A method for constructing a recombinant coryneform bacterium for fermentative synthesis of a poly-beta-(1,6)-N-acetylglucosamine polysaccharide

Abstract

The application discloses a construction method of recombinant corynebacterium glutamicum for fermentative synthesis of poly-beta-(1,6)-N-acetylglucosamine polysaccharide, and belongs to the technical field of bioengineering. The recombinant corynebacterium glutamicum for fermentative synthesis of PNAG is constructed by using synthetic biology and genetic engineering technology for the first time, and can utilize cheap carbon source and nitrogen source, is suitable for high-density batch feeding fermentation, and reduces the production cost of PNAG; the yield of the fermentation for 42 hours can reach 6.82 g / L, and has industrial application potential.

Description

Technical Field

[0001] This invention relates to a method for constructing recombinant Corynebacterium glutamicum that synthesizes polyβ-(1,6)-N-acetylglucosamine polysaccharide through fermentation, belonging to the field of bioengineering technology. Background Technology

[0002] Poly-β-(1,6)-N-acetylglucosamine (PNAG) is a polysaccharide formed by the tandem linkage of N-acetylglucosamine through β1-6 glycosidic bonds. It exists on the surface of many pathogenic bacteria, fungi, and parasites, providing protection for pathogenic organisms. Because PNAG protects pathogens from the immune system, polysaccharide antigens derived from PNAG can be used as vaccines to stimulate the immune system to acquire antibodies. PNAG has been shown to have broad 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 has completed Phase I human trials. This vaccine can induce antibodies to kill a range of pathogens expressing PNAG, showing good efficacy and tolerability, with no serious adverse events observed.

[0003] However, current methods for synthesizing PNAG mainly fall into two categories: biofilm extraction (e.g., culturing Staphylococcus aureus to synthesize PNAG) and chemical synthesis. Biofilm extraction requires culturing pathogenic bacteria (such as Staphylococcus aureus), typically using serum culture media, which suffers from drawbacks such as high production costs, extremely low fermentation density, limited PNAG yield, and high biological risks. Chemical synthesis involves complex reaction processes, is costly, and may generate byproducts and cause environmental pollution, making large-scale PNAG production highly impractical. With the continuous development of biotechnology, microbial fermentation for pharmaceutical preparation has become an indispensable new direction for the pharmaceutical industry due to its advantages of low cost, high efficiency, and environmental friendliness. Developing strains that meet pharmaceutical safety requirements and facilitate industrial production is the primary problem to be solved in achieving PNAG synthesis; however, there are currently no reports of fermentation synthesis of PNAG polysaccharides. Summary of the Invention

[0004] To address the aforementioned problems, this invention uses the industrially safe strain Corynebacterium glutamicum as the host (chassis) cell and recombinantly expresses the synthase subunit combination required for PNAG synthesis, thereby endowing Corynebacterium glutamicum with the ability to synthesize PNAG. Based on this, genetic engineering technology is used to increase the supply rate of the PNAG synthesis monomer UDP-N-acetylglucosamine, achieving the synthesis of high-purity PNAG.

[0005] This invention provides a recombinant Corynebacterium glutamicum capable of synthesizing polyβ-(1,6)-N-acetylglucosamine polysaccharide, expressing the synthase subunit combination required for the synthesis of polyβ-(1,6)-N-acetylglucosamine polysaccharide (PNAG), and expressing 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 the polyβ-(1,6)-N-acetylglucosamine polysaccharide comprises either (a) or (b) a gene combination:

[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 in 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, and pAPE12.

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

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

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

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

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

[0020] In one implementation, the fermentation process also involves feeding additional materials.

[0021] In one embodiment, the feed is glucose, which is added 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 thereof, in the preparation of PNAG or PNAG-containing pharmaceutical chemicals.

[0023] Beneficial effects:

[0024] 1. This invention is the first to use synthetic biology and genetic engineering technology to construct a recombinant Corynebacterium glutamicum that can be fermented to produce PNAG. It can utilize inexpensive carbon and nitrogen sources, is suitable for high-density fed-batch fermentation, and reduces the production cost of PNAG.

[0025] 2. Corynebacterium glutamicum is a widely used industrial microorganism with extremely high biosafety and batch-to-batch fermentation stability, laying the foundation for stable fermentation of PNAG;

[0026] 3. The *Corynebacterium glutamicum* strain constructed in this invention does not produce endogenous polysaccharides, and the PNAG synthesized from it has high purity. The fermentation cycle of strain *C. glutamicum* pEC-IcaABCD-pXMJ19-glmSMU is short. Three parallel measurements were set up for yield and glucose consumption rate determination, and the average value of the results was taken. The PNAG yield after 42 hours of fermentation can reach 6.82±0.5 g / L; the PNAG prepared by strain *C. glutamicum* pEC-IcaABCD-pXMJ19-glmSMU is less prone to precipitation, and the yield after 72 hours of fermentation can reach 3.2±0.5 g / L. Attached Figure Description

[0027] Figure 1 Metabolic pathways and related enzymes in the production of PNAG by recombinant Corynebacterium glutamicum.

[0028] Figure 2 : Batch fermenter production of recombinant Corynebacterium glutamicum PNAG.

[0029] Figure 3 The process for purifying PNAG from the fermentation broth of Corynebacterium glutamicum.

[0030] Figure 4 NMR spectroscopy image of PNAG produced by recombinant Corynebacterium glutamicum. Detailed Implementation

[0031] Strain: Corynebacterium glutamicum ATCC 13032, plasmids: pXMJ19 and pEC-XK99E, both of which are commercially available plasmids.

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

[0033] BHI: Brain and heart extract 37g / L, sorbitol 91g / L.

[0034] Fermentation medium: glucose 40 g / L, corn steep liquor powder 20 g / L, (NH4)2SO4 30 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MgSO4 25 g / L, MOPS (3-morpholinopropanesulfonic acid) 42 g / L.

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

[0036] NMR identification of PNAG: The lyophilized sample was dissolved in 5M hydrochloric acid, then adjusted to neutral with sodium hydroxide, NaCl was removed by dialysis, water molecules were removed by concentration, and then deuterium oxide was added. The H of PNAG was then measured. 1 Spectrum.

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

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

[0039] The Corynebacterium glutamicum expression plasmid pEC-XK99E was double-digested with EcoRI / KpnI. The IcaA, IcaB, IcaC, and IcaD gene fragments were ligated into the digested pEC-XK99E in one step using the Gibson Assembly kit. The resulting recombinant plasmid was named pECXK99E-IcaABCD.

[0040] The plasmid pECXK99E-IcaABCD was electroporated into *Corynebacterium glutamicum* ATCC13032 using an electroporator. The electroporation conditions were 1.5 kV, 5 ms (electroporation cuvette width 1 mm), repeated once. Recombinant bacteria were screened on BHI plates containing 25 mg / L kanamycin and named *C. glutamicum* pEC-IcaABCD.

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

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

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

[0044] *Pseudomonas putida* KT2440 cells were inoculated into 3 ml LB broth and cultured at 30°C and 220 rpm for 24 h. 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 *Pseudomonas putida* genomic DNA 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 using a PCR amplification system and program. Restriction enzyme sites were selected to digest plasmid pXMJ19, obtaining linear plasmid pXMJ19. Gibson assembly was performed using the amplified fragments glmU, glmS, and glmM with linear plasmid pXMJ19. The Gibson assembly reaction system was transformed into JM109 competent cells. Transformants were selected for plasmid sequencing. After sequence alignment, the recombinant plasmid pXMJ19-glmU-glmM-glmS was successfully constructed. It was then electroporated into *C. glutamicum* pEC-IcaABCD constructed in Example 1 and *C. glutamicum* pEC-IcaACD constructed in Example 2, respectively. The resulting strains were named *C. glutamicum* pEC-IcaABCD-pXMJ19-glmSMU* and *C. glutamicum* pEC-IcaACD-pXMJ19-glmSMU*, respectively.

[0045] Table 1 Primers and Sequences

[0046]

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

[0048] The recombinant Corynebacterium glutamicum pEC-IcaABCD-pXMJ19-glmSMU and recombinant Corynebacterium glutamicum pEC-IcaACD-pXMJ19-glmSMU single clones constructed in Example 3 were inoculated into 5 ml of BHI medium and cultured overnight at 200 rpm and 30°C. After 10 h, they were transferred to 250 ml Erlenmeyer flasks (containing 25 ml of fermentation medium) at a 1% inoculation rate. They were then cultured at 28°C and 200 rpm for 10 h. Finally, they were inoculated into 5 L fermenters at a 10% inoculation rate and fermented at 30°C for 72 h. During the first 18 h of fermentation, the glucose content in the fermenter was maintained at 10-15 g / L by feeding glucose, and the pH was controlled to neutral by feeding ammonia. Samples were taken periodically to determine the yield of soluble PNAG in the fermentation broth. Figure 2 As shown, strain pEC-IcaABCD-pXMJ19-glmSMU can produce 6.82 g / L of PNAG after 42 h of fermentation. Over time, PNAG in the fermentation broth gradually forms a solid precipitate, resulting in a decrease in the content of soluble PNAG in the fermentation broth. Strain pEC-IcaACD-pXMJ19-glmSMU can produce 3.2 g / L of PNAG after 72 h of fermentation, and both strains remain in a soluble state.

[0049] Example 5: Extraction of PNAG

[0050] Reference Figure 3The extraction process involved collecting the fermentation broth prepared according to the method in Example 4, treating it with 2% NaOH for 1 hour to remove proteins, adjusting the pH to neutral with 2% hydrochloric acid, centrifuging to remove impurities, and precipitating with 4 times the volume of ethanol to obtain crude PNAG. The PNAG was then washed three times with ethanol and ultrapure water to obtain pure PNAG. The obtained PNAG was subjected to NMR analysis using an AVANCE NEO 600MHz Bruker NMR spectrometer and standard MestReNova acquisition software. The polysaccharide solution was concentrated to a volume of 1-2 mL using a centrifuge tube (Amicon® Ultra filter, 3kDa MWCO), followed by the addition of 99.9% D₂O (D113904Aladdin) to dilute the H₂O in the solution. This step was repeated 9 times. Spectra were recorded at 25°C, and one-dimensional 1H-NMR spectra (600MHz) were recorded in normal acquisition mode through 1024 scans. The results are as follows: Figure 4 As shown, the NMR results indicate that the purified product is single, without impurity peaks, with a flat baseline, and the PNAG main signal is obvious, proving that the sample has high purity.

[0051] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Recombinant Corynebacterium glutamicum, characterized in that, Gene combinations expressing (a) or (b): (a) Genes IcaA, IcaB, IcaC, and IcaD; (b) Genes IcaA, IcaC, and IcaD; It also expresses the UDP-N-acetylglucosamine synthesis-related gene: glutamine-fructose-6-phosphate aminotransferase GlmS. 、 Glucose phosphotransferase GlmM and UDP-N-acetylglucosamine pyrophosphorylase / glucose-1-phosphoacetyltransferase bifunctional enzyme GlmU.

2. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that, Vectors expressing the gene combination or expressing GlmS, GlmM, and GlmU include pXMJ19, pECXK99E, pEC-XT99A, pEKEx1, pEKEx2, pVWEx1, pVWEx2, pZ8-1, pECTAC-K99, or pAPE12.

3. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that, The IcaA, IcaC, and IcaD genes were expressed using pEC-XK99E as a vector, and the GlmS, GlmM, and GlmU genes were expressed using pXMJ19 as a vector.

4. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that, The IcaA, IcaB, IcaC and IcaD genes were expressed using pEC-XK99E as a vector, and the GlmS, GlmM and GlmU genes were expressed using pXMJ19 as a vector.

5. The recombinant Corynebacterium glutamicum according to any one of claims 1 to 4, characterized in that, Corynebacterium glutamicum ATCC13032 was used as the host cell.

6. A method for preparing poly-N-acetylglucosamine, characterized in that, Fermentation is carried out using the recombinant Corynebacterium glutamicum according to any one of claims 1 to 5.

7. The method according to claim 6, characterized in that, The fermentation is carried out at 28~30℃ for at least 16 hours.

8. The use of the recombinant Corynebacterium glutamicum according to any one of claims 1 to 5, or the method according to any one of claims 6 to 7, in the preparation of PNAG or PNAG-containing pharmaceutical chemicals.

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