing Raoulbacterium ROBF60-DERBS strain is disclosed in the document "BF60 to efficiently synthesize 2,5-furandicarboxylic acid" (doi:10.1002 / bit.26725). The ornithine-solubilizing Raoulbacterium ROBF60-DERBS strain is available to the public through the School of Bioengineering, Qilu University of Technology.

[0032] The culture media involved in the examples are as follows:

[0033] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder water.

[0034] TB medium: tryptone 12 g / L, yeast extract 24 g / L, dipotassium hydrogen phosphate 12.54 g / L, potassium dihydrogen phosphate 2.31 g / L, glycerol 4 mL / L, balance water.

[0035] High-performance liquid chromatography (HPLC) methods for the determination of HMF, HMFCA, FFCA and FDCA:

[0036] After the sample is diluted a certain factor, 12000× g Centrifuge for 10 min, filter through a 0.22 μm aqueous filter membrane, and then perform analysis. Detection conditions: Shimadzu LC-20A, VWD detector; detection wavelength: 268 nm; column: Aminex HPX-87H (300 × 7.8 mm); mobile phase: 10 mM H2SO4 solution; flow rate: 0.6 mL / min; column temperature: 60℃; injection volume: 20 μL.

[0037] Figure 1 This is a process route diagram for the biocatalytic synthesis of FDCA from HMF.

[0038] Example 1

[0039] This invention is the first to identify PQQ-dependent membrane-bound glucose dehydrogenase (PQQ-GDH), a key enzyme in the FDCA synthesis pathway that catalyzes the further oxidation of HMFCA; the nucleotide sequence of the gene encoding PQQ-GDH is SEQ ID NO.1, and the amino acid sequence of PQQ-GDH is SEQ ID NO.2.

[0040] Construction of recombinant plasmids

[0041] Ornithine-free Raoulbacterium ROBF60-DERBS strain was streaked onto LB agar and incubated overnight at 30°C. Single colonies were picked and inoculated into liquid LB agar and incubated overnight at 30°C and 200 rpm. Plasmid extraction was performed to obtain plasmid pACYC-trc-hmfH. Using plasmid pACYC-trc-hmfH as a template, the linear pACYC-trc-hmfH plasmid fragment was amplified by PCR using pACYCDuet-1-F and pACYCDuet-1-R primers. The results of agarose gel electrophoresis are shown in the figure below. Figure 2 In section A, using the genome of *Rauvolfia ornithine-lysinophila* ROBF60-DERBS as a template, the target fragment PQQ-GDH was amplified by PCR using PQQ-GDH-F and PQQ-GDH-R (containing 20 bp homologous sequences) as primers. The agarose gel electrophoresis results are shown in [Figure A]. Figure 2 In step B, the target fragment PQQ-GDH was ligated with the linear pACYC-trc-hmfH plasmid fragment using a one-step cloning kit (C112-02, Nanjing Novizan Biotechnology Co., Ltd.) in a metal bath at 37°C for 30 min to achieve recombination and ligation. 10 μL of the ligation product was then added to... E. coli In DH5α competent cells, gently tap the tube wall to mix, incubate on ice for 30 min, heat shock at 42℃ for 90 s, quickly cool on ice for 2 min, add 900 μL of antibiotic-free LB liquid medium, and incubate on a shaker at 37℃ and 200 rpm for 1 h. Centrifuge at 5000 rpm for 3 min, discard 900 μL of supernatant, resuspend the bacterial cells in the remaining medium, and spread evenly on LB agar plates containing 20 mg / L chloramphenicol. Invert the plates and incubate at 37℃ for 16 h.

[0042] Single colonies with normal morphology were selected from the colonies grown on the plate and subjected to colony PCR amplification. Preliminary verification was then performed by gel electrophoresis. Bacterial solutions with band sizes consistent with theoretical values ​​were then sent to Beijing Qingke Biotechnology Co., Ltd. for DNA sequencing verification, ultimately yielding the correctly constructed recombinant bacteria. E. coli The recombinant plasmid pACYCDuet-1-hmfH-PQQ-GDH was obtained by running / pACYCDuet-1-hmfH-PQQ-GDH.

[0043] The primer sequences used above are as follows:

[0044] The primer sequence for pACYCDuet-1-F is SEQ ID NO.3, the primer sequence for pACYCDuet-1-R is SEQ ID NO.4, the primer sequence for PQQ-GDH-F is SEQ ID NO.5, and the primer sequence for PQQ-GDH-R is SEQ ID NO.6.

[0045] The PCR reaction system consisted of 1 μL each of forward and reverse primers, 25 μL of 2×Phanta Max Master Mix, 1 μL of template, and double-distilled water to a final volume of 50 μL.

[0046] PCR conditions were as follows: pre-denaturation at 95℃ for 5 min; followed by 30 cycles (95℃ for 15 s, 55℃ for 15 s, 72℃ for 2 min), extension at 72℃ for 5 min; and finally storage at 4℃.

[0047] Example 2

[0048] The preparation of ornithine-free Raoulbacterium ROBF60-DERBS competent cells is as follows:

[0049] (1) Take out the glycerol tube of the ornithine-releasing Raoulbacterium ROBF60-DERBS strain from the ultra-low temperature freezer at -80℃ and thaw it at room temperature; use an inoculation loop to dip the bacterial solution in the glycerol tube, streak it on LB solid medium, invert the plate, and incubate overnight at 30℃.

[0050] (2) Pick a single colony with an inoculation loop and inoculate it into a 50 mL Erlenmeyer flask containing 10 mL of LB medium. Place the flask in a constant temperature shaker at 30℃ and rotate at 200 rpm overnight to obtain the seed culture.

[0051] (3) Take the above seed culture and inoculate it into 50 mL of sterile LB liquid medium at an inoculation rate of 1%. Place it in a constant temperature shaker at 30℃ and rotate at 200 rpm until the bacterial cell OD reaches the target value. 600 When the coefficient of performance reaches 0.6~0.8, stop the culture.

[0052] (4) In a clean bench, transfer 30 mL of the above bacterial solution to a sterile 50 mL centrifuge tube and place it on ice for more than 10 min.

[0053] (5) Set the centrifuge parameters to 4℃ and 8000 rpm. After centrifuging the bacterial solution for 5 min, discard the supernatant and collect the bacterial cells in a clean bench.

[0054] (6) In a clean bench, add 50 mL of pre-cooled sterile water to the collected bacterial cells, resuspend the bacterial cells by blowing and aspiration, and then centrifuge at 4°C and 8000 rpm for 5 min in a centrifuge. Discard the supernatant and collect the bacterial cells.

[0055] (7) Add 50 mL of pre-cooled sterile 10% glycerol to the collected bacterial cells, resuspend the bacterial cells by pipetting, centrifuge and discard the supernatant, and retain the bacterial cells;

[0056] (8) Finally, add 500 μL of 10% glycerol that has been pre-chilled on ice, resuspend and mix the bacterial cells by pipetting, and then add them to sterile 1.5 mL centrifuge tubes that have been pre-chilled on ice. Aliquot the mixture into 100 μL per tube and store it in a -80℃ freezer for later use.

[0057] Example 3

[0058] Construction of recombinant ornithine-degrading Raoultella

[0059] The recombinant plasmid pACYCDuet-1-hmfH-PQQ-GDH obtained in Example 1 was electroporated into the ornithine-neutralizing Raoulbacterium ROBF60-DERBS competent cells prepared in Example 2. The specific steps are as follows:

[0060] (1) Take the 100 μL competent cells prepared in Example 2 out of the -80°C freezer and place them on ice to thaw;

[0061] (2) Add 10 μL of the recombinant plasmid pACYCDuet-1-hmfH-PQQ-GDH constructed in Example 1 to ROBF60-DERBS competent cells of *R. ornithine-releasing*. Gently mix with a pipette and continue to incubate on ice for 10 min. Then, add the competent cells to an electroporation cuvette after the ice bath for electroporation (2 mm, 2500 V, 5 ms). Immediately after electroporation, add antibiotic-free LB liquid medium. Then, place the transformation solution in a 30°C shaking culture for 30 min. Spread the solution on an LB agar plate containing 20 mg / L chloramphenicol. After colony PCR verification, the recombinant *R. ornithine-releasing* was obtained. R. ornithinolytica / pACYCDuet-1-hmfH-PQQ-GDH, abbreviated as Recombinant Ornithine-Solubilizing Raoulbacterium.

[0062] Example 4

[0063] Preparation of recombinant ornithine-resolving Raoulbacterium resting cells

[0064] (1) The single colony of the recombinant ornithine-releasing Raoulbacterium obtained in Example 3 was inoculated into liquid LB medium and cultured at 30°C and 220 rpm for 12 h to obtain seed culture solution;

[0065] (2) The seed culture solution was transferred to TB medium at an inoculation rate of 1% (V / V) and cultured at 30℃ and 220 rpm until OD. 600 When the concentration of protein was 0.6, IPTG was added to induce protein expression. The final concentration of IPTG was 0.2 mM. After culturing at 30°C for 24 h, the cells were collected by centrifugation at 4°C and 8000 rpm for 15 min. The cells were then washed twice with 50 mM sodium phosphate buffer (pH 8.0) and resuspended to obtain recombinant ornithine-releasing Raoultella resting cells.

[0066] Example 5

[0067] Whole-cell catalytic preparation of 2,5-furandicarboxylic acid

[0068] Using the recombinant ornithine-releasing Raoultella lataniae resting cells obtained in Example 4 as a biocatalyst and HMF as a substrate, FDCA was catalyzed for production. The specific steps are as follows:

[0069] Recombinant ornithine-degrading Raoultella resting cells were resuspended in 50 mM sodium phosphate buffer (pH 8.0) to OD. 600 =100 to obtain a bacterial suspension. HMF was added to the bacterial suspension to a final concentration of 300 mM. The reaction system was 10 mL, and 0.5 g CaCO3 was added to neutralize the acidic products. The reaction was catalyzed at 30℃ for 144 h, and the yield of FDCA reached 270.5 mM. Figure 3 The molar conversion rate was 90.2%. FDCA was prepared by whole-cell catalysis using the originating strain ROBF60-DERBS resting cells according to the same method, achieving a yield of 232.5 mM (…). Figure 4 The yield of recombinant ornithine-resolving Raoulbacterium FDCA was 16.3% higher than that of the original strain ROBF60-DERBS.

Claims

1. A glucose dehydrogenase PQQ-GDH, characterized in that, Its amino acid sequence is SEQ ID NO.

2.

2. A gene encoding a gene, characterized in that, The glucose dehydrogenase PQQ-GDH of claim 1 is encoded.

3. The encoding gene as described in claim 2, characterized in that, The nucleotide sequence of the encoding gene is SEQ ID NO.

1.

4. A recombinant expression vector, characterized in that, It includes the encoding gene SEQ ID NO.1 as described in claim 3.

5. A recombinant ornithine-lysinophilic Raoulbacterium, characterized in that, It includes the recombinant expression vector as described in claim 4.

6. The recombinant ornithine-lysinophilic Raoulbacterium as described in claim 5, characterized in that, The recombinant expression vector described in claim 4 was transformed into ROBF60-DERBS of *Rauvolfia ornithine-solulucosides* to obtain recombinant *Rauvolfia ornithine-solulucosides*.

7. The method for constructing the recombinant ornithine-releasing Raoulbacterium as described in claim 5, characterized in that, Includes the following steps: The recombinant plasmid pACYCDuet-1-hmfH-PQQ-GDH containing the target gene SEQ ID NO.1 was electroporated into ROBF60-DERBS competent cells of *Rauvolus ornithine-lysinus*, and positive clones were screened to obtain recombinant *Rauvolus ornithine-lysinus*.

8. The construction method as described in claim 7, characterized in that, The construction method of recombinant plasmid pACYCDuet-1-hmfH-PQQ-GDH includes the following steps: Using plasmid pACYC-trc-hmfH as a template, the linear pACYC-trc-hmfH plasmid fragment was amplified by PCR using pACYCDuet-1-F and pACYCDuet-1-R as primers. Using the genome of ROBF60-DERBS of Ornithine-eating Raouli as a template, the target fragment PQQ-GDH was amplified by PCR using PQQ-GDH-F and PQQ-GDH-R as primers. The target fragment PQQ-GDH was then ligated into the linear pACYC-trc-hmfH plasmid fragment using a one-step cloning kit to obtain the recombinant plasmid pACYCDuet-1-hmfH-PQQ-GDH. The plasmid pACYC-trc-hmfH was extracted from ornithine-lysin-ROBF60-DERBS. The primer sequence for pACYCDuet-1-F is SEQ ID NO.3, the primer sequence for pACYCDuet-1-R is SEQ ID NO.4, the primer sequence for PQQ-GDH-F is SEQ ID NO.5, and the primer sequence for PQQ-GDH-R is SEQ ID NO.

6.

9. The use of the glucose dehydrogenase PQQ-GDH of claim 1, the recombinant expression vector of claim 4, or the recombinant ornithine-degrading Raoulbacterium of claim 5 in the preparation of 2,5-furandicarboxylic acid.

10. The method for preparing 2,5-furandicarboxylic acid from recombinant ornithine-releasing Raoultella as described in claim 5, characterized in that, Includes the following steps: Using a recombinant ornithine-releasing Raoultella resting cell suspension, the OD of the suspension was... 600 To prepare 2,5-furandicarboxylic acid, 5-hydroxymethylfurfural was added to the bacterial suspension to a final concentration of 150-400 mM, and CaCO3 was added at a concentration of 30-80 g / L. The mixture was then converted at 25-37℃ and 150-220 rpm for 50-150 h.

Images