Construction method of recombinant corynebacterium glutamicum for fermentation synthesis of poly beta-(1, 6)-N-acetylglucosamine polysaccharide

Abstract

The invention discloses a construction method of recombinant corynebacterium glutamicum for fermentation synthesis of poly beta-(1, 6)-N-acetylglucosamine polysaccharide, and belongs to the technical field of bioengineering. The recombinant corynebacterium glutamicum capable of preparing the PNAG through fermentation is constructed by using synthetic biology and genetic engineering technologies for the first time, cheap carbon sources and nitrogen sources can be utilized, the recombinant corynebacterium glutamicum is suitable for high-density fed-batch fermentation, and the generation cost of the PNAG is reduced; the yield after 42 hours of fermentation can reach 6.82 g / L, and the method has industrial application potential.

Description

Technical Field

[0001] The present invention relates to a method for constructing a recombinant Corynebacterium glutamicum capable of fermentatively synthesizing poly-β-(1,6)-N-acetylglucosamine polysaccharide, belonging to the field of bioengineering technology. Background Art

[0002] Poly-β-(1,6)-N-acetylglucosamine polysaccharide (Poly-N-acetylglucosamine, abbreviated as PNAG) is a polysaccharide formed by the tandem connection of N-acetylglucosamine through β1-6 glycosidic bonds, and exists on the surfaces of many pathogenic bacteria, fungi, and parasites, providing a protective function for pathogenic organisms. Since PNAG can protect pathogenic bacteria from being killed by the immune system, the polysaccharide antigen derived from PNAG can be used as a vaccine to stimulate the immune system to obtain antibodies. It has been proven that PNAG has a wide range of immunomodulatory and protective effects and is expected to play an important role in various medical and biotechnology fields in the future. For example, the PNAG vaccine (AV0328) developed by Alopexx Company has completed the first-phase human trial. This vaccine can induce the production of antibodies to kill a series of pathogens expressing PNAG, showing good effects and good tolerance, and no serious adverse events have been observed.

[0003] However, at the present stage, the synthesis methods of PNAG are mainly divided into the extraction method of pathogenic microorganism biofilm (such as culturing Staphylococcus aureus to synthesize PNAG) and chemical synthesis method. The biofilm separation method requires culturing pathogenic bacteria (such as Staphylococcus aureus, etc.), and the culture usually uses serum medium, which has defects such as high production cost, extremely low fermentation density of the strain, limited PNAG production, and high biological risks; while the chemical synthesis method involves complex reaction processes, high costs, and may produce by-products and environmental pollution problems, and the feasibility of large-scale production of PNAG is very low. With the continuous development of biotechnology, the preparation of pharmaceutical products by microbial fermentation has become an indispensable new development direction in the pharmaceutical industry due to its advantages such as low cost, high efficiency, and environmental friendliness. Developing strains that meet the requirements of pharmaceutical safety and contribute to industrial production is the primary problem to be solved for the synthesis of PNAG. However, there is currently no report on the fermentative synthesis of PNAG polysaccharide. Summary of the Invention

[0004] In order to solve the above problems, the present invention uses the industrially safe strain Corynebacterium glutamicum as the host (chassis) cell to recombinantly express the synthase subunit combination required for PNAG synthesis, so as to endow Corynebacterium glutamicum with the ability to synthesize PNAG. On this basis, through genetic engineering technology, the supply rate of the PNAG synthesis monomer UDP-N-acetylglucosamine is increased to achieve the synthesis of high-purity PNAG.

[0005] The present invention provides a recombinant Corynebacterium glutamicum with the ability to synthesize poly-β-(1,6)-N-acetylglucosamine polysaccharide, which expresses a combination of synthase subunits required for the synthesis of poly-β-(1,6)-N-acetylglucosamine polysaccharide (PNAG), and expresses genes related to the synthesis of the monomer UDP-N-acetylglucosamine required for PNAG synthesis.

[0006] In one embodiment, the combination of synthase subunits required for the synthesis of poly-β-(1,6)-N-acetylglucosamine polysaccharide includes the gene combination of (a) or (b):

[0007] (a) Genes IcaA, IcaB, IcaC, and IcaD;

[0008] (b) Genes IcaA, IcaC, and IcaD.

[0009] In one embodiment, the nucleotide sequences of genes IcaA, IcaB, IcaC, and IcaD are shown as SEQ ID NO.1 to SEQ ID NO.4 respectively.

[0010] In one embodiment, the vectors expressing the gene combination include, but are not limited to, pEC-XK99E, pXMJ19, pECXK99E, pEC-XT99A, pEKEx1, pEKEx2, pVWEx1, pVWEx2, pZ8-1, pECTAC-K99, pAPE12.

[0011] In one embodiment, the recombinant Corynebacterium glutamicum also expresses glutamine-fructose-6-phosphate aminotransferase, phosphoglucomutase GlmM, and UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase bifunctional enzyme derived from Pseudomonas putida KT2440.

[0012] In one embodiment, the nucleotide sequence encoding the glutamine-fructose-6-phosphate aminotransferase GlmS is shown as SEQ ID NO.5, the nucleotide sequence encoding the phosphoglucomutase GlmM is shown as SEQ ID NO.6, and the nucleotide sequence encoding the UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase bifunctional enzyme GlmU is shown as SEQ ID NO.7.

[0013] In one embodiment, the vectors expressing GlmS, GlmM, and GlmU include, but are not limited to, pXMJ19, pECXK99E, pEC-XT99A, pEKEx1, pEKEx2, pVWEx1, pVWEx2, pZ8-1, pECTAC-K99, or pAPE12.

[0014] In one embodiment, the recombinant Corynebacterium glutamicum expresses the IcaA, IcaC, and IcaD genes using pEC-XK99E as a vector, and expresses the GlmS, GlmM, and GlmU genes using pXMJ19 as a vector.

[0015] In one embodiment, the recombinant Corynebacterium glutamicum expresses the IcaA, IcaB, IcaC, and IcaD genes using pEC-XK99E as a vector, and expresses the GlmS, GlmM, and GlmU genes using pXMJ19 as a vector.

[0016] In one embodiment, Corynebacterium glutamicum ATCC 13032 is used as a host cell.

[0017] The present invention also provides a method for preparing poly-N-acetylglucosamine, which uses the recombinant Corynebacterium glutamicum for fermentation.

[0018] In one embodiment, the fermentation is carried out at 28 - 30 °C for at least 16 h.

[0019] In one embodiment, the fermentation is carried out at 30 °C for 24 - 72 h.

[0020] In one embodiment, feeding is also carried out during the fermentation process.

[0021] In one embodiment, the feeding is to add glucose to control the glucose concentration in the fermentation system to 10 - 15 g / L.

[0022] The present invention also provides the application of the recombinant Corynebacterium glutamicum, or the method in the preparation of PNAG or pharmaceutical chemicals containing PNAG.

[0023] Beneficial effects:

[0024] 1. The present invention constructs a recombinant Corynebacterium glutamicum capable of fermenting and preparing PNAG for the first time using synthetic biology and genetic engineering techniques. It can utilize cheap carbon sources and nitrogen sources, is suitable for high-density fed-batch fermentation, and reduces the production cost of PNAG.

[0025] 2. Corynebacterium glutamicum is an industrially widely used microorganism with extremely high biosafety and fermentation stability between batches, laying a foundation for the stable fermentation of PNAG.

[0026] 3. The Corynebacterium glutamicum constructed in the present invention does not produce endogenous polysaccharides, and the PNAG synthesized by it has high purity. The fermentation cycle of the strain C.glutamicum pEC-IcaABCD-pXMJ19-glmSMU is short. For the determination of the yield and the glucose consumption rate, 3 parallels were set, and the measurement results were averaged. The PNAG yield at 42 h of fermentation can reach 6.82 ± 0.5 g / L; the PNAG prepared from the strain C.glutamicum pEC-IcaABCD-pXMJ19-glmSMU is not prone to precipitation, and the yield at 72 h of fermentation can reach 3.2 ± 0.5 g / L. Description of the Drawings

[0027] Figure 1 : Metabolic pathway of recombinant Corynebacterium glutamicum for producing PNAG and related enzymes.

[0028] Figure 2 : Batch fermentation tank yield diagram of PNAG of recombinant Corynebacterium glutamicum.

[0029] Figure 3 : Process for purifying PNAG from the fermentation broth of recombinant Corynebacterium glutamicum.

[0030] Figure 4 : NMR identification diagram of PNAG produced by recombinant Corynebacterium glutamicum. Detailed Embodiments

[0031] Strains: Corynebacterium glutamicum ATCC 13032, plasmids: pXMJ19, pEC-XK99E, all of which are commercial plasmids.

[0032] LB medium: 5 g / L of yeast powder, 10 g / L of peptone, 10 g / L of sodium chloride.

[0033] BHI: 37 g / L of brain heart infusion, 91 g / L of sorbitol.

[0034] Fermentation medium: 40 g / L of glucose, 20 g / L of corn steep liquor powder, (NH 4 ) 2 SO 4 30 g / L, KH 2 PO 4 1 g / L, K 2 HPO 4 1 g / L, MgSO 4 25 g / L, MOPS (3-morpholinopropanesulfonic acid) 42 g / L.

[0035] Determination of the yield of PNAG: Take an appropriate amount of fermentation broth, centrifuge at 2000 rpm for 10 min, take the supernatant, add 2% NaOH, heat at 60 °C for 2 h, adjust the pH to neutral with concentrated hydrochloric acid, and then centrifuge at 10000 rpm for 10 min to remove the precipitate. Add 4 volumes of pre-cooled ethanol, place at -20 °C for alcohol precipitation for 6 hours, centrifuge at 10000 rpm for 10 min, discard the supernatant, evaporate the ethanol at room temperature to dry the precipitate, add 5 mL of ultrapure water, soak for 2 h, centrifuge to remove the supernatant, add 5 mL of ultrapure water again, wash until the precipitate is colorless, then place in a freeze dryer and freeze for 48 h, take out and measure the dry weight.

[0036] NMR identification of PNAG: Dissolve the freeze-dried sample in 5M hydrochloric acid, then adjust to neutral with sodium hydroxide, dialyze to remove NaCl, concentrate to remove water molecules, then add deuterium oxide, and measure the H spectrum of PNAG. 1 spectrum.

[0037] Example 1: Construction of recombinant plasmid pECXK99E-IcaABCD and recombinant Corynebacterium glutamicum C.glutamicum pEC-IcaABCD

[0038] Using the IcaA, IcaB, IcaC, and IcaD genes of Staphylococcus as templates, codon optimization was performed on the TWIST website with Corynebacterium glutamicum ATCC 13032 as the expression host. The IcaA gene shown in SEQ ID NO.1, the IcaB gene shown in SEQ ID NO.2, the IcaC gene shown in SEQ ID NO.3, and the IcaD gene shown in SEQ ID NO.4 were obtained by gene synthesis respectively.

[0039] Double-digest the Corynebacterium glutamicum expression plasmid pEC-XK99E with EcoRI / KpnI, and use the Gibson Assembly kit to ligate the IcaA, IcaB, IcaC, and IcaD gene fragments to the digested pEC-XK99E in one step. The obtained recombinant plasmid was named pECXK99E-IcaABCD;

[0040] Use an electroporator to electrotransform the plasmid pECXK99E-IcaABCD into Corynebacterium glutamicum ATCC13032 by electroporation. The electroporation conditions are voltage 1.5 KV, 5 ms, (the width of the electroporation cuvette is 1 mm), and repeat once. Screen the recombinant bacteria on a BHI plate containing 25 mg / L kanamycin. This recombinant bacterium was named C.glutamicum pEC-IcaABCD.

[0041] Example 2: Construction of recombinant plasmid pECXK99E-IcaACD and recombinant Corynebacterium glutamicum C.glutamicum pEC-IcaACD

[0042] The specific implementation method is the same as that of Example 1, except that the Corynebacterium glutamicum expression plasmid pEC-XK99E is digested with EcoRI / KpnI, and the IcaA, IcaC, and IcaD gene fragments are ligated into the digested pEC-XK99E in one step using the Gibson Assembly kit. The obtained recombinant plasmid is named pECXK99E-IcaACD. The constructed recombinant plasmid was transformed into Corynebacterium glutamicum ATCC13032 by the same method as in Example 1, and the strain with correct verification was named C.glutamicum pEC-IcaACD.

[0043] Example 3: Recombinant plasmid pXMJ19-glmSMU and recombinant Corynebacterium glutamicum C.glutamicum pEC-IcaABCD-pXMJ19-glmSMU

[0044] Pseudomonas putida KT2440 was inoculated into 3 ml of LB liquid medium and cultured at 30 °C and 220 rpm for 24 h. The cells were collected, and genomic DNA was extracted using a cell genomic DNA extraction kit. Primers glmS-F / glmS–R, glmM-F / glmM–R, and glmU-F / glmU-R were designed. Using the extracted genomic DNA of Pseudomonas putida as a template, the glmU gene (nucleotide sequence shown in SEQ ID NO.5), glmS gene (nucleotide sequence shown in SEQ ID NO.6), and glmM gene (nucleotide sequence shown in SEQ ID NO.7) were amplified by PCR amplification system and procedure. The plasmid pXMJ19 was digested at the selected restriction sites to obtain the linear plasmid pXMJ19. The amplified fragments glmU, glmS, and glmM were subjected to Gibson assembly reaction with the linear plasmid pXMJ19. The Gibson assembly reaction system was transformed into JM109 competent cells. The transformants were selected for plasmid sequencing reaction and compared with the sequence. The recombinant plasmid pXMJ19-glmU-glmM-glmS was successfully constructed and was electrotransformed into the Corynebacterium glutamicum C.glutamicum pEC-IcaABCD constructed in Example 1 and the Corynebacterium glutamicum C.glutamicum pEC-IcaACD constructed in Example 2, respectively. The obtained strains were named C.glutamicum pEC-IcaABCD-pXMJ19-glmSMU and C.glutamicum pEC-IcaACD-pXMJ19-glmSMU.

[0045] Table 1 Primers and Sequences

[0046]

[0047] Example 4: Fermentation of Recombinant Corynebacterium glutamicum for the Synthesis of PNAG

[0048] The monoclonal strains of recombinant Corynebacterium glutamicum pEC-IcaABCD-pXMJ19-glmSMU and recombinant Corynebacterium glutamicum pEC-IcaACD-pXMJ19-glmSMU constructed in Example 3 were respectively inoculated into 5 ml of BHI medium and cultured overnight at 30 °C with shaking at 200 rpm. After 10 h, they were respectively transferred to 250-ml Erlenmeyer flasks (with a liquid volume of 25 ml of fermentation medium) at an inoculation amount of 1%. They were cultured at 200 rpm and 28 °C for 10 h, and then inoculated into a 5-L fermenter at an inoculation amount of 10% and fermented at 30 °C for 72 h. Glucose was fed to maintain the glucose content in the fermenter at 10 - 15 g / L at 18 h of fermentation. Ammonia water was fed to control the pH to neutral. Samples were taken at regular intervals to determine the yield of soluble PNAG in the fermentation broth. As Figure 2 shown, the PNAG yield of the strain pEC-IcaABCD-pXMJ19-glmSMU reached 6.82 g / L after 42 h of fermentation. As time went by, the PNAG in the fermentation broth gradually formed solid precipitates, resulting in a decreasing trend in the content of soluble PNAG in the fermentation broth; the PNAG yield of the strain pEC-IcaACD-pXMJ19-glmSMU reached 3.2 g / L after 72 h of fermentation, and all remained in a soluble state.

[0049] Example 5: Extraction of PNAG

[0050] Refer to Figure 3Extraction process: Collect the fermentation broth prepared by the method of Reference Example 4, treat it with 2% NaOH for 1 hour to remove proteins, adjust the pH to neutral with 2% hydrochloric acid, centrifuge to remove impurities, and precipitate with 4 volumes of ethanol to obtain the crude product of PNAG. Wash the obtained PNAG three times with ethanol and ultrapure water respectively to obtain the pure product of PNAG. Perform nuclear magnetic resonance detection on the obtained PNAG, and use a nuclear magnetic resonance spectrometer (AVANCE NEO 600MHz, Bruker, Germany) and standard MestReNova acquisition software to measure the nuclear magnetic resonance (NMR) spectrum. The polysaccharide solution was concentrated to a volume of 1 - 2 mL using a concentrator centrifuge tube (Amicon°Ultra filter, 3 kDa MWCO), and then 99.9% D2O (D113904, Aladdin) was added to dilute the H2O in the solution. This step was repeated 9 times. The spectrum was recorded at 25 °C, and a one-dimensional 1H-NMR spectrum (600 MHz) was recorded in the normal acquisition mode through 1024 scans. The results are as Figure 4 shown. The nuclear magnetic resonance results show that the purified product is single, without impurity peaks, the baseline is flat, and the main signals of PNAG are obvious, proving that the sample has a high purity.

[0051] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A recombinant Corynebacterium glutamicum, characterized in that It expresses the synthase subunit combination required for the synthesis of poly-β-(1,6)-N-acetylglucosamine polysaccharide, and expresses genes related to UDP-N-acetylglucosamine synthesis, and has the ability to synthesize poly-β-(1,6)-N-acetylglucosamine polysaccharide.

2. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that Gene combinations expressing (a) or (b): (a) Genes IcaA, IcaB, IcaC, and IcaD; (b) Genes IcaA, IcaC and IcaD.

3. The recombinant Corynebacterium glutamicum according to any one of claims 1 to 3, characterized in that Glutamine-fructose-6-phosphate aminotransferase GlmS, phosphoglucomutase GlmM, and UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphate acetyltransferase bifunctional enzyme GlmU were also expressed.

4. The recombinant Corynebacterium glutamicum according to claim 3, characterized in that Vectors expressing the gene combination or expressing GlmS, GlmM and GlmU include but are not limited to pXMJ19, pECXK99E, pEC-XT99A, pEKEx1, pEKEx2, pVWEx1, pVWEx2, pZ8-1, pECTAC-K99 or pAPE12.

5. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that pEC-XK99E was used as a vector to express IcaA, IcaC and IcaD genes, and pXMJ19 was used as a vector to express GlmS, GlmM and GlmU genes.

6. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that pEC-XK99E was used as a vector to express IcaA, IcaB, IcaC and IcaD genes, and pXMJ19 was used as a vector to express GlmS, GlmM and GlmU genes.

7. The recombinant Corynebacterium glutamicum according to any one of claims 1 to 6, characterized in that Corynebacterium glutamicum ATCC 13032 was used as the host cell.

8. A method for preparing poly-N-acetylglucosamine, characterized in that: The recombinant Corynebacterium glutamicum described in any one of claims 1 to 7 is used for fermentation.

9. The method according to claim 8, characterized in that The fermentation is carried out at 28-30° C. for at least 16 hours.

10. Use of the recombinant Corynebacterium glutamicum according to any one of claims 1 to 7, or the method according to any one of claims 8 to 9 in the preparation of PNAG or pharmaceutical chemicals containing PNAG.

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