Escherichia coli with high fructose chondroitin yield and production method thereof

Abstract

The invention discloses Escherichia coli with high fructose chondroitin yield and a production method of the Escherichia coli, and belongs to the technical field of microorganisms. On the basis of Escherichia coli K4, a coding gene of a key enzyme in a fructose chondroitin synthesis route is mutated, and a strain for improving the fructose chondroitin synthesis capability is constructed. The yield of fructose chondroitin in a 5 L fermentation tank reaches 10 g / L, the yield of fructose chondroitin in a 100 L fermentation tank reaches 15-20 g / L, the obtained final product is uniform in structure and stable in quality, the production cost is effectively reduced, the environmental pollution risk is reduced, and the strain is suitable for large-scale industrial production and application.

Description

Technical Field

[0001] The invention relates to an Escherichia coli with high fructose chondroitin production and a production method thereof, belonging to the technical field of microorganisms. Background Art

[0002] Chondroitin sulfate is a sulfated glycosaminoglycan widely found in animal tissues, in which the chondroitin skeleton is formed by linear alternating connections of glucuronic acid (GlcUA) and N-acetylgalactosamine (GalNAc) via 1,3-bonds and 1,4-bonds, respectively. Currently, chondroitin sulfate is mainly extracted directly from animal tissues. Due to the long production cycle and dispersed distribution of raw materials, cumbersome process steps and long cycle, and unstable product quality and yield caused by raw materials, the production of chondroitin sulfate using microbial fermentation with mild conditions and abundant raw materials 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 fructosyl-modified chondroitin skeleton. Fructose chondroitin is synthesized by fermentation of K4 strain and then the fructosyl group is removed by acid hydrolysis and other methods to prepare chondroitin. As an emerging chondroitin synthesis strategy, it is green, environmentally friendly, economical and efficient.

[0003] In recent years, researchers at home and abroad have systematically studied the production of fructose chondroitin by microbial fermentation from the perspectives of biochemical engineering and metabolic engineering. The patent with the announcement number CN104974973B discloses the technology of using recombinant strains of E. coli to produce chondroitin by fermentation. The vector is used to introduce a chondroitin derived from the E. coli K4 strain into E. coli such as K5 that can produce UDP-glucuronic acid. kfoA and kfoC Genes, among which kfoA The gene encodes UDP-glucose-4-epimerase, kfoC The gene encodes chondroitin synthase, the vector contains an antibiotic resistance gene, and the chondroitin production reaches 52.6 mg / L. The patent with announcement number CN102869782B discloses the technology of using recombinant Escherichia coli K12 or Escherichia coli B or wild campestris to ferment and produce chondroitin. kfoD , orf3 ( kfoI )、 kfoE ,or orfI ( kfoH ) into Escherichia coli or Xanthomonas sp., using a vector to introduce one or more functional genes in kfoA , kfoC、kfoF , kfoG , kfoB , kpsF , kpsE , kpsD , kpsU , kpsC , kpsS , kpsM or kpsT One or more genes in the recombinant strain are expressed, and the recombinant strain is cultured in a medium with glycerol as the carbon source for 24-72 hours to secrete 15-50 g / L of non-fructosylated chondroitin from the cells. Patent No. CN103228781B discloses the use of genetic engineering to inactivate the glycosyltransferase gene that adds fructose residues to the main chain of chondroitin. kfoE A recombinant derivative of Escherichia coli K4 (ΔkfoE / kan) that can directly produce fructosylated chondroitin skeleton was obtained. R , DSM23578 and ΔkfoE, DSM23644), but the chondroitin production of this strain has not been reported because the plasmids carried affect bacterial growth and the instability of plasmid replication will also cause unstable chondroitin production. glmS and glmM The copy number of the gene, glmS Encodes aminotransferase, glmM The enzyme encoded phosphoglucosamine mutase, thereby strengthening the synthesis of the precursor UDP-GalNAc, solving the problem of plasmid instability, and increasing the yield of fructose chondroitin. The recombinant strain was fermented in a 7.5 L fermenter, and the yield of fructose chondroitin reached 7.12 g / L. However, the yield of both fructose chondroitin and the chondroitin skeleton without fructosylation failed to meet the needs of industrial production and could not replace the direct extraction method. Further increasing the yield of chondroitin or its analog fructose chondroitin has become an urgent problem to be solved. Summary of the invention

[0004] In view of the problems in the prior art that the recombinant strain containing plasmids has unstable plasmids and the yield of chondroitin or its analogues is low, the present invention provides a plasmid-free strain with high yield of chondroitin skeleton or its analogues.

[0005] The invention provides an engineered Escherichia coli bacterium, which expresses one or more of the following mutants: a phosphotransferase mutant, a phosphoglucosamine mutase mutant, a glutamate synthetase mutant, a NADH oxidoreductase mutant and a lipid transport system substrate binding protein mutant.

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

[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 synthetase mutant contains the amino acid sequence 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; and 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 is Escherichia coli DH001, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 26, 2024, with the deposit number: CGMCC No.32081, the deposit address is Beijing, China, and the classification name is Escherichia coli.

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

[0010] In one embodiment, the Escherichia coli DH001 has a gene with a nucleotide sequence as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, and 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-throw starter culture containing live cells of Escherichia coli DH001.

[0013] The present invention also provides a method for improving the fructose chondroitin production capacity of Escherichia coli, comprising (a) or (b): (a) expressing one or more of the following mutants in Escherichia coli: a phosphoenolpyruvate (PEP) phosphotransferase mutant, a phosphoglucosamine mutase mutant, a glutamate synthetase large subunit mutant, a glutamate synthetase small subunit mutant, a NADH oxidoreductase mutant, and a lipid transport system substrate binding protein mutant; (b) Mutating the coding sequence of one or more of the following proteins of Escherichia coli: phosphoenolpyruvate (PEP)-dependent phosphotransferase glvB, phosphoglucosamine mutase glmM, glutamate synthetases gltB and gltD, NADH oxidoreductase hcr, and lipid transport system substrate binding protein mlaC.

[0014] In one embodiment, the method is to glvB , glmM , gltB , gltD , hcr and mlaC Gene replacement in situ.

[0015] In one embodiment, the Escherichia coli includes but is not limited to Escherichia coli K4 (ATCC 23502).

[0016] In one embodiment, the mutation is to mutate the arginine (R) at position 224 of phosphoenolpyruvate-dependent phosphotransferase (glvB) to cysteine ​​(C), and the amino acid sequence of the obtained mutant is shown in SEQ ID NO.1.

[0017] In one embodiment, the mutation is to mutate the 128th alanine (A) of phosphoglucosamine mutase (glmM) to threonine (T), and the amino acid sequence of the obtained mutant is shown in SEQ ID NO.3.

[0018] In one embodiment, the mutation is to mutate the proline (P) at position 223 of the large subunit gltB of glutamate synthetase to serine (S), and the amino acid sequence of the obtained mutant is shown in SEQ ID NO.5.

[0019] In one embodiment, the mutation is to mutate the 190th proline (P) of the small subunit gltD of glutamate synthetase to serine (S), and to mutate the 286th glutamate (E) to lysine (K). The amino acid sequence of the obtained mutant is shown in SEQ ID NO.7.

[0020] In one embodiment, the mutation is to mutate the threonine (T) at position 174 of NADH oxidoreductase hcr to isoleucine (I), and the amino acid sequence of the obtained mutant is shown in SEQ ID NO.9.

[0021] In one embodiment, the mutation is to mutate the 88th alanine (A) of the phospholipid transport system substrate binding protein mlaC to valine (V), and the 140th proline (P) to serine (S), and the amino acid sequence of the obtained mutant is shown in SEQ ID NO.11.

[0022] In one embodiment, the nucleotide sequence of the gene glvB encoding the phosphotransferase mutant is shown in SEQ ID NO.2; the nucleotide sequence of the gene glmM encoding the phosphoglucosamine mutase mutant is shown in SEQ ID NO.4. The nucleotide sequence of the gene gltB encoding the glutamate synthetase large subunit mutant is shown in SEQ ID NO.6; the nucleotide sequence of the gene gltD encoding the glutamate synthetase small subunit mutant is shown in SEQ ID NO.8; the nucleotide sequence of the gene hcr encoding the NADH oxidoreductase mutant is shown in SEQ ID NO.10; the nucleotide sequence of the gene mlaC encoding the phospholipid transport system substrate binding protein mutant is shown in SEQ ID NO.12.

[0023] In one embodiment, the starting strain of the recombinant Escherichia coli includes but is not limited to Escherichia coli K4.

[0024] The present invention also provides a method for producing fructose chondroitin by fermentation using the Escherichia coli DH001.

[0025] In one embodiment, the method comprises fermenting the Escherichia coli in a culture medium for 40 h to 48 h, and collecting fructose chondroitin in the fermentation product.

[0026] In one embodiment, the carbon source in the culture medium includes but is not limited to glucose.

[0027] In one embodiment, the nitrogen source in the culture medium is an organic nitrogen source and / or an inorganic nitrogen source; optionally, the organic nitrogen source may be yeast extract powder, and the inorganic nitrogen source may be ammonium sulfate.

[0028] In one embodiment, the nitrogen source is yeast extract and ammonium sulfate.

[0029] In one embodiment, the culture medium also contains inorganic salts and trace elements necessary for the growth of the strain, including but not limited to phosphorus, magnesium, iron, etc.

[0030] In one embodiment, the inorganic salt and trace elements are selected from one or more of potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and ferrous sulfate heptahydrate.

[0031] In one embodiment, the culture medium contains: glucose, yeast extract, ammonium sulfate, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate.

[0032] In one embodiment, the fermentation is carried out at 30-37°C.

[0033] In one embodiment, glucose is fed during the fermentation.

[0034] In one embodiment, the method may further include the step of recovering fructose chondroitin from the fermentation broth.

[0035] In one embodiment, the recovery is followed by purification.

[0036] In one embodiment, the recovery can be recovered and purified 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; the recovery step and the purification step can be performed sequentially, continuously or discontinuously, or can be performed simultaneously or by combining into one step, but the step mode is not limited thereto.

[0037] The present invention also provides the use of the Escherichia coli DH001, or the composition, or the method in preparing fructose chondroitin or related products thereof.

[0038] In one embodiment, the related products include but are not limited to chondroitin and chondroitin sulfate.

[0039] Beneficial effects: (1) The present invention constructs a strain DH001 with improved chondroitin synthesis ability by mutating key enzyme genes in the chondroitin synthesis pathway (phosphoenolpyruvate-dependent phosphotransferase glvB, phosphoglucosamine mutase glmM, glutamate synthases gltB and gltD, NADH oxidoreductase hcr and lipid transport system substrate binding protein mlaC) based on Escherichia coli K4 (ATCC23502).

[0040] (2) The strain constructed in the present invention has excellent production performance and genetic stability. After 8 generations of subculture, the fructose chondroitin production in a 5L fermentation tank is maintained at above 8 g / L.

[0041] (3) The present invention provides a method for producing fructose chondroitin by fermentation using strain DH001. In a 5 L fermenter, the yield of fructose chondroitin can reach 10 g / L. The fermentation scale is expanded to a 100 L fermenter for high-density fermentation. The production cycle is reasonably controllable, and the yield of fructose chondroitin reaches 15-20 g / L. The final product has a uniform structure and stable quality, which effectively reduces the production cost and the risk of environmental pollution, and is suitable for large-scale industrial production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the synthesis pathway diagram of fructose chondroitin (K4CPS).

[0043] Figure 2 This is the chondroitin production of the recombinant strain in shake flask fermentation in Example 2.

[0044] Figure 3 The results of gene sequencing of the mutants of the recombinant strain FCH5 in Example 2; R224C ; b, glmM A128T ; c, gltB P223S ; d, hcr T174I ; e,gltD P190S / E286K ; f, mlaC A88V / P140S .

[0045] Figure 4 This is the HPLC analysis spectrum of chondroitin disaccharide after enzymatic hydrolysis of sodium chondroitin sulfate standard.

[0046] Figure 5 This is the HPLC analysis spectrum of chondroitin disaccharide after acid hydrolysis and enzymatic hydrolysis of the fermentation product in Example 4.

[0047] Figure 6 This is the monosaccharide composition analysis spectrum of fructose chondroitin in Example 4; wherein, a, monosaccharide standard; b, sample. DETAILED DESCRIPTION

[0048] 1. Sequence information: (1) SEQ ID NO.1 is the amino acid sequence of the glvB mutant; (2) SEQ ID NO.2 is glvB mutated gene nucleotide sequence; (3) SEQ ID NO.3 is the amino acid sequence of the glmM mutant; (4) SEQ ID NO.4 is glmM mutated gene nucleotide sequence; (5) SEQ ID NO.5 is the amino acid sequence of the gltB mutant; (6) SEQ ID NO.6 is gltB mutated gene nucleotide sequence; (7) SEQ ID NO.7 is the amino acid sequence of the gltD mutant; (8) SEQ ID NO.8 is gltD mutated gene nucleotide sequence; (9) SEQ ID NO.9 is the amino acid sequence of the hcr mutant; (10) SEQ ID NO.10 is hcr mutated gene nucleotide sequence; (11) SEQ ID NO.11 is the amino acid sequence of the mlaC mutant; (12) SEQ ID NO.12 is mlaC mutated gene nucleotide sequence; (13) SEQ ID NO.13 is a knockout Escherichia coli glvB sgRNA sequence of the gene; (14) SEQ ID NO.14 is integrated glvB sgRNA sequence of the mutated gene; (15) SEQ ID NO.15 is a knockout Escherichia coli glmM sgRNA sequence of the gene; (16) SEQ ID NO.16 is integrated glmM sgRNA sequence of the mutated gene; (17) SEQ ID NO.17 is a knockout Escherichia coli gltB sgRNA sequence of the gene; (18) SEQ ID NO.18 is integrated gltB sgRNA sequence of the mutated gene; (19) SEQ ID NO.19 is a knockout Escherichia coli gltD sgRNA sequence of the gene; (20) SEQ ID NO.20 is integrated gltD sgRNA sequence of the mutated gene; (21) SEQ ID NO.21 is a knockout Escherichia coli hcr sgRNA sequence of the gene; (22) SEQ ID NO.22 is integrated hcr sgRNA sequence of the mutated gene; (23) SEQ ID NO.23 is a knockout Escherichia coli mlaC sgRNA sequence of the gene; (24) SEQ ID NO.24 is integrated mlaCsgRNA sequence for the mutated gene.

[0049] Unless otherwise specified, amino acid mutants are named using the accepted IUPAC single-letter amino acid abbreviations. For example, R224C represents a mutant obtained by mutating the arginine (R) at position 224 to cysteine ​​(C) based on the wild type.

[0050] (II) Determination method: Fructose chondroitin production detection: According to the analytical method on page 1595 of Part II of the Chinese Pharmacopoeia 2020, take 10 mL of fermentation broth to a 50 mL centrifuge tube, add pure glacial acetic acid to adjust the pH to pH 3.8, place in an 85 °C water bath for acid hydrolysis for 4 h, cool to room temperature after the acid hydrolysis, use 6 M sodium hydroxide (NaOH) to adjust the pH to between 7.0-8.0, and transfer to a 25 mL volumetric flask to volume. 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)chloromethane buffer, mix well, place in a 37 °C water bath for 1 h, boil in a boiling water bath for 5 minutes after the enzymatic hydrolysis, cool in cold water and centrifuge, take 20 μL of the supernatant and load it on a HypersilSAX column (Waters) for HPLC analysis, and the detection wavelength is 232 nm. The quantification was carried out by external standard method, and the sum of the peak areas of ΔDi-0S, ΔDi-4S and ΔDi-6S after enzymatic hydrolysis of 10 g / L sodium chondroitin sulfate standard (China Food and Drug Inspection Institute, 4386 / 10 / 12, batch number 140792-202003) was taken as the peak area of ​​the reference substance; the peak area of ​​ΔDi-0S after enzymatic hydrolysis of the sample was taken as the peak area of ​​the sample, and the chondroitin content was calculated (sample peak area / reference substance peak area × standard concentration 10 g / L × dilution factor 2.5); when calculating the fructose chondroitin content, the chondroitin content was multiplied by the coefficient 1.43 (coefficient = fructose chondroitin disaccharide molecular weight ÷ chondroitin disaccharide molecular weight, i.e. 540÷378=1.43).

[0051] Glucose determination method: The fermentation broth was centrifuged at 10,000 r / min for 5 min, the supernatant was diluted 20 times, and the glucose was determined using a biosensor analyzer M-100 (Silman Technology).

[0052] 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, and the operations performed are all known in the art or performed according to the user manuals of commercial products.

[0053] Example 1 Construction of recombinant strains The Crispr-Cas9 system was used to edit the gene of Escherichia coli K4 (ATCC23502). glvB The codon encoding the 224th amino acid in the gene was mutated from CGT to TGT, resulting in the E. coli FCH1 strain. glmM The codon encoding the 128th amino acid in the gene was mutated from GCC to ACC, resulting in the E. coli FCH2 strain. gltB The codon encoding the 223rd amino acid in the gene mutated from CCG to TCG, and gltD The codon CCG encoding the 190th amino acid in the gene mutated to TCG, and gltD The codon GAA encoding the 286th amino acid in the gene was mutated to AAA, and the Escherichia coli FCH3 strain was obtained; hcr The codon ACC encoding the 174th amino acid in the gene was mutated to ATC to obtain the Escherichia coli FCH4 strain; mlaC The codon GCT encoding the 88th amino acid in the gene was mutated to GTT, and the codon CCG encoding the 140th amino acid was mutated to TCG, thereby obtaining the E. coli FCH5 strain. The specific steps are: (1) Preparation of competent E. coli K4 / pCas 50 ng of pCas plasmid (purchased from Addgene) was transformed into E. coli K4 competent cells by heat shock at 42 °C for 90 seconds, coated with solid LB medium (containing 100 mg / L kanamycin (kana)), and placed in an incubator at 30 °C overnight to obtain E. coli K4 / pCas strain. Pick a single colony, inoculate it in 5 mL LB liquid medium (100 mg / L kana), add 50 μL 1 M L-arabinose (L-Ara) solution, and culture it at 30 °C 180 rpm overnight to obtain seed solution. Take 500 μL of seed solution and inoculate it in 50 mL LB liquid medium (100 mg / L kana), add 500 μL 1 M L-Ara, and culture it at 30 °C 180 rpm until OD 600 The concentration of the culture medium was 0.4-0.6, and the mixture was placed on ice for 5 min. In a clean bench, the bacterial solution was poured into a 50 mL sterile centrifuge tube, and centrifuged at 5000 rpm at 8°C for 10 min. The supernatant was discarded, and 20 mL of pre-cooled 100 mM CaCl2 was added to resuspend the bacteria. The mixture was placed on ice for 30 min, and centrifuged at 5000 rpm at 8°C for 10 min. The supernatant was discarded, and 0.5 mL of pre-cooled 100 mM CaCl2 solution and 60% glycerol were added to resuspend the bacteria. 50 μL of each tube was dispensed into sterile 1.5 mL EP tubes and stored at -80°C.

[0054] (2) Knockout of endogenous genes in Escherichia coli glvB To obtain the upstream homology arm fragment, the amplification system was as follows: 1 μL of 50 ng / μL E. coli K4 genomic DNA as a template, 1 μL of 10 μM primers glvB-F1 and glvB-R1 (Table 1), 25 μL of 2× PrimeSTAR MAX Premix, and 2 μL of ddH2O2. PCR reaction conditions: pre-denaturation at 98 °C for 5 min, followed by temperature cycling at 98 °C for 10 sec; 57 °C for 15 sec; 72 °C for 1 min, for a total of 30 cycles; the termination temperature was 4 °C. After PCR amplification, the correct PCR product was verified by 1% agarose gel electrophoresis, and the upstream homology arm fragment was purified using a PCR Purification Kit.

[0055] The downstream homology arm fragment was obtained by referring to the upstream homology arm fragment, and the primers were glvB-F2 and glvB-R2 (Table 1). Overlap PCR was used to obtain Donor DNA, and the amplification system was as follows: 1 μL of the upstream and downstream homology arm fragments of 50 ng / μL as templates, 1 μL of 10 μM primers glvB-F1 and glvB-R2, 25 μL of 2× PrimeSTAR MAX Premix, and 2 μL of ddH2O2. PCR reaction conditions: pre-denaturation at 98 °C for 5 min, then enter the temperature cycle of 98 °C for 10 sec; 57 °C for 15 sec; 72 °C for 1 min, for a total of 30 cycles; the termination temperature was 4 °C.

[0056] According to the CRISPR / Cas 9 gene editing principle, the specific guide SgRNA sequence shown in SEQ ID NO.13 was synthesized by Suzhou GeneWeizhi Biotechnology Co., Ltd. and connected to the pTarget plasmid to obtain the pTarget-ΔglvB plasmid.

[0057] Take competent cells carrying pCas plasmid from -80 ℃, place on ice for 2 min, add 500 ng Donor DNA and 50 ng pTarget-ΔglvB, mix gently, and place on ice for 30 min. After the rest, heat shock at 42 ℃ for 90 sec and place on ice for 2 min. After the rest, add 1 mL of pre-cooled LB medium to the competent cells, place on a shaker at 30 ℃, incubate at 180 rpm for 2 h, take 100 μL and spread on LB solid medium (50 mg / L streptomycin (strep) and 100 mg / L kana), and culture overnight at 30 ℃.

[0058] Several single clones were picked from the transformation plate as templates, and colony PCR was performed to verify positive clones using glvB-TF / glvB-TR as primers. The size of the amplified fragment was 1762 bp.

[0059] The positive clones were inoculated into 5 mL LB liquid medium (100 mg / L Kana), and IPTG with a final concentration of 0.1 mM was added to induce the pCas plasmid to eliminate the pTarget-ΔglvB plasmid. The culture was carried out at 30°C, 180rpm for 16 h, and then streaked onto LB solid medium (100 mg / L kana). The culture was carried out overnight at 30°C. Single colonies were picked (only half of the size of the single colony was picked here) and numbered. The colonies were inoculated into the corresponding numbered areas on LB solid medium (50 mg / L strep) according to the numbers, and cultured overnight at 30°C. The single colonies that could not grow were the strains that successfully eliminated the pTarget-ΔglvB plasmid.

[0060] (3) Integration of mutant genes glvB R224C The pTarget-ΔglvB-eliminated strain prepared in step (2) was re-prepared as competent cells carrying the pCas plasmid.

[0061] Using Escherichia coli K4 genomic DNA as a template, the upstream and downstream homology arms were amplified using primer pairs glvB-F3 / glvB-R3 and glvB-F4 / glvB-R4; synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. glvB R224C Gene fragment (SEQ ID NO.2); the above downstream homology arms, glvB R224C The gene sequence was used as a template, and glvB-F3 / glvB-R4 was used as a template. Overlap PCR was used to fuse the three fragments to obtain Donor DNA. The PCR system and method refer to step (2).

[0062] According to the CRISPR / Cas 9 gene editing principle, the specific guide SgRNA sequence shown in SEQ ID NO. 14 was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and connected to the pTarget plasmid to obtain pTarget-glvB R224C Plasmid.

[0063] The remaining operations refer to step (2). The primers used to identify positive clones were glvB-TF / glvB-TR, and the amplified fragment size was 3073 bp.

[0064] Subsequent gene editing referred to the above operation procedures to edit the glmM, gltB, gltD, hcr, and mlaC genes. The relevant primers are shown in Table 1. The sizes of the amplified fragments identified by PCR for the positive clones of glmM, gltB, gltD, hcr, and mlaC knockout / mutation gene integration were 1687 / 2721 bp, 1167 / 2427 bp, 1734 / 2808 bp, 1371 / 2340 bp, and 1180 / 1816 bp, respectively.

[0065] After all gene editing is completed, the pCas plasmid is eliminated to complete the strain construction.

[0066] Table 1 Primers and sequences

[0067] Example 2 Shake flask fermentation of recombinant strains FCH1, FCH2, FCH3, FCH4, and FCH5 The E. coli FCH1, FCH2, FCH3, FCH4, and FCH5 strains constructed in Example 1 were fermented in shake flasks. A single clone of each recombinant strain was picked and inoculated in a 250 mL shake flask with an angle baffle containing 50 mL of seed culture medium (formula as shown in Table 2), and cultured at 37 ° C and 200 rpm for 10 h. The seed solution was transferred into a 500 mL shake flask with an angle baffle containing 100 mL of fermentation medium (formula as shown in Table 3) at a 1% inoculation amount (v / v). The fermentation conditions were: speed 200 rpm, temperature 37 ° C, initial pH controlled at 7.5, and fermentation for 48 h. Each strain was repeated three times. After the fermentation was completed, the fermentation broth was collected and the fructose chondroitin production was determined by HPLC. E. coli K4 (ATCC 23502) was used as a control.

[0068] Table 2 Seed culture medium formula

[0069] Table 3 Fermentation medium formula

[0070] The results of fructose chondroitin production by shake flask fermentation of each strain are as follows Figure 2 As shown, after 48 h of fermentation, the fructose chondroitin production of the starting strain K4, 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 production of the recombinant strains was higher than that of the starting strains, among which the production of FCH5 was 484.6% higher than that of the starting strain.

[0071] Example 3 Genotype analysis of recombinant strains The genome analysis of the Escherichia coli FCH5 strain constructed in Example 1 was performed in the following steps: (1) Polymerase chain reaction-single-strand conformation polymorphism analysis (PCR-SSCP) The genome of strain FCH5 was extracted, and PCR amplification was performed 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 refer to step (2) of Example 1. After high-temperature denaturation and ice bath, the PCR amplification product was subjected to SSCP electrophoresis using 8% non-denaturing polyacrylamide gel, with the synthetic mutant gene as the positive control, the native wild-type gene as the negative control, and water as the blank control. Since the mutant gene has a single or multiple base mutations, its conformation is different from that of the native gene, so its mobility in SSCP electrophoresis is different, and the final fragment band is inconsistent with the negative control fragment position, but consistent with the positive control position, indicating that it is a strain with successful allelic replacement.

[0072] (2) Sequencing The PCR amplification product in step (1) was connected to the pMD19-T vector for sequencing. The mutant bases were compared by sequence alignment to verify that the allelic replacement of the strain was successful. The results are as follows Figure 3 shown.

[0073] The strain FCH5 with the correct genotype edited after the above verification was deposited and named DH001.

[0074] Example 4 Fermentation of the recombinant strain in a 5 L fermenter The recombinant E. coli DH001 was fermented in a 5L fermenter to produce fructose-chondroitin, with E. coli K4 (ATCC23502) as a control. The specific steps were as follows: Escherichia coli K4 (ATCC 23502) and recombinant strain DH001 were inoculated into LB liquid medium and activated overnight at 37°C, and inoculated into a 500 mL shake flask with a corner baffle containing 100 mL of the seed medium shown in Table 2 at a 1% inoculation rate (v / v), and cultured at 37°C and 200 rpm for 10 h. The seed solution was transferred into a 5 L T&J Intelli-FermA tabletop fermenter (purchased from Shanghai Dibier Company) containing the fermentation medium shown in Table 3 at a 10% inoculation rate (v / v), and 0.2 g / L defoamer was added. Fermentation was carried out for 48 h according to the control process shown in Table 4. Each strain was repeated three times. After the fermentation was completed, the fermentation broth was collected and the fructose chondroitin production was determined by HPLC. The results are shown in Table 5.

[0075] Table 4 Fermentation control process

[0076] Table 5 Fructose chondroitin fermentation results

[0077] Example 5 Stability verification of recombinant strain DH001 The recombinant strain DH001 was fermented with fructose chondroitin according to Example 4. After the fermentation, an appropriate amount of fermentation liquid was taken, diluted with sterile water, and then coated on an LB solid plate. It was cultured at 37 ° C until a single colony grew. After picking a single colony and identifying it by PCR, the next generation of fructose chondroitin fermentation was carried out. The culture was continued for 8 generations, and the fructose chondroitin yield in the fermentation liquid of each generation was measured. The results showed that the fructose chondroitin yield remained above 8 g / L after 8 generations.

[0078] Example 6 Fermentation of recombinant strain DH001 in a 100 L fermenter (1) Seed tank fermentation: After recombinant E. coli DH001 was activated in LB medium according to the method of Example 3, it was inoculated into a 30 L seed tank at a 5% inoculation rate. The seed medium formula is shown in Table 6. The culture was carried out for 8-10 h until the OD 600 Reach 2.5~5.

[0079] Table 6 Seed tank culture medium formula

[0080] (2) Fermentation in a 100 L fermenter The seed liquid prepared in step (1) was transferred into a 100 L fermenter at an inoculation rate of 10%. The fermentation medium formula was as shown in Table 7. The stirring rate was 300 rpm, the ventilation rate was 1 vvm, the dissolved oxygen correction was 100%, and the dissolved oxygen was controlled at 15%-30% throughout the process. The pH was controlled at 7.0-8.0 using 25% (w / v) ammonia water. The ventilation rate was 1 vvm. 40%-60% mass fraction of glucose was added as the carbon source flow feed 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 was controlled to 0.5-1 g / L according to the DO requirement. The fermentation was carried out for 42-48 h according to the control process shown in Table 4 until the OD 600 When the temperature reaches 110-135, release the tank, collect the fermentation liquid, and measure the fructose chondroitin production.

[0081] Table 7 Fermentation medium formula

[0082] (3) Fructose chondroitin production detection The fructose chondroitin content in the fermentation broth was detected by HPLC. The chondroitin disaccharide spectrum after enzymatic hydrolysis of sodium chondroitin sulfate standard was as follows: Figure 4 As shown in the figure, the chondroitin disaccharide spectrum of the fermentation product fructose chondroitin after acid hydrolysis and enzymatic hydrolysis is as follows Figure 5 As shown, it was calculated that the content of chondroitin disaccharide in the fermentation broth was 12.15±1.67 g / L, and the fructose chondroitin production was 17.37±2.39 g / L.

[0083] (4) Analysis of fructose chondroitin monosaccharide composition The monosaccharide composition of fructose chondroitin recovered and purified from the fermentation broth was determined by HPLC based on PMP (1-phenyl-3-methyl-5-pyrazolone) pre-column derivatization.

[0084] Hydrolysis: Dissolve 2 mg of fructose chondroitin in 2 mL of 2 M trifluoroacetic acid (TFA), place in a 100 ℃ water bath for hydrolysis for 2 h, adjust the pH to 7.0 with 6 M NaOH, and transfer to a 10 mL volumetric flask to make up to volume.

[0085] Derivatization: Take 200 μL of sample, add 100 μL of 0.3 M NaOH and 100 μL of 0.5 M PMP-methanol, vortex to mix, place in a 70 ℃ water bath for hydrolysis for 30 min, cool to room temperature, add 100 μL of 0.3 M hydrochloric acid (HCl) to neutralize NaOH, and vortex to mix.

[0086] Extraction: Add 1 mL of chloroform for extraction, centrifuge at 5000 rpm for 3 min, collect the supernatant, repeat the extraction three times, and remove as much excess PMP reagent as possible.

[0087] Detection: 10 μL of sample was loaded on HPLC with 0.1 M ammonium acetate-acetonitrile (volume ratio 83: 17) as the mobile phase, column temperature 35 °C, detection wavelength 254 nm, and the retention time of the reference standard was used to determine the monosaccharide composition. Figure 6 As shown in the figure, the fructose chondroitin obtained by fermentation is composed of monosaccharides such as fructose, glucuronic acid and N-acetylgalactose.

[0088] Although the present invention is disclosed as above in terms of preferred embodiments, it is not limited to the above embodiments. Any person familiar with the technology may make various changes and modifications without departing from the spirit and scope of the present invention. The scope of protection of the present invention shall be based on the definition of the claims.

Claims

1. An engineered Escherichia coli, characterized in that: One or more of the following mutants are expressed: a phosphotransferase mutant, a phosphoglucosamine mutase mutant, a glutamate synthetase mutant, a NADH oxidoreductase mutant, and a lipid transport system substrate binding protein mutant.

2. The Escherichia coli engineered bacteria according to claim 1, characterized in that The Escherichia coli engineered bacteria uses Escherichia coli K4 as a starting strain.

3. The Escherichia coli engineered bacteria according to claim 1 or 2, characterized in that: 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 synthetase mutant contains the amino acid sequence 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; and the lipid transport system substrate binding protein mutant contains the amino acid sequence shown in SEQ ID NO.

11.

4. The engineered Escherichia coli according to claim 1, characterized in that: The engineered Escherichia coli bacteria is Escherichia coli DH001, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 26, 2024, with the deposit number: CGMCC No.32081, the deposit address is Beijing, China, and the classification name is Escherichia coli.

5. A composition containing the engineered Escherichia coli bacteria according to any one of claims 1 to 4.

6. A method for improving the production capacity of fructose chondroitin in Escherichia coli, characterized in that: Include (a) or (b): (a) expressing one or more of the following mutants in Escherichia coli: a phosphotransferase mutant, a phosphoglucosamine mutase mutant, a glutamate synthetase mutant, a NADH oxidoreductase mutant and a lipid transport system substrate binding protein mutant; (b) Mutating the coding sequence of one or more of the following proteins of Escherichia coli: phosphotransferase, phosphoglucosamine mutase, glutamate synthetase, NADH oxidoreductase and lipid transport system substrate binding protein.

7. The method according to claim 6, characterized in that The mutation is selected from one or more of the following: (1) Mutation of arginine 224 at position phosphotransferase to cysteine; (2) Mutating alanine 128 of phosphoglucosamine mutase to threonine; (3) Mutation of proline 223 in the large subunit of glutamate synthetase to serine; (4) Mutating the proline 190th position of the small subunit of glutamate synthetase to serine and the glutamate 286th position to lysine; (5) Mutation of threonine 174 at position NADH oxidoreductase to isoleucine; (6) The alanine at position 88 of the phospholipid transport system substrate binding protein was mutated to valine, and the proline at position 140 was mutated to serine.

8. The method according to claim 6 or 7, characterized in that: The Escherichia coli includes but is not limited to Escherichia coli K4.

9. A method for preparing fructose chondroitin by fermentation, characterized in that: The engineered Escherichia coli bacteria according to any one of claims 1 to 4 are fermented in a culture medium for 40 h to 48 h, and fructose chondroitin in the fermentation product is collected.

10. The method according to claim 9, characterized in that The carbon source in the culture medium includes, but is not limited to, glucose; the nitrogen source includes, but is not limited to, yeast extract and ammonium sulfate.

11. The method according to claim 9 or 10, characterized in that: The method may further include the step of recovering fructose chondroitin from the fermentation broth.

12. Use of the engineered Escherichia coli according to any one of claims 1 to 4, or the composition according to claim 5, or the method according to any one of claims 6 to 11 in the preparation of fructose chondroitin or its related products.

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