An Escherichia coli for producing high-yield chondroitin sulfate and a production method thereof

Abstract

The present invention discloses an Escherichia coli for producing high-yield fructose chondroitin and its production method, belonging to the technical field of microbiology. Based on Escherichia coli K4, the present invention mutates the encoding genes of key enzymes in the fructose chondroitin synthesis pathway to construct a strain with improved fructose chondroitin synthesis ability. The yield of fructose chondroitin of this strain reaches 10 g / L in a 5 L fermenter and 15 - 20 g / L in a 100 L fermenter. The obtained end products have uniform structures and stable quality, effectively reducing production costs and the risk of environmental pollution, and are suitable for large-scale industrial production applications.

Description

Technical Field

[0001] The present invention relates to an Escherichia coli for producing high-yield fructose chondroitin and a production method thereof, belonging to the field of microbial technology. Background Art

[0002] Chondroitin sulfate is a sulfated glycosaminoglycan widely present in animal tissues. The chondroitin backbone is formed by the linear alternating connection of glucuronic acid (GlcUA) and N-acetyl-D-galactosamine (GalNAc) via 1,3-bonds and 1,4-bonds, respectively. Currently, chondroitin sulfate is mainly directly extracted from animal tissues. Due to problems such as the long production cycle and scattered distribution of raw materials, the cumbersome and long process steps, and the instability of product quality and yield caused by raw materials, using the microbial fermentation method with mild conditions and rich raw materials to produce chondroitin sulfate is a more attractive alternative. Chondroitin analogs are attached to the cell walls of certain prokaryotic microorganisms in the form of capsular polysaccharides. For example, the capsular polysaccharide of Escherichia coli K4 contains a chondroitin backbone modified with fructose groups. Using the K4 strain to ferment and synthesize fructose chondroitin and then removing the fructose groups by methods such as acid hydrolysis to prepare chondroitin is a new chondroitin synthesis strategy, which has the characteristics of environmental protection and high efficiency.

[0003] In recent years, researchers at home and abroad have systematically studied the production of fructose chondroitin by microbial fermentation from aspects such as biochemical engineering and metabolic engineering. The patent with the publication number CN104974973B discloses the technology of using a recombinant Escherichia coli strain to ferment and produce chondroitin. By introducing, in Escherichia coli capable of producing UDP-glucuronic acid such as K5, genes derived from Escherichia coli K4 strain using a vector, kfoA and kfoC genes, where kfoA gene encodes UDP-glucose-4-epimerase, kfoC gene encodes chondroitin synthase, and the vector contains an antibiotic resistance gene, and the chondroitin yield reaches 52.6 mg / L. The patent with the publication number CN102869782B discloses the technology of using recombinant Escherichia coli K12 or Escherichia coli B or Xanthomonas campestris to ferment and produce chondroitin. By using a vector to introduce kfoD , orf3 ( kfoI ), kfoE , or orfI ( kfoH ) in one or more functional genes of Escherichia coli or Xanthomonas campestris, kfoA , kfoC, kfoF , kfoG , kfoB ,kpsF , kpsE , kpsD , kpsU , kpsC , kpsS , kpsM or kpsT One or more genes among, and culturing the recombinant strain in a medium with glycerol as a carbon source for 24 - 72 hours to secrete 15 - 50 g / L of non-fucosylated chondroitin from the cells. The patent with the publication number CN103228781B discloses using genetic engineering means to inactivate the glycosyltransferase gene that adds fucose residues to the chondroitin backbone kfoE , obtaining recombinant derivatives of Escherichia coli K4 (ΔkfoE / kan R , DSM23578 and ΔkfoE, DSM23644) that can directly produce a chondroitin backbone without fucosylation. However, due to the plasmid carried affecting the growth of the bacterial cells, the instability of plasmid replication also causes unstable chondroitin production, and the chondroitin production of this strain has not been reported. The patent with the publication number CN115895987B increases glmS and glmM gene copy numbers in the Escherichia coli genome, glmS encoding aminotransferase, glmM encoding phosphoglucosamine mutase, thereby strengthening the synthesis of the precursor UDP-GalNAc, solving the problem of plasmid instability, increasing the production of fucosylated chondroitin. Using the recombinant strain for fermentation in a 7.5 L fermenter, the fucosylated chondroitin production reaches 7.12 g / L. However, neither the fucosylated chondroitin nor the chondroitin backbone without fucosylation can meet the industrial production requirements and cannot replace the direct extraction method. Further increasing the production of chondroitin or its analogue fucosylated chondroitin has become an urgent problem to be solved. SUMMARY OF THE INVENTION

[0004] Aiming at the problems of plasmid instability and low production of chondroitin or its analogues in the existing recombinant strains containing plasmids, the present invention provides a plasmid-free strain with high production of chondroitin backbone or its analogues.

[0005] The present invention provides an engineered Escherichia coli strain expressing one or more of the following mutants: phosphotransferase mutant, phosphoglucosamine mutase mutant, glutamate synthase mutant, NADH oxidoreductase mutant, and lipid transport system substrate-binding protein mutant.

[0006] In one embodiment, the engineered Escherichia coli strain uses Escherichia coli K4 (ATCC 23502) as the starting strain and expresses one or more of the following mutants: phosphotransferase mutant, phosphoglucosamine mutase mutant, glutamate synthase mutant, NADH oxidoreductase mutant, and lipid transport system substrate-binding protein mutant.

[0007] In one embodiment, the phosphotransferase mutant contains the amino acid sequence shown in SEQ ID NO.1; the phosphoglucosamine mutase mutant contains the amino acid sequence shown in SEQ ID NO.3; the glutamate synthase mutant contains the amino acid sequences shown in SEQ ID NO.5 and SEQ ID NO.7; the NADH oxidoreductase mutant contains the amino acid sequence shown in SEQ ID NO.9; the lipid transport system substrate-binding protein mutant contains the amino acid sequence shown in SEQ ID NO.11.

[0008] In one embodiment, the engineered Escherichia coli strain is Escherichia coli DH001, which was deposited with the General Microbiological Center of the China National Culture Collection Center on September 26, 2024, with the deposit number: CGMCC No. 32081, the deposit address is Beijing, China, and the taxonomic name is Escherichia coli.

[0009] In one embodiment, Escherichia coli DH001 expresses the protein shown by one or more of the amino acid sequences in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11.

[0010] In one embodiment, Escherichia coli DH001 has the genes with nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12.

[0011] The present invention also provides a composition containing the Escherichia coli DH001.

[0012] In one embodiment, the composition includes, but is not limited to, a direct vat set containing live cells of Escherichia coli DH001.

[0013] The present invention also provides a method for improving the chondroitin fructose production ability of Escherichia coli, including (a) or (b):

[0014] (a) Express one or more of the following mutants in Escherichia coli: phosphoenolpyruvate (PEP) phosphotransferase mutant, phosphoglucosamine mutase mutant, glutamine synthetase large subunit mutant, glutamine synthetase small subunit mutant, NADH oxidoreductase mutant, and lipid transport system substrate-binding protein mutant;

[0015] (b) Mutate the coding sequences of one or more of the following proteins in Escherichia coli: PEP-dependent phosphotransferase glvB, phosphoglucosamine mutase glmM, glutamine synthetase gltB and gltD, NADH oxidoreductase hcr, and lipid transport system substrate-binding protein mlaC.

[0016] In one embodiment, the method is to perform in situ replacement of the glvB , glmM , gltB , gltD , hcr and mlaC genes on the Escherichia coli genome.

[0017] In one embodiment, the Escherichia coli includes, but is not limited to, Escherichia coli K4 (ATCC 23502).

[0018] In one embodiment, the mutation is to mutate the arginine (R) at position 224 of PEP-dependent phosphotransferase (glvB) to cysteine (C), and the amino acid sequence of the obtained mutant is as shown in SEQ ID NO.1.

[0019] In one embodiment, the mutation is to mutate the alanine (A) at position 128 of phosphoglucosamine mutase (glmM) to threonine (T), and the amino acid sequence of the obtained mutant is as shown in SEQ ID NO.3.

[0020] In one embodiment, the mutation is to mutate the proline (P) at position 223 of the glutamine synthetase large subunit gltB to serine (S), and the amino acid sequence of the obtained mutant is as shown in SEQ ID NO.5.

[0021] In one embodiment, the mutation is to mutate the proline (P) at position 190 of the glutamine synthetase small subunit gltD to serine (S), and mutate the glutamate (E) at position 286 to lysine (K), and the amino acid sequence of the obtained mutant is as shown in SEQ ID NO.7.

[0022] In one embodiment, the mutation is the substitution of threonine (T) at position 174 of NADH oxidoreductase hcr with isoleucine (I), and the amino acid sequence of the obtained mutant is as shown in SEQ ID NO.9.

[0023] In one embodiment, the mutation is the substitution of alanine (A) at position 88 of the phospholipid transport system substrate-binding protein mlaC with valine (V), and the substitution of proline (P) at position 140 with serine (S), and the amino acid sequence of the obtained mutant is as shown in SEQ ID NO.11.

[0024] In one embodiment, the nucleotide sequence of the gene glmB encoding the phosphotransferase mutant is as shown in SEQ ID NO.2; the nucleotide sequence of the gene glmM encoding the phosphoglucosamine mutase mutant is as shown in SEQ ID NO.4. The nucleotide sequence of the gene gltB encoding the large subunit mutant of glutamate synthase is as shown in SEQ ID NO.6; the nucleotide sequence of the gene gltD encoding the small subunit mutant of glutamate synthase is as shown in SEQ ID NO.8; the nucleotide sequence of the gene hcr encoding the NADH oxidoreductase mutant is as shown in SEQ ID NO.10; the nucleotide sequence of the gene mlaC encoding the phospholipid transport system substrate-binding protein mutant is as shown in SEQ ID NO.12.

[0025] In one embodiment, the starting strain of the recombinant Escherichia coli includes but is not limited to Escherichia coli K4.

[0026] The present invention also provides a method for fermenting and producing fructosaminoglycan using the Escherichia coli DH001.

[0027] In one embodiment, the method is to ferment the Escherichia coli in a medium for 40 h to 48 h, and collect the fructosaminoglycan in the fermentation product.

[0028] In one embodiment, the carbon source in the medium includes but is not limited to glucose.

[0029] In one embodiment, the nitrogen source in the medium is an organic nitrogen source and / or an inorganic nitrogen source; optionally, the organic nitrogen source can be yeast extract powder, and the inorganic nitrogen source can be ammonium sulfate.

[0030] In one embodiment, the nitrogen source is yeast extract powder and ammonium sulfate.

[0031] In one embodiment, the medium also contains inorganic salts and trace elements necessary for the growth of the strain, including but not limited to phosphorus, magnesium, iron, etc.

[0032] In one embodiment, the inorganic salts and trace elements are selected from one or more of potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and ferrous sulfate heptahydrate.

[0033] In one embodiment, the culture medium contains: glucose, yeast extract powder, ammonium sulfate, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate.

[0034] In one embodiment, the fermentation is carried out at 30 - 37 °C.

[0035] In one embodiment, glucose is supplemented during the fermentation process.

[0036] In one embodiment, the method may further include the step of recovering fructose chondroitin from the fermentation broth.

[0037] In one embodiment, purification is carried out after the recovery.

[0038] In one embodiment, the recovery can be carried out using centrifugation, filtration, ultrafiltration, ethanol precipitation, and various forms of chromatography such as gel filtration chromatography (molecular sieve) method, adsorption chromatography, ion exchange chromatography or a combination thereof for recovery and purification; the recovery step and the purification step can be carried out partially sequentially continuously or discontinuously, or can be carried out simultaneously or by combining into one step, but the step manner is not limited thereto.

[0039] The present invention also provides the use of Escherichia coli DH001, or the composition, or the method in the preparation of fructose chondroitin or its related products.

[0040] In one embodiment, the related products include but are not limited to chondroitin and chondroitin sulfate.

[0041] Beneficial effects:

[0042] (1) By mutating the key enzyme genes (phosphoenolpyruvate-dependent phosphotransferase glvB, phosphoglucosamine mutase glmM, glutamate synthase gltB and gltD, NADH oxidoreductase hcr, and lipid transport system substrate-binding protein mlaC) in the fructose chondroitin synthesis pathway on the basis of Escherichia coli K4 (ATCC23502), the present invention constructs a strain DH001 with improved fructose chondroitin synthesis ability.

[0043] (2) The strain constructed by the present invention has excellent production performance and genetic stability, and the yield of fructose chondroitin at the 5 L fermenter level remains above 8 g / L after 8 passages.

[0044] (3)The present invention provides a method for fermenting and producing fructosyl chondroitin using strain DH001. In a 5 L fermenter, the yield of fructosyl chondroitin can reach 10 g / L. When the fermentation scale is enlarged to a 100 L fermenter for high-density fermentation, the production cycle is reasonably controllable, and the yield of fructosyl chondroitin reaches 15 - 20 g / L. The obtained end product has a uniform structure and stable quality, effectively reducing the production cost and the risk of environmental pollution, and is suitable for large-scale industrial production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a synthetic pathway diagram of fructosyl chondroitin (K4CPS).

[0046] Figure 2 It is the chondroitin yield of the recombinant strain in shake flask fermentation in Example 2.

[0047] Figure 3 It is the sequencing result of the mutant gene of the recombinant strain FCH5 in Example 2; among them, a, glvB R224C ; b, glmM A128T ; c, gltB P223S ; d, hcr T174I ; e, gltD P190S / E286K ; f, mlaC A88V / P140S .

[0048] Figure 4 It is the HPLC analysis chromatogram of chondroitin disaccharide after enzymatic hydrolysis of sodium chondroitin standard.

[0049] Figure 5 It is the HPLC analysis chromatogram of chondroitin disaccharide after acid hydrolysis and enzymatic hydrolysis of the fermentation product in Example 4.

[0050] Figure 6 It is the monosaccharide composition analysis chromatogram of fructosyl chondroitin in Example 4; among them, a, monosaccharide standard; b, sample. DETAILED DESCRIPTION OF THE INVENTION

[0051] (I) Sequence information:

[0052] (1) SEQ ID NO.1 is the amino acid sequence of the glvB mutant;

[0053] (2) SEQ ID NO.2 is the glvB nucleotide sequence of the mutant gene;

[0054] (3) SEQ ID NO.3 is the amino acid sequence of the glmM mutant;

[0055] (4) SEQ ID NO.4 is the glmM nucleotide sequence of the mutant gene;

[0056] (5) SEQ ID NO.5 is the amino acid sequence of the gltB mutant;

[0057] (6) SEQ ID NO.6 is gltB the nucleotide sequence of the mutant gene;

[0058] (7) SEQ ID NO.7 is the amino acid sequence of the gltD mutant;

[0059] (8) SEQ ID NO.8 is gltD the nucleotide sequence of the mutant gene;

[0060] (9) SEQ ID NO.9 is the amino acid sequence of the hcr mutant;

[0061] (10) SEQ ID NO.10 is hcr the nucleotide sequence of the mutant gene;

[0062] (11) SEQ ID NO.11 is the amino acid sequence of the mlaC mutant;

[0063] (12) SEQ ID NO.12 is mlaC the nucleotide sequence of the mutant gene;

[0064] (13) SEQ ID NO.13 is the sgRNA sequence for knocking out the gene in Escherichia coli glvB ;

[0065] (14) SEQ ID NO.14 is the sgRNA sequence for integrating glvB the mutant gene;

[0066] (15) SEQ ID NO.15 is the sgRNA sequence for knocking out the gene in Escherichia coli glmM ;

[0067] (16) SEQ ID NO.16 is the sgRNA sequence for integrating glmM the mutant gene;

[0068] (17) SEQ ID NO.17 is the sgRNA sequence for knocking out the gene in Escherichia coli gltB ;

[0069] (18) SEQ ID NO.18 is the sgRNA sequence for integrating gltB the mutant gene;

[0070] (19) SEQ ID NO.19 is the sgRNA sequence for knocking out the gene in Escherichia coli gltD ;

[0071] (20) SEQ ID NO.20 is the sgRNA sequence for integratinggltD sgRNA sequence of the mutant gene;

[0072] (21) SEQ ID NO.21 is the sgRNA sequence for knocking out the gene in Escherichia coli hcr ;

[0073] (22) SEQ ID NO.22 is the sgRNA sequence for integrating hcr the mutant gene;

[0074] (23) SEQ ID NO.23 is the sgRNA sequence for knocking out the gene in Escherichia coli mlaC ;

[0075] (24) SEQ ID NO.24 is the sgRNA sequence for integrating mlaC the mutant gene.

[0076] Unless otherwise specified, amino acid mutants are named using the accepted IUPAC single-letter amino acid abbreviations. For example, R224C represents the mutant obtained by mutating arginine (R) at position 224 to cysteine (C) on the basis of the wild type.

[0077] (II) Measurement method:

[0078] Yield detection of fructosylated chondroitin: According to the analysis method on page 1595 of Part II of the Chinese Pharmacopoeia (2020 Edition), take 10 mL of the fermentation broth into a 50 mL centrifuge tube, add pure glacial acetic acid to adjust the pH to 3.8, place it in a water bath at 85 °C for acid hydrolysis for 4 h. After the acid hydrolysis is completed, cool it to room temperature, use 6 M sodium hydroxide (NaOH) to adjust the pH to between 7.0 and 8.0, and transfer it to a 25 mL volumetric flask for volume fixation. Take 100 μL of the acid-hydrolyzed sample and mix it with 100 μL of chondroitin sulfate ABC enzyme (SIGMA-Aldrich, C2905-10UN), add 800 μL of tris(hydroxymethyl)aminomethane buffer, mix well, place it in a water bath at 37 °C for reaction for 1 h. After the enzymatic hydrolysis is completed, place it in a boiling water bath for 5 minutes, cool it with cold water and then centrifuge. Take 20 μL of the supernatant and load it onto a Hypersil SAX chromatographic column (Waters) for HPLC analysis, and the detection wavelength is 232 nm. Quantitative analysis is carried out by the external standard method. The sum of the peak areas of ΔDi-0S, ΔDi-4S, and ΔDi-6S after enzymatic hydrolysis of the chondroitin sulfate sodium standard product (National Institutes for Food and Drug Control, 4386 / 10 / 12, batch number 140792-202003) at 10 g / L is used as the control product peak area; the peak area of ΔDi-0S after enzymatic hydrolysis of the sample is used as the sample peak area, and the chondroitin content is calculated (sample peak area / control product peak area × standard product concentration 10 g / L × dilution factor 2.5); when calculating the fructosylated chondroitin content, multiply the chondroitin content by a coefficient of 1.43 (coefficient = molecular weight of fructosylated chondroitin disaccharide ÷ molecular weight of chondroitin disaccharide, that is, 540 ÷ 378 = 1.43).

[0079] Glucose determination method: After centrifuging the fermentation broth at 10000 r / min for 5 min, take the supernatant and dilute it 20 times, and use a biosensor analyzer M-100 (Sillerman Technology) for determination.

[0080] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products, or can be prepared by known methods. The operations carried out are known in the art, or are carried out according to the user manuals of commercially available products.

[0081] Example 1 Construction of recombinant strains

[0082] The Escherichia coli K4 (ATCC23502) was genetically edited using the Crispr-Cas9 system, and the glvB codon encoding the 224th amino acid of the gene was mutated from CGT to TGT to obtain the Escherichia coli FCH1 strain; then the glmM codon encoding the 128th amino acid of the gene was mutated from GCC to ACC to obtain the Escherichia coli FCH2 strain; then the gltB codon encoding the 223rd amino acid of the gene was mutated from CCG to TCG, and the gltDThe codon CCG encoding the 190th amino acid of the gene was mutated to TCG, and gltD the codon GAA encoding the 286th amino acid of the gene was mutated to AAA to obtain Escherichia coli strain FCH3; then hcr the codon ACC encoding the 174th amino acid of the gene was mutated to ATC to obtain Escherichia coli strain FCH4; then mlaC the codon GCT encoding the 88th amino acid of the gene was mutated to GTT, and the codon CCG encoding the 140th amino acid was mutated to TCG to obtain Escherichia coli strain FCH5. The specific steps are as follows:

[0083] (1)Preparation of Escherichia coli K4 / pCas competent cells

[0084] 50 ng of pCas plasmid (purchased from Addgene) was transformed into Escherichia coli K4 competent cells by heat shock at 42 °C for 90 sec, and spread on solid LB medium (containing 100 mg / L kanamycin (kana)), and incubated overnight at 30 °C in an incubator to obtain Escherichia coli K4 / pCas strain. A single colony was picked and inoculated into 5 mL of LB liquid medium (100 mg / L kana), and 50 μL of 1 M L-arabinose (L-Ara) solution was added, and cultured overnight at 30 °C and 180 rpm to obtain a seed solution. 500 μL of the seed solution was inoculated into 50 mL of LB liquid medium (100 mg / L kana), 500 μL of 1 M L-Ara was added, and cultured at 30 °C and 180 rpm until the OD 600 was 0.4 - 0.6, ice-bathed for 5 min, the bacterial solution was poured into a 50 mL sterile centrifuge tube in a laminar flow hood, centrifuged at 5000 rpm at 8 °C for 10 min, the supernatant was discarded, 20 mL of pre-cooled 100 mM CaCl2 was added to resuspend the cells, ice-bathed for 30 min, centrifuged at 5000 rpm at 8 °C for 10 min, the supernatant was discarded, 0.5 mL of pre-cooled 100 mM CaCl2 solution and 60% glycerol were added to resuspend the cells, and 50 μL of each was aliquoted into sterile 1.5 mL EP tubes and stored at -80 °C.

[0085] (2)Knockout of Escherichia coli endogenous gene glvB

[0086] Obtain the upstream homologous arm fragment. Amplification system: 1 μL of 50 ng / μL Escherichia coli K4 genomic DNA as template, 1 μL each of 10 μM primers glvB-F1 and glvB-R1 (Table 1), 25 μL of 2×PrimeSTAR MAX Premix, 22 μL of ddH2O. PCR reaction conditions: Pre-denaturation at 98 °C for 5 min, then enter the temperature cycle: 98 °C for 10 sec; 57 °C for 15 sec; 72 °C for 1 min, for a total of 30 cycles; termination temperature is 4 °C. After PCR amplification, verify the correct PCR product by 1% agarose gel electrophoresis, and purify the upstream homologous arm fragment using the PCR Purification Kit.

[0087] The method for obtaining the downstream homologous arm fragment refers to that of the upstream homologous arm fragment, and the primers are glvB-F2 and glvB-R2 (Table 1). Use Overlap PCR to obtain Donor DNA. Amplification system: 1 μL each of 50 ng / μL upstream and downstream homologous arm fragments as template, 1 μL each of 10 μM primers glvB-F1 and glvB-R2, 25 μL of 2×PrimeSTAR MAX Premix, 22 μL of ddH2O. PCR reaction conditions: Pre-denaturation at 98 °C for 5 min, then enter the temperature cycle: 98 °C for 10 sec; 57 °C for 15 sec; 72 °C for 1 min, for a total of 30 cycles; termination temperature is 4 °C.

[0088] According to the CRISPR / Cas 9 gene editing principle, use the specific guiding SgRNA sequence shown in SEQ ID NO.13, which is synthesized by Suzhou Genewiz Biotechnology Co., Ltd. and ligated to the pTarget plasmid to obtain the pTarget-ΔglvB plasmid.

[0089] Take the competent cells carrying the pCas plasmid from -80 °C, let them stand on ice for 2 min, add 500 ng of Donor DNA and 50 ng of pTarget-ΔglvB, mix gently, and let them stand on ice for 30 min. After standing, perform heat shock at 42 °C for 90 sec and then let them stand on ice for 2 min. After standing, add 1 mL of pre-cooled LB medium to the competent cells, incubate at 30 °C and 180 rpm on a shaker for 2 h, take 100 μL and spread it on the LB solid medium (50 mg / L streptomycin (strep) and 100 mg / L kana), and culture overnight at 30 °C.

[0090] Pick several monoclonal colonies on the transformation plate as templates, and use glvB-TF / glvB-TR as primers to perform colony PCR to verify positive clones. The amplified fragment size is 1762 bp.

[0091] Inoculate the positive clones into 5 mL of LB liquid medium (100 mg / L Kana), add IPTG with a final concentration of 0.1 mM to induce the elimination of the pTarget-ΔglvB plasmid by the pCas plasmid, culture at 30 °C and 180 rpm for 16 h, streak on LB solid medium (100 mg / L kana), culture overnight at 30 °C, pick single colonies (only pick half of the single colony size here) and number them, inoculate according to the numbers on the corresponding numbered areas of LB solid medium (50 mg / L strep), culture overnight at 30 °C, and the single colonies that cannot grow are the strains with successful elimination of the pTarget-ΔglvB plasmid.

[0092] (3)Integrate mutant genes glvB R224C

[0093] Prepare the competent cells carrying the pCas plasmid from the pTarget-ΔglvB strains prepared in step (2) again.

[0094] Using Escherichia coli K4 genomic DNA as a template, amplify the upstream and downstream homologous arms using primer pairs glvB-F3 / glvB-R3 and glvB-F4 / glvB-R4 respectively; synthesized by Genewiz Suzhou Co., Ltd. glvB R224C Gene fragment (SEQ ID NO.2); with the upstream and downstream homologous arms, glvB R224C Using the gene sequence as a template, and using glvB-F3 / glvB-R4 as a template, perform Overlap PCR to fuse the three fragments to obtain Donor DNA. The PCR system and method refer to step (2).

[0095] According to the CRISPR / Cas 9 gene editing principle, use the specific guiding SgRNA sequence shown in SEQ ID NO. 14, synthesize and ligate it to the pTarget plasmid by Genewiz Suzhou Co., Ltd. to obtain the pTarget-glvB R224C plasmid.

[0096] The rest of the operations refer to step (2). The positive clone identification primers are glvB-TF / glvB-TR, and the amplified fragment size is 3073 bp.

[0097] For subsequent gene editing, refer to the above operation process to edit the glmM, gltB, gltD, hcr, and mlaC genes. The relevant primers are shown in Table 1. The amplified fragment sizes for the glmM, gltB, gltD, hcr, and mlaC gene knockout / mutation gene integration positive clone PCR identification are 1687 / 2721 bp, 1167 / 2427 bp, 1734 / 2808 bp, 1371 / 2340 bp, and 1180 / 1816 bp, respectively.

[0098] After all gene editing is completed, eliminate the pCas plasmid to complete the strain construction.

[0099] Table 1 Primers and Sequences

[0100]

[0101] Example 2 Shake Flask Fermentation of Recombinant Strains FCH1, FCH2, FCH3, FCH4, and FCH5

[0102] Perform shake flask fermentation on the Escherichia coli FCH1, FCH2, FCH3, FCH4, and FCH5 strains constructed in Example 1. Pick monoclonal colonies of each recombinant strain and inoculate them into 250 mL shake flasks with corner baffles containing 50 mL of seed medium (formula shown in Table 2), and culture at 37 °C and 200 rpm for 10 h. Transfer the seed liquid to 500 mL shake flasks with corner baffles containing 100 mL of fermentation medium (formula shown in Table 3) at an inoculation amount of 1% (v / v). The fermentation conditions are: rotation speed 200 rpm, temperature 37 °C, initial pH controlled at 7.5, and ferment for 48 h. Repeat each strain three times. After fermentation is completed, collect the fermentation broth and determine the yield of fructosaminoglycan by HPLC method. Use Escherichia coli K4 (ATCC 23502) as a control.

[0103] Table 2 Seed Medium Formula

[0104]

[0105] Table 3 Fermentation Medium Formula

[0106]

[0107] The fructosaminoglycan production results of the shake flask fermentation of each strain are as Figure 2As shown, after 48 h of fermentation, the yields of fructosyl chondroitin of the starting strain K4 and the recombinant strains FCH1, FCH2, FCH3, FCH4, and FCH5 were 0.156 g / L, 0.275 g / L, 0.498 g / L, 0.749 g / L, 0.824 g / L, and 0.912 g / L, respectively. The yields of the recombinant strains were all higher than that of the starting strain. Among them, the yield of FCH5 was 484.6% higher than that of the starting strain.

[0108] Example 3 Genotype analysis of recombinant strains

[0109] The genomic analysis of the Escherichia coli FCH5 strain constructed in Example 1 was carried out. The specific steps were as follows:

[0110] (1) Polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP)

[0111] The genome of strain FCH5 was extracted. PCR amplifications were carried out with primer pairs glvB-TF / glvB-TR, glmM-TF / glmM-TR, gltB-TF / gltB-TR, gltD-TF / gltD-TR, hcr-TF / hcr-TR, and mlaC-TF / mlaC-TR, respectively. The PCR system and method referred to step (2) of Example 1. After the PCR amplification products were denatured at high temperature and ice-bathed, SSCP electrophoresis was carried out using 8% non-denaturing polyacrylamide gel. The synthesized mutant gene was used as the positive control, the original wild-type gene was used as the negative control, and water was used as the blank control. Since there were single or multiple base mutations in the mutant gene, resulting in different conformations from the original gene, their mobilities in SSCP electrophoresis were different. Finally, the fragment bands were inconsistent with the positions of the negative control fragments and were consistent with the positions of the positive control, indicating that it was a strain with successful allelic replacement.

[0112] (2) Sequencing

[0113] The PCR amplification products in step (1) were ligated to the pMD19-T vector for sequencing. By aligning the mutant bases in the sequences, the successful allelic replacement of the strain was verified. The results were as Figure 3 shown.

[0114] The strain FCH5 with correct genotype editing verified above was subjected to strain preservation and named DH001.

[0115] Example 4 Fermentation of recombinant strains in a 5 L fermenter

[0116] The recombinant Escherichia coli DH001 was fermented in a 5 L fermenter to produce fructosyl-chondroitin, with Escherichia coli K4 (ATCC23502) as the control. The specific steps were as follows:

[0117] Escherichia coli K4 (ATCC 23502) and the recombinant strain DH001 were separately inoculated into LB liquid medium and activated overnight at 37 °C. They were then inoculated into a 500 mL shake flask with angled baffles containing 100 mL of the seed medium shown in Table 2 at an inoculation amount of 1% (v / v), and cultured at 37 °C and 200 rpm for 10 h. The seed liquid was transferred into a 5 L T&J Intelli-Ferm A bench-top fermenter (purchased from Shanghai Dibeier Co., Ltd.) containing the fermentation medium shown in Table 3 at an inoculation amount of 10% (v / v). 0.2 g / L of antifoaming agent was added, and fermentation was carried out for 48 h according to the control process shown in Table 4. Each strain was repeated three times. After fermentation was completed, the fermentation broth was collected, and the yield of fructosaminoglycan was determined by HPLC. The results are shown in Table 5.

[0118] Table 4 Fermentation control process

[0119]

[0120] Table 5 Fermentation results of fructosaminoglycan

[0121]

[0122] Example 5 Verification of the stability of the recombinant strain DH001

[0123] The recombinant strain DH001 was used for fructosaminoglycan fermentation according to Example 4. After fermentation was completed, an appropriate amount of the fermentation broth was taken, diluted with sterile water, and spread on an LB solid plate. It was cultured at 37 °C until single colonies grew. Single colonies were picked, identified by PCR, and then used for the next generation of fructosaminoglycan fermentation. Fermentation was carried out by continuous subculture for 8 generations, and the yield of fructosaminoglycan in the fermentation broth of each generation was determined. The results showed that the yield of fructosaminoglycan remained above 8 g / L after 8 subcultures.

[0124] Example 6 Fermentation of the recombinant strain DH001 in a 100 L fermenter

[0125] (1) Seed tank fermentation: After the recombinant Escherichia coli DH001 was activated in LB medium according to the method of Example 3, it was inoculated into a 30 L seed tank at an inoculation amount of 5%. The formula of the seed medium is shown in Table 6, and it was cultured for 8 - 10 h until the OD 600 reached 2.5 - 5.

[0126] Table 6 Formula of the seed tank medium

[0127]

[0128] (2) Fermentation in a 100 L fermenter

[0129] Transfer the seed liquid prepared in step (1) into a 100 L fermenter at an inoculation amount of 10%. The formula of the fermentation medium is shown in Table 7. The stirring rate is 300 rpm, the aeration rate is 1 vvm, the dissolved oxygen correction is 100%, the dissolved oxygen is controlled at 15%-30% throughout the process, the pH is controlled at 7.0-8.0 using 25% (w / v) ammonia water, the aeration rate is 1 vvm, and glucose with a mass fraction of 40%-60% is used as the carbon source for fed-batch feeding to make the glucose concentration in the fermentation system 1-2 g / L within the initial 12 h of fermentation. After 12 h of fermentation, the glucose concentration is controlled at 0.5-1 g / L in combination with the DO requirement. Ferment for 42-48 h according to the control process shown in Table 4 until the OD 600 reaches 110-135, then discharge the tank, collect the fermentation broth, and measure the yield of fructosaminoglycan.

[0130] Table 7 Formula of the fermentation medium

[0131]

[0132] (3)Detection of the yield of fructosaminoglycan

[0133] Detect the content of fructosaminoglycan in the fermentation broth by HPLC method. The chondroitin disaccharide chromatogram after enzymatic hydrolysis of sodium chondroitin sulfate standard is as Figure 4 shown, and the chondroitin disaccharide chromatogram after acid hydrolysis and enzymatic hydrolysis of the fermentation product fructosaminoglycan is as Figure 5 shown. After calculation, the content of chondroitin disaccharide in the fermentation broth is 12.15±1.67 g / L, and the yield of fructosaminoglycan is 17.37±2.39 g / L.

[0134] (4)Analysis of the monosaccharide composition of fructosaminoglycan

[0135] Use the pre-column derivatization HPLC method based on PMP (1-phenyl-3-methyl-5-pyrazolone) to determine the monosaccharide composition of fructosaminoglycan recovered and purified from the fermentation broth.

[0136] Hydrolysis: Dissolve 2 mg of fructosaminoglycan in 2 mL of 2 M trifluoroacetic acid (TFA), place it in a water bath at 100 °C for hydrolysis for 2 h, adjust the pH to 7.0 with 6 M NaOH, and transfer it to a 10 mL volumetric flask for constant volume.

[0137] Derivatization: Take 200 μL of the sample, add 100 μL of 0.3 M NaOH and 100 μL of 0.5 M PMP-methanol, vortex and mix evenly, place it in a water bath at 70 °C for hydrolysis for 30 min, cool to room temperature, add 100 μL of 0.3 M hydrochloric acid (HCl) to neutralize NaOH, and vortex and mix evenly.

[0138] Extraction: Add 1 mL of chloroform for extraction, centrifuge at 5000 rpm for 3 min, collect the supernatant, and repeat the extraction three times to remove as much excess PMP reagent as possible.

[0139] Detection: Load 10 μL of the sample onto HPLC. The mobile phase is 0.1 M ammonium acetate - acetonitrile (volume ratio 83:17), the column temperature is 35 °C, the detection wavelength is 254 nm, and the retention time of the reference standard is used to determine the monosaccharide composition. The results are as Figure 6 shown. The fructose chondroitin obtained by fermentation consists of monosaccharides such as fructose, glucuronic acid, and N-acetylgalactose.

[0140] Although the present invention is disclosed above with preferred embodiments, it is not limited to the above embodiments. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. The protection scope of the present invention shall be defined by the claims.

Claims

1. An engineered Escherichia coli strain, characterized in that, Using Escherichia coli K4 as the starting strain, the phosphoenolpyruvate-dependent phosphotransferase gene glvB was knocked out, and a phosphotransferase mutant with the amino acid sequence shown in SEQ ID NO.1 was expressed.

2. The engineered Escherichia coli according to claim 1, wherein The glucosamine phosphomutase gene glmM was also knocked out, and a glucosamine phosphomutase mutant with the amino acid sequence shown in SEQ ID NO.3 was expressed.

3. The engineered Escherichia coli according to claim 2, characterized in that, The glutamate synthase genes gltB and gltD were also knocked out, and glutamate synthase mutants with the amino acid sequences shown in SEQ ID NO.5 and SEQ ID NO.7 were expressed.

4. The engineered Escherichia coli according to claim 3, characterized in that, The NADH oxidoreductase gene hcr was also knocked out, and an NADH oxidoreductase mutant with the amino acid sequence shown in SEQ ID NO.9 was expressed.

5. The engineered Escherichia coli according to claim 4, characterized in that, The lipid transport system substrate-binding protein gene mlaC was also knocked out, and a lipid transport system substrate-binding protein mutant with the amino acid sequence shown in SEQ ID NO.11 was expressed.

6. Escherichia coli ( Escherichia coli ), strain DH001, was deposited at the China General Microbiological Culture Collection Center on September 26, 2024, with the deposit number CGMCC No. 32081, at the address of Beijing, China, and is classified as Escherichia coli.

7. A composition comprising the engineered Escherichia coli of any one of claims 1 to 5 or Escherichia coli DH001 of claim 6.

8. A method for improving the production capacity of chondroitin fructose by Escherichia coli, characterized in that, Using Escherichia coli K4 as the starting strain, the phosphoenolpyruvate-dependent phosphotransferase gene glvB was knocked out, and a phosphotransferase mutant with the amino acid sequence shown in SEQ ID NO.1 was expressed.

9. The method according to claim 8, wherein The glucosamine phosphomutase gene glmM was also knocked out, and a glucosamine phosphomutase mutant with the amino acid sequence shown in SEQ ID NO.3 was expressed.

10. The method according to claim 9, characterized in that, The glutamate synthase genes gltB and gltD were also knocked out, and glutamate synthase mutants with the amino acid sequences shown in SEQ ID NO.5 and SEQ ID NO.7 were expressed.

11. The method according to claim 10, characterized in that, The NADH oxidoreductase gene hcr was also knocked out, and an NADH oxidoreductase mutant with the amino acid sequence shown in SEQ ID NO.9 was expressed.

12. The method according to claim 11, wherein The coding gene of the lipid transport system substrate-binding protein mlaC was also knocked out, and a lipid transport system substrate-binding protein mutant with the amino acid sequence shown in SEQ ID NO.11 was expressed.

13. A method for fermentatively preparing fructose chondroitin, characterized in that, The engineered Escherichia coli of any one of claims 1 to 5 or Escherichia coli DH001 of claim 6 was fermented in a medium for 40 h to 48 h, and chondroitin sulfate was collected from the fermentation product.

14. The method according to claim 13, wherein The carbon source in the medium includes glucose; the nitrogen source includes yeast extract powder and ammonium sulfate.

15. The method according to claim 13 or 14, characterized in that, The method further includes the step of recovering chondroitin sulfate from the fermentation broth.

16. Use of the engineered Escherichia coli of any one of claims 1 to 5, or Escherichia coli DH001 of claim 6, or the composition of claim 7, or the method of any one of claims 8 to 15 in the preparation of chondroitin sulfate.

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