Chondroitin polymerase mutant, recombinant engineering bacteria and application thereof

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a chondroitin polymerase mutant, a recombinant engineering bacterium and application thereof. The chondroitin polymerase mutant is obtained by gene mining and protein engineering modification based on chondroitin polymerase from Comamonas thiooxydans, and finally the recombinant engineering bacterium is constructed. The chondroitin polysaccharide yield of the engineering bacterium is 6.25 g / L after 48 h fermentation in a flask, which is 11.6 times of that of endogenous chondroitin polymerase of E. coli K4. The application provides a more efficient microbial strain for green and low-cost large-scale biological manufacturing of chondroitin polysaccharide, and has great industrial application potential.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and specifically relates to a chondroitin polymerase mutant, recombinant engineered bacteria and their applications, particularly the nucleotide sequence encoding the mutant, the recombinant expression vector, and the recombinant engineered bacteria containing the sequence / recombinant expression vector and their application in the fermentation preparation of chondroitin polysaccharide and in increasing the yield of chondroitin polysaccharide. Background Technology

[0002] Chondroitin and its sulfated derivative chondroitin sulfate (CS) are glycosaminoglycans (GAGs) composed of D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) linked by alternating β-1,3 and β-1,4 glycosidic bonds. They play an important role in maintaining cartilage strength, toughness, and joint function, and are widely used in the pharmaceutical and health product fields, such as osteoarthritis treatment, ophthalmic disease treatment, and nutritional supplements.

[0003] Currently, chondroitin sulfate is mainly produced industrially by extracting polysaccharides from the cartilage tissue of animals (such as pigs, cattle, and sharks). However, traditional animal tissue extraction methods have many drawbacks: animal cartilage resources are limited, greatly affected by seasons and regions, and there is a risk of potential contamination by exogenous pathogens, resulting in unstable quality; furthermore, purification processes after extraction are costly. To overcome the shortcomings of animal extraction processes, enzymatic synthesis and whole-cell microbial biosynthesis have become the mainstream research and development directions in the industry in recent years.

[0004] Chondroitin polymerase is a key enzyme in the synthesis of chondroitin and belongs to the glycosyltransferase family. This enzyme catalyzes the polymerization of UDP-N-acetylgalactosamine (UDP-GalNAc) and UDP-glucuronic acid (UDP-GlcA) to synthesize chondroitin polysaccharide chains. Currently, publicly available natural chondroitin polymerases are mainly derived from strains such as *E. coli* K4 and *Pasteurella multocida*. Among these, the chondroitin polymerase derived from *E. coli* K4 is the most thoroughly studied polymerase (US20090305357A1). However, the catalytic efficiency of existing chondroitin backbone polymerases derived from *E. coli* K4 (KfoC) cannot meet the demands of large-scale industrial synthesis of chondroitin polysaccharides, resulting in limited yield per unit time and severely restricting the synthesis of chondroitin polysaccharides.

[0005] Currently, the main strategies for constructing high-chondroitin-producing strains largely revolve around optimizing the host cell metabolic network, modifying the strain genome rather than the core rate-limiting polymerase. These strategies can be mainly divided into the following approaches:

[0006] Firstly, by overexpressing genes involved in UDP sugar precursor supply such as ugd, glmU, and kfoA, the supply of UDP-GlcA and UDP-GalNAc in the cell can be increased, thereby strengthening the precursor sugar metabolism pathway (CN106497845A, CN108841771A, CN101679955A, etc.). However, this modification approach can only alleviate the problem of insufficient substrate supply and cannot solve the fundamental bottleneck of low polymerase catalytic efficiency and weak glycan elongation ability.

[0007] Secondly, global transcriptional regulation of the strain can be modified, such as overexpressing the transcription activator rfaH, knocking out genes competing for polysaccharide synthesis pathways, and modifying membrane transport proteins (Cimini D, et al. Homologous overexpression of rfaH in E. coli K4 improves the production of chondroitin-like capsular polysaccharide [J]. Microbial Cell Factories, 2013, 12 (1):46.). However, this technology can only improve the transmembrane transport efficiency of polysaccharides and cannot improve the glycosyl transfer catalytic activity of polymerases.

[0008] Third, the whole genome of intact Escherichia coli K4 strain is randomly mutated using mutagen and then the strain is screened. However, this technology can only obtain randomly mutated strains of the whole genome, and cannot accurately locate the kfoC functional site. The mutant strains have multiple unknown genomic mutations, complex genetic background, poor fermentation stability, and it is difficult to screen for target strains with only polymerase amino acid mutations.

[0009] Fourth, the chondroitin polysaccharide synthesis pathway was reconstructed using heterologous hosts such as Bacillus subtilis (CN104388372), Pichia pastoris (CN118773031A), and Corynebacterium glutamicum (CN113684166). Although the expression elements of the host cells were optimized, the catalytic activity of the wild-type chondroitin polymerase itself was not improved. Therefore, the increase in chondroitin polysaccharide yield after fermentation was limited.

[0010] In summary, most existing technologies for modifying engineered bacteria are based on the metabolic level of the host cell, ignoring the key factor that the polymerase itself is the core of the rate-limiting factor for glycan elongation. Few literature or patents report a complete technical solution for constructing recombinant engineered bacteria that produce high yields of chondroitin polysaccharides by using chondroitin polymerase protein molecules as the core of modification and enhancing the catalytic activity of chondroitin polymerase through targeted and site-specific compound mutations.

[0011] Therefore, there is an urgent need in this field to discover new chondroitin polymerases and to modify the structure of chondroitin polymerases through protein engineering to obtain mutants with improved performance. Based on this, efficient recombinant engineered bacteria for chondroitin polysaccharides can be constructed. This is of great significance for improving the efficiency of industrial preparation of chondroitin polysaccharides and the development of the chondroitin market. Summary of the Invention

[0012] To address the above technical problems, this invention provides a chondroitin polymerase mutant, a recombinant engineered bacterium, and their applications.

[0013] This invention uses chondroitin polymerase from E. coli K4 as the starting gene. Through gene mining and phylogenetic tree construction, chondroitin polymerases from multiple different sources (Comamonas thiooxydans, Thiosulfatimonas sediminis, Enterobacteriaceae, and Aeromonas bivalvium) were screened. Highly active chondroitin polymerases were further screened and subjected to site-directed mutagenesis. Finally, these polymerases were expressed in E. coli K4 chassis strains to synthesize chondroitin polysaccharides. Results showed that the polymerase mutants of this invention produced a 11.6-fold higher capacity for synthesizing fructose chondroitin compared to the E. coli K4-derived chondroitin polymerase kfoC. All polymerase mutants exhibited superior performance in chondroitin polysaccharide synthesis.

[0014] The technical solution provided by this invention is as follows:

[0015] A first aspect of the present invention is to provide a chondroitin polymerase mutant, said chondroitin polymerase mutant being obtained by site-directed mutagenesis based on any one of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, wherein the mutation site is selected from at least one of the following positions: position 244, position 272, position 280, position 299, position 310, position 313, position 402, position 413, position 444, and position 493.

[0016] Preferably, the mutation site is selected from at least one of the following: cysteine ​​at position 244 is replaced with alanine, arginine at position 272 is replaced with leucine, leucine at position 280 is replaced with methionine, arginine at position 299 is replaced with glycine, alanine at position 310 is replaced with threonine, serine at position 313 is replaced with glutamic acid, arginine at position 402 is replaced with serine, valine at position 413 is replaced with phenylalanine, alanine at position 444 is replaced with histidine, and valine at position 493 is replaced with cysteine.

[0017] Preferably, the chondroitin polymerase mutant is obtained by site-directed mutagenesis based on the amino acid sequence shown in SEQ ID NO:1.

[0018] As a further preferred embodiment, the chondroitin polymerase mutant is obtained by performing at least one of the following mutations based on the amino acid sequence shown in SEQ ID NO:1:

[0019] The cysteine ​​at position 244 is replaced with alanine C244A (SEQ ID NO:9);

[0020] The cysteine ​​at position 244 is replaced with alanine, and the arginine at position 272 is replaced with leucine C244A-R272L (SEQ ID NO:10).

[0021] The cysteine ​​at position 244 is replaced with alanine, the arginine at position 272 is replaced with leucine, and the arginine at position 299 is replaced with glycine C244A-R272L-R299G (SEQ ID NO:11);

[0022] The cysteine ​​at position 244 is replaced with alanine, the arginine at position 272 is replaced with leucine, the arginine at position 299 is replaced with glycine, and the valine at position 413 is replaced with phenylalanine C244A-R272L-R299G-V413F (SEQ ID NO:12).

[0023] The cysteine ​​at position 244 is replaced with alanine, the arginine at position 272 is replaced with leucine, the arginine at position 299 is replaced with glycine, the valine at position 413 is replaced with phenylalanine, and the alanine at position 444 is replaced with histidine C244A-R272L-R299G-V413F-A444H (SEQ ID NO:13);

[0024] The following substitutions are made: cysteine ​​at position 244 is replaced with alanine, arginine at position 272 is replaced with leucine, arginine at position 299 is replaced with glycine, valine at position 413 is replaced with phenylalanine, alanine at position 444 is replaced with histidine, and valine at position 493 is replaced with cysteine. The substitution is C244A-R272L-R299G-V413F-A444H-V493C (SEQ ID NO:14).

[0025] A second aspect of the present invention is to provide a gene encoding any of the chondroitin polymerase mutants described above.

[0026] Furthermore, expression cassettes, expression vectors, or recombinant engineered bacteria containing the chondroitin polymerase mutant gene also fall within the scope of protection of this invention.

[0027] A third aspect of the present invention is to provide a recombinant engineered Escherichia coli K4 strain, wherein, preferably, the endogenous chondroitin polymerase gene kfoC of the Escherichia coli K4 is replaced with a gene encoding any of the chondroitin polymerases described above.

[0028] As a further preferred embodiment, the engineered bacteria also overexpress at least one of UDP-glucosamine isomerase and UDP-glucose dehydrogenase.

[0029] As a further preferred embodiment, the engineered bacteria are enabled to directly secrete non-fructosyltransferase chondroitin polysaccharide by knocking out the fructosyltransferase gene kfoE.

[0030] Furthermore, the application of the expression cassette, expression vector, recombinant engineered bacteria, and recombinant Escherichia coli K4 engineered bacteria provided in this invention in the fermentation preparation of chondroitin polysaccharide is also a key technical content that this invention aims to protect.

[0031] The present invention has the following advantages and effects compared with the prior art:

[0032] (1) In this invention, chondroitin polymerases from multiple sources were screened, and a chondroitin polymerase with higher enzyme activity than that of Escherichia coli K4 was obtained, namely the chondroitin polymerase derived from Commonas thiooxydans. Based on this, a highly active chondroitin polymerase mutant was obtained.

[0033] (2) This invention obtained recombinant engineered bacteria by integrating the gene of a chondroitin polymerase mutant obtained through site-directed mutation into Escherichia coli K4 strain, achieving efficient synthesis of chondroitin polysaccharide in E. coli K4 recombinant engineered bacteria. Experimental results showed that the synthesis efficiency of chondroitin polysaccharide by this recombinant engineered bacteria was 2.45~6.34 g / L, and its highest synthesis efficiency was 11.6 times that of the endogenous chondroitin polymerase kfoC in E. coli K4. The modification of chondroitin polymerase and strain in this invention significantly improved the production efficiency of chondroitin polysaccharide, thus providing a more efficient route for the production and preparation of chondroitin polysaccharide. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the principle of engineered bacteria synthesizing chondroitin polysaccharide provided by the present invention.

[0035] Figure 2 This is a high-performance liquid chromatogram of chondroitin polysaccharide synthesized by the CtCS-M5 strain in Example 2 of the present invention;

[0036] Figure 3 This is a high-performance liquid chromatogram of chondroitin polysaccharide synthesized by the CtCS-M6 strain in Example 2 of the present invention. Detailed Implementation

[0037] To enable those skilled in the art to better understand the present invention, the invention will now be further described in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention.

[0038] I. Main reagents and consumables used in this invention:

[0039] High-fidelity DNA polymerase (Phanta Max Super-Fidelity DNA polymerase P525) was purchased from Nanjing Novizan Biotechnology Co., Ltd.; FastDigest DpnI DNA restriction endonuclease was purchased from Thermo Fisher Scientific; pTrcHis A plasmid, a known E. coli expression vector with a size of 4414 bp and a Trc promoter, and ampicillin resistance, was purchased from Novogen; E. coli DH5α competent cells were purchased from Angyu Biotechnology Co., Ltd.; agar powder was purchased from Solarbio; tryptophan and yeast extract were purchased from OXOID.

[0040] II. Culture medium and preparation

[0041] LB medium: Each 100 mL of LB medium contains 1 g tryptone, 0.5 g yeast extract, and 1 g NaCl, with a pH of 7.0–7.2.

[0042] Preparation method: Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1 L of distilled water. Do not adjust the pH. Autoclave at 121°C for 20 min. If preparing solid culture medium, add 1.8 g agar powder to 100 mL of medium.

[0043] Shake-flask fermentation medium: Each 100 mL of fermentation medium contains 0.5 g yeast extract, 0.3 g tryptone, 0.2 g KH2PO4, 0.97 g K2HPO4, 0.12 g (NH4)2SO4, 0.15 g MgSO4·7H2O and 1.5 g glucose, pH 7.8.

[0044] Unless otherwise specified, the reagents used in the following embodiments of the present invention are all conventional reagents in the art, which can be commercially available or prepared according to conventional methods in the art, and are of laboratory purity grade. Unless otherwise specified, the methods used in the following embodiments are all conventional methods in the art, and the experimental conditions used are all conventional experimental conditions in the art, which can be referred to in relevant experimental manuals or manufacturer's instructions.

[0045] Example 1

[0046] This embodiment uses the chondroitin polymerase from E. coli K4 as the starting gene. Through gene mining and phylogenetic tree construction, four chondroitin polymerases from different sources (Comamonas thiooxydans, Thiosulfatimonas sediminis, Enterobacteriaceae, and Aeromonas bivalvium) were screened. Using CRISPR gene editing technology, the chondroitin polymerases from different sources were integrated into the E. coli K4 strain. After constructing the recombinant strain, fermentation medium was dispensed into 250 mL shake flasks at 30 mL volumes. After inoculation at a 2% inoculum, fermentation was carried out at 37°C for 48 h. The chondroitin polysaccharide content of the fermentation broth was determined by HPLC. The effects of chondroitin polymerases from different sources on the synthesis of chondroitin polysaccharides are shown in Table 1.

[0047] Table 1. Efficiency of chondroitin polymerase synthesis of chondroitin polysaccharides from different sources Chondroitin polymerase name Chondroitin polymerase amino acid sequence Chondroitin polymerase encoding gene source Chondroitin polysaccharide yield (g / L) Relative synthesis efficiency kfoC / / E. coli K4 0.54±0.05 100% CtCS SEQ ID NO:1 SEQ ID NO:2 Comamonas thiooxydans 1.68±0.05 311% TsCS SEQ ID NO:3 SEQ ID NO:4 Thiosulfatimonas sediminis 1.03±0.07 190% EnCS SEQ ID NO:5 SEQ ID NO:6 Enterobacteriaceae 1.34±0.14 248% AbCS SEQ ID NO:7 SEQ ID NO:8 Aeromonas bivalvium 1.43±0.09 265%

[0048] As shown in Table 1, the chondroitin polymerase derived from Commonas thiooxydans has the highest activity relative to Escherichia coli K4. Therefore, this invention is based on the chondroitin polymerase derived from Commonas thiooxydans.

[0049] Specifically, this invention uses a whole-genome synthesis method to obtain the chondroitin polymerase gene, and uses gene editing technology to obtain recombinant engineered bacteria containing the chondroitin polymerase gene. The specific steps are as follows:

[0050] Obtaining the S1 chondroitin polymerase mutant gene

[0051] The chondroitin polymerase gene and polymerase mutant gene of Comamonas thiooxydans were obtained by Genscript Biotech Inc. and ligated into plasmid pTrcHis A.

[0052] The chondroitin polymerase mutants were obtained by making the following mutations based on the amino acid sequence shown in SEQ ID NO:1:

[0053] The cysteine ​​at position 244 is replaced with alanine C244A (SEQ ID NO:9);

[0054] The cysteine ​​at position 244 is replaced with alanine, and the arginine at position 272 is replaced with leucine C244A-R272L (SEQ ID NO:10).

[0055] The cysteine ​​at position 244 is replaced with alanine, the arginine at position 272 is replaced with leucine, and the arginine at position 299 is replaced with glycine C244A-R272L-R299G (SEQ ID NO:11);

[0056] The cysteine ​​at position 244 is replaced with alanine, the arginine at position 272 is replaced with leucine, the arginine at position 299 is replaced with glycine, and the valine at position 413 is replaced with phenylalanine C244A-R272L-R299G-V413F (SEQ ID NO:12).

[0057] The cysteine ​​at position 244 is replaced with alanine, the arginine at position 272 is replaced with leucine, the arginine at position 299 is replaced with glycine, the valine at position 413 is replaced with phenylalanine, and the alanine at position 444 is replaced with histidine C244A-R272L-R299G-V413F-A444H (SEQ ID NO:13);

[0058] The following substitutions are made: cysteine ​​at position 244 is replaced with alanine, arginine at position 272 is replaced with leucine, arginine at position 299 is replaced with glycine, valine at position 413 is replaced with phenylalanine, alanine at position 444 is replaced with histidine, and valine at position 493 is replaced with cysteine. The substitution is C244A-R272L-R299G-V413F-A444H-V493C (SEQ ID NO:14).

[0059] Obtaining the chondroitin polymerase mutant: The pTrcHis A plasmid containing the above chondroitin polymerase mutant was amplified by PCR, digested with DpnI, and transformed into DH5α competent cells. After obtaining plates containing single colonies, single colonies were picked from LB plates and inoculated into LB liquid medium containing Amp resistance (100 μg / mL). The culture was carried out overnight at 37°C and 200 rpm. After preservation (sterile glycerol final concentration 15%), the culture was sent to Genewiz (Tianjin, China) for sequencing to identify whether the initial chondroitin polymerase mutation was successful.

[0060] Construction of S2 Escherichia coli K4 engineered bacteria containing chondroitin polymerase or its mutant

[0061] Construction of the target plasmid: The N20 sequence was predicted using the CHOPCHOP website, the target plasmid was amplified by PCR, and transformed into DH5α competent cells. Single colonies were picked and sent to Genewiz (China, Tianjin) Co., Ltd. for sequencing.

[0062] Donor DNA amplification: Using pTrcHis A plasmid containing the chondroitin polymerase gene and the E. coli K4 genome as templates, the Donor DNA fragment was amplified by PCR;

[0063] Replacing E. coli K4 endogenous chondroitin polymerase kfoC with gene editing technology: E. coli K4 (containing pCas9) competent cells were removed from a -80℃ ultra-low temperature freezer and placed on ice. After thawing, the target plasmid and Donor DNA were added to the competent cells. The electroporation instrument parameters were set to Ec 2, and the Pulse button was pressed. Immediately after electroporation, 800 μL of antibiotic-free LB liquid medium was added. The cells were incubated at 37℃ and 200 rpm for 1 h. After incubation, 100 μL of the bacterial culture was aspirated and spread onto LB medium plates containing Kana resistance (50 μg / mL) and Spe resistance (50 μg / mL). The plates were incubated upside down at 37℃ overnight.

[0064] After successfully gene-editing the strain and eliminating the target plasmid and pCas9 plasmid, the strain was inoculated into test tubes containing 5 mL of antibiotic-free LB liquid medium and cultured overnight at 37°C and 200 rpm in a shaker. This strain was used as an expression strain for chondroitin polymerase and its mutants. Sterile glycerol was added to the bacterial culture to a final concentration of 15%, and the culture was stored for a long time in an ultra-low temperature freezer at -80°C.

[0065] Example 2

[0066] This embodiment mainly uses the recombinant Escherichia coli K4 engineered bacteria obtained in Example 1 to synthesize fructose chondroitin. A schematic diagram illustrating the principle of recombinant engineered bacteria synthesizing fructose chondroitin in this embodiment is shown below. Figure 1 As shown.

[0067] In this embodiment, the method for producing chondroitin using recombinant engineered bacteria specifically includes the following steps:

[0068] (1) Activation of recombinant engineered strains

[0069] Take the glycerol bacteria stored at -80℃, thaw them rapidly in a 37℃ water bath, and then add 50 μL of glycerol bacteria to 5 mL of antibiotic-free LB medium in a clean bench. Incubate overnight at 37℃ and 200 rpm.

[0070] (2) Fermentation of recombinant engineered strains

[0071] Determination of OD of bacterial culture 600 Inoculate at a rate of 2%; dispense 30 mL of fermentation medium into sterile Erlenmeyer flasks. After inoculation, transfer the flasks to a shaker at 37°C and incubate at 200 rpm. Measure OD every 4 hours. 600 Cultured for 48 hours.

[0072] The method for determining the concentration of chondroitin polysaccharide in this embodiment is as follows:

[0073] Take 48 h of fermentation broth, centrifuge at 12000 rpm for 10 min, then take about 900 μL of supernatant into a 2 mL centrifuge tube, add 100 μL of 50% trichloroacetic acid (TCA) solution, mix well, centrifuge at 13000 rpm for 5 min, take 100 μL of 4M trifluoroacetic acid (TFA) solution into a 2 mL centrifuge tube, add 100 μL of the supernatant after centrifugation, repeatedly pipette tip, react at 99℃ in a constant temperature mixer for 2 h, after the reaction is completed, cool with cold water, centrifuge at 12000 rpm for 30~60 s, dry in a nitrogen blower, add 1 mL of ultrapure water after drying, vortex mix, centrifuge at 12000 rpm for 10 min.

[0074] Take 50 mg of chondroitin reference standard, dissolve and dilute it in ultrapure water to a solution containing approximately 3 mg per mL. Take 100 μL of 4M TFA solution into a 2 mL centrifuge tube, add 100 μL of the reference standard solution, repeatedly pipette the tube, and react at 99°C in a constant temperature mixer for 2 h. After the reaction is complete, cool with cold water, centrifuge at 12000 rpm for 30-60 s, and dry in a nitrogen blower. After drying, add 1 mL of ultrapure water to each tube, vortex to mix, and centrifuge at 12000 rpm for 10 min.

[0075] Instrument parameters: Column: ChromCore 300 SCX (250*4.6 mm, 5 μm); Flow rate: 0.8 mL / min; Column temperature: 30℃; Detector: Differential detector; Injection volume: 20 μL; Run time: 35 min; Mobile phase: Mobile phase A: pH 3.0010mM ammonium formate buffer + acetonitrile (95:5).

[0076] The yield and relative synthesis efficiency of chondroitin polysaccharide synthesized by the recombinant Escherichia coli K4 engineered strain provided by this invention are shown in Table 2. The high-performance liquid chromatograms of the chondroitin polysaccharide products obtained from the mutants CtCS-M5 and CtCS-M6 are shown in Table 2. Figure 2 , Figure 3 As shown.

[0077] Table 2. Chondroitin polymerase CtCS mutant strains' efficiency in synthesizing chondroitin polysaccharides Chondroitin polymerase mutant recombinant strain Chondroitin polymerase amino acid sequence Chondroitin polysaccharide yield (g / L) Relative synthesis efficiency CtCS SEQ ID NO:1 1.68±0.05 100% CtCS-M1 SEQ ID NO:9 2.27±0.05 135% CtCS-M2 SEQ ID NO:10 2.81±0.05 167% CtCS-M3 SEQ ID NO:11 3.20±0.13 190% CtCS-M4 SEQ ID NO:12 3.68±0.08 219% CtCS-M5 SEQ ID NO:13 4.03±0.16 240% CtCS-M6 SEQ ID NO:14 6.25±0.16 372%

[0078] Table 2 combined Figures 2-3 The results showed that the peak elution times of the chondroitin polysaccharide products obtained by recombinant strains CtCS-M5 and CtCS-M6 were 9.207 min and 9.346 min, respectively, and the chondroitin polysaccharide yields were 4.03 g / L and 6.25 g / L, respectively. The synthesis efficiencies were 240% and 372% of those of wild-type CtCS, respectively. In particular, the chondroitin polysaccharide synthesis efficiency of recombinant strain CtCS-M6 was the best, and its synthesis efficiency was 11.6 times that of E. coli K4 endogenous chondroitin polymerase kfoC.

[0079] The experimental results of this invention show that the chondroitin polysaccharide synthesis efficiency of each chondroitin polymerase mutant is in the range of 2.27~6.25 g / L, which demonstrates that the above-mentioned recombinant engineered bacteria have good synthesis effect in the chondroitin polysaccharide synthesis pathway.

[0080] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A chondroitin polymerase mutant, characterized in that, The sequence was obtained by site-directed mutagenesis based on the amino acid sequence shown in SEQ ID NO:

1. The mutagenesis is as follows: The cysteine ​​at position 244 is replaced with alanine, and the amino acid sequence is shown in SEQ ID NO:9; Alternatively, cysteine ​​at position 244 can be replaced with alanine, and arginine at position 272 can be replaced with leucine, with the amino acid sequence shown in SEQ ID NO:

10. Alternatively, cysteine ​​at position 244 can be replaced with alanine, arginine at position 272 with leucine, and arginine at position 299 with glycine, with the amino acid sequence shown in SEQ ID NO:11; Alternatively, cysteine ​​at position 244 can be replaced with alanine, arginine at position 272 with leucine, arginine at position 299 with glycine, and valine at position 413 with phenylalanine, as shown in SEQ ID NO:

12. Alternatively, cysteine ​​at position 244 can be replaced with alanine, arginine at position 272 with leucine, arginine at position 299 with glycine, valine at position 413 with phenylalanine, and alanine at position 444 with histidine, as shown in SEQ ID NO:

13. Alternatively, cysteine ​​at position 244 can be replaced with alanine, arginine at position 272 with leucine, arginine at position 299 with glycine, valine at position 413 with phenylalanine, alanine at position 444 with histidine, and valine at position 493 with cysteine, as shown in SEQ ID NO:

14.

2. The gene encoding a chondroitin polymerase mutant as described in claim 1.

3. An expression cassette, expression vector, or recombinant engineered bacteria containing the chondroitin polymerase mutant gene as described in claim 2.

4. The application of the expression cassette, expression vector, and recombinant engineered bacteria as described in claim 3 in the fermentation preparation of chondroitin polysaccharide.

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