WO2021077581A1 - Engineered yeast for fermentation production of chondroitin sulfate, and use thereof - Google Patents

Abstract

An engineered yeast for fermentation production of chondroitin sulfate, and use thereof, relating to the technical field of bio-engineering. Using synthetic biology technology and genetic engineering means, and with Pichia pastoris GS115 and Saccharomyces cerevisiae as starting strains, genes related to a chrondroitin sulfate synthesis pathway are expressed heterologously in cells: the genes kfoC and kfoA from Escherichia coli K4, the chondroitin sulfate transferase genes C4ST and C6ST from mice, the UDP-glucose dehydrogenase gene tuaD from Bacillus subtilis, and the ATP sulfurylase gene MET13 from Saccharomyces cerevisiae, to obtain a production strain that synthesizes chrondroitin sulfate A (CSA), chrondroitin sulfate C (CSC), and chrondroitin sulfate E (CSE). For the first time, a microorganism fermentation carbon source is used to synthesize different forms of chrondroitin sulfate.

Description

Yeast engineering bacteria for fermentative production of chondroitin sulfate and its application Technical field

The invention relates to a yeast engineering strain for fermentative production of chondroitin sulfate and its application, and belongs to the technical field of bioengineering.

Background technique

Chondroitin sulfate (CS) is a proteoglycan widely distributed in cartilage tissue and has important biological functions. Its backbone is a linear polysaccharide formed by alternately connecting D-glucuronic acid and N-acetylgalactosamine. Chondroitin sulfate is formed by the sulfation modification of chondroitin by sulfate transferase. According to the position of sulfation modification, chondroitin sulfate can be divided into the following four types: Chondroitin sulfate A (4-O-sulfation) , Chondroitin sulfate C (6-O-sulfated), chondroitin sulfate D (2,6-di-O-sulfated) and chondroitin sulfate E (4,6-di-O-sulfated). Due to its excellent biocompatibility, CS has a wide range of applications, such as medical and health, health care products, food and cosmetics. The form of sulfation is closely related to its biological activity and application field. Chondroitin sulfate A and chondroitin sulfate C are often used to treat arthritis, and chondroitin sulfate E can promote the neurite outgrowth of primary neurons.

The current commercial chondroitin sulfate relies heavily on animal tissue extraction. This method has many problems, such as long raw material cycle, potential animal virus infection, environmental pollution, highly heterogeneous structure of chondroitin sulfate extracted from tissues, potential pathogenic factors, and keratan persulfate, which reduces the activity of medicines and even leads to loss Live and wait. In order to obtain chondroitin sulfate with good structural uniformity and biosafety, the synthesis of chondroitin sulfate by microorganisms can effectively avoid the above problems. However, there is currently no report on the synthesis of chondroitin sulfate in microbial cells using carbon sources directly in microbial cells.

Summary of the invention

The purpose of the present invention is to overcome the problems in the prior art, realize the directional production of chondroitin sulfate by the microbial method, overcome various drawbacks brought about by the traditional tissue extraction method, and the inefficiency of the conventional enzymatic method to catalyze the synthesis of chondroitin sulfate. And cumbersome.

To achieve the above-mentioned purpose of the invention, the technical solution of the invention is as follows:

The first object of the present invention is to provide a genetically engineered bacteria producing chondroitin sulfate, which uses yeast as a host to express (a), (b) or (c); or contains (d), ( e) or the nucleotide sequence of (f); wherein,

(a) Chondroitin synthase, UDP-N-acetylglucosamine C4 isomerase, UDP-glucose dehydrogenase, chondroitin 4-O-sulfatase C4ST, ATP sulfurylase;

(b) Chondroitin synthase, UDP-N-acetylglucosamine C4 isomerase, UDP-glucose dehydrogenase, chondroitin 6-O-sulfatase C6ST, ATP sulfurylase;

(c) Chondroitin synthase, UDP-N-acetylglucosamine C4 isomerase, UDP-glucose dehydrogenase, chondroitin 4-O-sulfatase C4ST, chondroitin 6-O-sulfatase C6ST and ATP Sulfurylase

(d) DNA sequences containing kfoC, kfoA, tuaD, C4ST and MET13;

(e) DNA sequences containing kfoC, kfoA, tuaD, C6ST and MET13;

(f) DNA sequences containing kfoC, kfoA, tuaD, C4ST, C6ST and MET13.

In one embodiment, said kfoC is a gene encoding chondroitin synthase (Genbank accession number BAC00523.1); said KfoA is encoding UDP-N-acetylglucosamine C4 isomerase (Genbank accession number BAC00525 .1) gene; said tuaD is a gene encoding UDP-glucose dehydrogenase (Genbank accession number is NP_391438.1); said C4ST is encoding chondroitin 4-O-sulfatase (Genbank accession number is NP_067414. 2), the C6ST is the gene encoding chondroitin 6-O-sulfatase (Genbank accession number is BAA29054.1); the MET13 is the gene encoding ATP sulfurylase (Genbank accession number is NP_011390.2) gene.

In one embodiment, the KfoC and KfoA are derived from Escherichia coli K4; the tuaD is derived from Bacillus subtilis 168; the C4ST and C6ST are derived from Mus musculus; the ATP sulfurylase MET13 From Saccharomyces cerevisiae.

In one embodiment, the gene sequences of kfoC, kfoA, tuaD, C4ST, and C6ST are shown in SEQ ID NOs. 1 to 5, respectively.

In one embodiment, the yeast is Pichia pastoris or Saccharomyces cerevisiae and a mutant or mutagenic strain thereof.

In one embodiment, the yeast is Pichia GS115 or Saccharomyces cerevisiae S-CA.

In one embodiment, the gene is expressed by a plasmid, or is integrated and expressed on the genome of the host.

In one embodiment, the plasmid includes but is not limited to: pGAPZB, pAO815, pRS303, pRS304 or pRS303.

The second object of the present invention is to provide a method for constructing the genetically engineered bacteria producing chondroitin sulfate, which connects (a), (b) or (c) to the genome of yeast; wherein,

(a) DNA sequence containing: kfoC, kfoA, tuaD and C4ST;

(b) DNA sequences containing kfoC, kfoA, tuaD and C6ST genes;

(c) DNA sequences containing kfoC, kfoA, tuaD, C4ST and C6ST.

In one embodiment, the method for constructing Pichia pastoris that produces chondroitin sulfate includes the following steps: the specific steps are as follows:

(1) The kfoC, kfoA, and tuaD genes were assembled to pGAPZB vector using Gibson assembly to construct pGAPZB-kfoC-T2A-kfoA-T2A2-tuaD plasmid, wherein the sequences of T2A and T2A2 are as SEQ ID NO. 6, SEQ ID respectively Shown in NO.7;

(2) The pGAPZB-kfoC-T2A-kfoA-T2A2-tuaD prepared in step (1) was linearized and transformed into Pichia pastoris GS115 competent cells, and the positive strains were screened and named GS115/CAD;

(3) Amplify the hygromycin gene, replace the bleomycin resistance gene on the pGAPZB plasmid with the hygromycin gene, and obtain the modified plasmid named GAPHyg. Amplify the MET13 gene, connect it to the GAPHyg vector through one-step cloning and assembly, and construct the GAPHyg-MET13 plasmid;

(4) Linearize the plasmid GAPHyg-MET13 of step (3) and transform it into competent cells of GS115/CAD, select positive clones and name it GS115/CADM;

(5) The expression optimization of C4ST and C6ST: The original gene sequences of C4ST and C6ST were truncated at different lengths from the N end (20 amino acids, 40 amino acids, 60 amino acids, 80 amino acids), and truncated them to obtain The N-terminal of the strain with the highest enzyme activity is fused with different tag proteins (SUMO, TrxA, MBP) for optimized expression;

(6) Connect the optimized gene sequences C4ST and C6ST shown in SEQ ID NO. 4 or SEQ ID NO. 5 to the pAO815 vector by homologous recombination to construct pAO815-C4ST and pAO815-C6ST plasmids;

(7) After linearization of the plasmids pAO815-C4ST and pAO815-C6ST constructed in step (6), they were transferred into competent cells GS115/CADM, and positive clones were screened to obtain new strains named GS115/CADMC4 and GS115/CADMC6; Or connect the optimized gene sequences C4ST and C6ST in step (6) to the pAO815 vector using Gibson assembly to construct a pAO815-C4ST-P2A-C6ST plasmid, where the P2A sequence is shown in SEQ ID NO. 8; positive clones are screened to obtain The new strain was named GS115/CADMC4C6.

In one embodiment, the construction method of Saccharomyces cerevisiae producing chondroitin sulfate includes the following steps: the specific steps are as follows:

(1) Connect kfoC and kfoA to the pRS305 vector using Gibson assembly to construct a pRS305-kfoC-P2A-kfoA plasmid, where the P2A sequence is shown in SEQ ID NO.8;

(2) The obtained plasmid pRS305-kfoC-P2A-kfoA was transformed into S.cerevisiae CEN.PK2-1C haploid S. cerevisiae competent cells, and positive clones were screened and named as S. cerevisiae S-CA;

(3) Connect MET13 and the foreign gene tuaD to the pRS303 vector using Gibson assembly to construct a pRS303-tuaD-F2A-MET13 plasmid, where the F2A sequence is shown in SEQ ID NO.9;

(4) The prepared pRS303-tuaD-F2A-MET13 plasmid was integrated into the competent cell genome of Saccharomyces cerevisiae S-CA strain, and positive clones were screened and named as Saccharomyces cerevisiae S-CADM;

(5) Connect the genes C4ST and C6ST shown in SEQ ID NO.4 and SEQ ID NO.5 to the pRS304 vector by homologous recombination to construct pRS304-C4ST and pRS304-C6ST plasmids, and then transfer them into pRS304-C4ST and pRS304-C6ST plasmids. In S-CADM competent cells of Saccharomyces cerevisiae, select positive clones and name them S-CADMC4 and S-CADMC6; or link the optimized genes C4ST and C6ST to the pRS305 vector through Gibson assembly to construct pRS305-C4ST-T2A- The C6ST plasmid, in which the T2A sequence is shown in SEQ ID NO. 6, was transferred into S-CADM competent cells of Saccharomyces cerevisiae, and positive clones were screened and named S-CADMC4C6.

The third objective of the present invention is to provide the application of the strain in the production of chondroitin sulfate or its derivative products.

In one embodiment, the application is to use a medium containing 40-60 g/L glucose as a fermentation medium, inoculate the genetically engineered bacteria into the fermentation medium, and ferment for 80-120 h at 25-35°C.

In one embodiment, the fermentation medium further contains yeast extract, peptone, potassium phosphate buffer, MnSO ₄ and an amino acid mixture.

In one embodiment, the fermentation also controls the following conditions: 1-5 vvm aeration volume, 300-900 rpm to ensure that the dissolved oxygen is not less than 30%, pH is maintained at 5-7, and after 48 hours of fermentation, a constant flow rate of sterile 500g is added. /L glucose solution to ensure that the residual sugar is maintained at 1-2g/L.

The present invention also claims a composition containing the genetically engineered bacteria.

In one embodiment, the composition includes, but is not limited to, a cytoprotective agent.

The beneficial effects of the present invention:

1. The present invention uses yeast as a host for the first time, and strengthens the PAPS supply system by integrating and expressing genes in the synthesis pathway of chondroitin sulfate, thereby realizing the one-step synthesis of chondroitin sulfate in yeast.

2. The genetically engineered bacteria of Pichia pastoris and Saccharomyces cerevisiae of the present invention directly synthesize chondroitin sulfate by metabolizing glycerol, methanol or glucose, realizing the synthesis of chondroitin sulfate A, C, E of specific structure in microorganisms; genetically engineered bacteria GS115 /CADMC4 chondroitin sulfate A yields 125mg/L, genetically engineered strain GS115/CADMC6 chondroitin sulfate C yields 54mg/L, genetically engineered strain GS115/CADMC4C6 chondroitin sulfate E yields 36mg/L; genetically engineered strain S-CADMC4 The yield of chondroitin sulfate A was 75 mg/L, the yield of genetically engineered strain S-CADMC6 chondroitin sulfate C was 48 mg/L, and the yield of genetically engineered strain S-CADMC4C6 chondroitin sulfate E was 34 mg/L.

3. Compared with the chondroitin sulfate obtained by the traditional tissue extraction method, the chondroitin sulfate directly synthesized by the microbial cells in the present invention has a uniform product structure, no potential pathogenic factors, and the quality and safety of the product can be guaranteed.

4. The present invention uses microorganisms to directly synthesize chondroitin sulfate. Compared with other in vitro enzymatic synthesis of chondroitin sulfate, it simplifies the cumbersome operation, avoids the in vitro enzyme extraction, purification and post-catalysis process, and significantly improves production Efficiency reduces costs.

Description of the drawings

Figure 1 is a schematic diagram of the metabolic network of the genetically engineered strain GS115/CADMC4 chondroitin sulfate.

Figure 2 is a map of partially constructed recombinant plasmids, 2-1: pGAPZB-kfoC-T2A-kfoA-T2A2-tuaD, 2-2: pGAPHyg-MET13, 2-3: pAO815-C4ST, 2-4: pAO815-C6ST, 2 -5: pAO815-C4ST-P2A-C6ST.

Figure 3 shows the enzyme activity of Pichia pastoris expressing different truncated lengths of C4ST and C6ST; 1 to 5 represent the original length, truncated 20 amino acids, truncated 40 amino acids, truncated 60 amino acids, and truncated 80 amino acids, respectively Amino acids.

Figure 4 shows the effects of different fusion proteins on the activity of Pichia pastoris expressing C4ST and C6ST enzymes; among them, CK means control.

Figure 5 is an LC-MS image of chondroitin sulfate CSA produced by recombinant strain GS115/CADMC4.

Figure 6 is an LC-MS diagram of CSC produced by recombinant strain GS115/CADMC6.

Figure 7 is an LC-MS diagram of CSE produced by recombinant strain GS115/CADMC4C6.

Fig. 8 is an LC-MS diagram of CSA produced by recombinant strain S-CADMC4.

Figure 9 is an LC-MS diagram of CSC produced by recombinant strain S-CADC6.

Figure 10 is an LC-MS diagram of CSE produced by recombinant strain S-CADMC4C6.

Detailed ways

material:

1. Pichia pastoris GS115 and Saccharomyces cerevisiae CEN.PK2-1C (coded as S288C) were purchased from NTCC Type Culture Collection.

2. PrimeSTAR DNA polymerase, phosphorylase, DNA Marker, Solution I, AvrII and other enzyme reagents were purchased from TaKaRa (Dalian).

3. ClonExpress one-step directed cloning kit was purchased from Vazyme Biotech (Nanjing).

4. Glue recovery kit, EcoRI, NotI, KpnI and other fast cutting enzymes were purchased from Thermo Fisher Scientific.

5. Plasmid extraction kit was purchased from Bioengineering (Shanghai) Co., Ltd.

6. Various analytical reagents were purchased from Sinopharm Group.

7. For the preparation and transformation steps of GS115 competence, please refer to Thermo Fisher Invitrogen's Pichia EasyCompo Kit, S.cerevisiae CEN.PK2-1C Saccharomyces cerevisiae strain Competent preparation and transformation procedures refer to the "Yeast Genetics Method Experimental Guide (Second)" by Scientific Press Edition)" pages 98-99.

8. Medium: LB solid medium (g/L): peptone 10, yeast powder 5, sodium chloride 10, agar powder 20.

LB liquid medium (g/L): peptone 10, yeast powder 5, sodium chloride 10.

Seed medium (g/L): peptone 20, yeast powder 10, glucose 20.

Defective medium plate (g/L): Yeast inorganic nitrogen source medium 6.7, glucose 20; add histidine, tryptophan, leucine and uracil as needed to make the final concentration in the medium 50μg /mL, natural pH. Add 20g/L of agar powder when preparing the solid medium.

Pichia pastoris genetically engineered bacteria fermentation medium (g/L): glycerol 40, K ₂ SO ₄ 18, MgSO ₄ ·7H ₂ O 14.9, KOH 4.13, 85% H ₃ PO ₄ 26.7mL L ^–1 , CaSO ₄ · 2H ₂ O 0.93, 4.35mL·L ^–1 PTM1 trace element

Among them, PTM1(g·L ^–1 ): CuSO ₄ ·5H ₂ O 6, KI 0.09, MnSO ₄ ·H2O 3, H ₃ BO ₃ 0.02, MoNa ₂ O ₄ ·2H ₂ O 0.2, CoCl ₂ ·6H ₂ O 0.5 , ZnCl ₂ 20, FeSO ₄ ·7H ₂ O 65, Biotin 0.2, H ₂ SO ₄ 5.0 mL.

Saccharomyces cerevisiae genetically engineered bacteria fermentation medium (g/L): yeast powder 10; peptone 20; glucose 40; potassium phosphate buffer 100mmol/L, pH 6.0, MnSO _{4 2} , 100×amino acid mixture 10ml/L.

The 100×amino acid mixture is: L-histidine, L-glutamic acid, L-glutamine, L-methionine, L-lysine, L-leucine and L-isoleucine 0.5 each g is dissolved in 100ml of water together, filtered and sterilized.

The specific primers designed in the examples are shown in Table 1.

Table 1 Primer sequence list

Example 1: Construction of pGAPZB-kfoC-T2A-kfoA-T2A2-tuaD plasmid and strain GS115/CAD

(1) Take the synthetic genes kfoC, kfoA and tuaD (sequences shown in SEQ ID NO. 1 to 3 respectively) as templates, and use primers kfoC-F/kfoC-R, kfoA-F/kfoA-R, tuaD-F /tuaD-R were PCR amplified 3 genes, and Gibson assembly was used to connect to pGAPZB vector to construct pGAPZB-kfoC-T2A-kfoA-T2A2-tuaD plasmid, in which T2A and T2A2 sequences were designed on the primers, and their complete sequences were constructed into pGAPZB-kfoC-T2A-kfoA-T2A2-tuaD plasmids. It is SEQ ID NO. 6, SEQ ID NO. 7;

(2) Prepare Pichia pastoris GS115 competent cells, and transform the plasmid pGAPZB-kfoC-T2A-kfoA-T2A2-tuaD obtained above into competent cells after being linearized with the rapid digging enzyme AvrII, through bleomycin resistance The positive clones were screened on the plate, and the genes kfoC, kfoA and tuaD were integrated, and the strain was named GS115/CAD.

Example 2: Construction of pGAPHyg-MET13 plasmid

Extract the Saccharomyces cerevisiae GS115/CAD genome, design primers MET13-F/MET13-R to amplify the endogenous gene MET13, and use one-step cloning assembly to connect to the GAPZB modified vector GAPHyg to construct the GAPHyg-MET13 plasmid.

Example 3: Construction of GS115/CADM strain

Prepare GS115/CAD recombinant bacteria competent cells, linearize the GAPHyg-MET13 plasmid obtained in Example 2 with the quick-cut enzyme AvrII, and transform it into GS115/CAD recombinant bacteria competent cells, and screen with hygromycin resistance plate to get positive The clone is the GS115/CADM recombinant strain.

Example 4: Expression optimization of C4ST and C6ST

The N-terminals of the C4ST and C6ST sequences of genes were truncated with 20 amino acids as a length, and the truncated lengths were 20 amino acids, 40 amino acids, 60 amino acids, and 80 amino acids, respectively. Using the full-length sequence of C4ST and C6ST as templates, using primers C4ST-F, 20C4ST-F, 40C4ST-F, 60C4ST-F, 80C4ST-F, C4ST-R, C6ST-F, 20C6ST-F, 40C6ST-F, 60C6ST- F, 80 C6ST-F, C6ST-R were respectively amplified by PCR and truncated two genes of different lengths, cloned in one step and connected to pAO815 vector to construct a series of plasmids, and electrotransformed the constructed plasmids into Pichia pastoris, after fermentation and culture The purified enzyme is subjected to enzyme activity determination. The obtained strains with the highest enzyme activity were pAO815-60C4ST and pAO815-20C6ST, that is, when the N-terminal of C4ST protein was truncated by 60 amino acids and the N-terminal of C6ST protein was truncated by 20 amino acids, the corresponding enzyme activity was the highest, 26U/L, respectively. 10.5U/L (Figure 3).

Then based on the constructed pAO815-60C4ST and pAO815-20C6ST plasmids, SUMO (Genbank accession number is NP_010798.1), TrxA (Genbank accession number is AFG42725.1), MBP protein ( Genbank accession number is NP_418458.1). The constructed strain was fermented at 30°C for 96h, and purified according to (refer to the purification steps disclosed in A microbial-enzymatic strategy for producing chondroitin sulfate glycosaminoglycans ). The purified enzyme is subjected to enzyme activity determination. The results are shown in Figure 4, where C4ST has the highest enzyme activity when fused with SUMO protein, and its enzyme activity is 63.5 U/L. When C6ST is fused with MBP protein, the enzyme activity is the highest, and its enzyme activity is 33.5U/L.

Example 5: Construction of pAO815-C4ST-P2A-C6ST plasmid

Using C4ST, C6ST (sequences such as SEQ ID NO.4 and SEQ ID NO.5) as templates, and primers 2C4ST-F/2C4ST-R, 2C6ST-F/2C6ST-R, PCR amplification of C4ST and C6ST genes was performed, respectively. The C-terminal of the C4ST gene and the N-terminal of the C6ST gene were respectively added with a section of P2A short peptide, and then connected to the pAO815 vector by Gibson assembly, screened with ampicillin resistance plate, selected colony PCR to verify the correct strain for sequencing, and constructed The pAO815-C4ST-P2A-C6ST plasmid containing the P2A sequence. The P2A sequence is designed on the primer, and its complete sequence is SEQ ID NO.8.

Example 6: Construction of GS115/CADMC4, GS115/CADMC6 and GS115/CADMC4C6 strains

Prepare GS115/CADM recombinant bacteria competent cells according to the method described in Thermo Fisher Invitrogen's Pichia EasyCompo Kit, and linearize and recover the above plasmids pAO815-SUMO-60C4ST, pAO815-MBP-20C6ST and pAO815-C4ST-P2A-C6ST with the quick-cut enzyme SalI Then they were transferred into competent cells GS115/CADM, and positive clones were screened with histidine-deficient plates to obtain GS115/CADMC4, GS115/CADMC6 and GS115/CADMC4C6 strains.

Example 7: Fermentation of Pichia pastoris engineering bacteria to produce chondroitin sulfate

The recombinant strains GS115/CADMC4, GS115/CADMC6 and GS115/CADMC4C6 were subjected to 3-L fed-batch fermentation. Firstly, a single colony is obtained by dividing and streaking. Pick a single colony and inoculate it in 5ml YPD liquid medium, culture it at 30℃220rpm for 16-18h, and then transfer it to three bottles of 50mLYPD liquid medium according to the 10% inoculum. Cultivate at 220 rpm for about 24 hours, and then inoculate 15% in a 3-L fermentor containing 1L of fermentation medium. The fermentation temperature is controlled to 28°C, pH is 5.5, aeration volume is 4.0vvm, and the stirring speed is related to dissolved oxygen. Control the dissolved oxygen at 30%, and the stirring speed at 300-1000 rpm. After the glycerin in the fermentation medium is consumed, continue to starve and culture for 2-3 hours, and then feed 50% (v/v) glycerol (containing 12mL/L PTM1) with a constant flow rate of 20mL· h ^-1 ·L ^-1 , continue starvation culture for 2 hours after the end of the feed, enter the methanol induction phase, and the speed will not change. Methanol containing 12mL·L ^-1 PTM1 was used for induction and the final concentration was controlled at 18g/L. The rate of methanol flow and the final concentration of methanol in the culture medium were controlled by a methanol detector in real time.

Collect the bacteria obtained by fermentation, wash the bacteria with deionized water twice, then resuspend the bacteria, use high pressure homogenate to break the wall and centrifuge to obtain the intracellular supernatant. Place the intracellular supernatant in a 70°C water bath to heat and precipitate part of the protein, and collect the supernatant after centrifugation. Add 3 times the pre-cooled absolute ethanol to the supernatant to precipitate the chondroitin sulfate, stir well and centrifuge to obtain the precipitate. The precipitate was re-dissolved in deionized water, and 3 times of pre-cooled absolute ethanol was added to precipitate the chondroitin sulfate. The precipitate obtained by centrifugation, drying, and re-dissolved in 20mM Tris-HCl (pH8.0) is the chondroitin sulfate sample. Take 500μl chondroitin sulfate sample, add 5μl chondroitin sulfate lyase ABCI, and place it in a 37℃ water bath for 12h. The lysed solution was heated at 90°C for 10 minutes to inactivate and denature the protein. After centrifugation, the supernatant was taken for LC-MS detection.

LC-MS detection uses Acquity UPLC BEH Amide column (1.7μm, 2.1×100mm, Waters, MA, USA). The eluent A is acetonitrile, and the eluent B is ultrapure water. The pH value is adjusted to 10.4 with ammonia water. The elution gradient used is set as follows: 0-2 minutes, 5% B; 2-3 minutes, 5-30% B; 3-6 minutes, 30-60% B; 6-8 minutes, 60% B. The column temperature was maintained at 40°C, and the flow rate was 0.2 mL/min. Scan and monitor the mass range of m/z 100-800 in negative ion mode. The disaccharide molecules of chondroitin sulfate A and chondroitin sulfate C should have a mass-to-charge ratio of 458 in the negative ion mode, and the disaccharide molecules of chondroitin sulfate E should have a mass-to-charge ratio of 538 in the negative ion mode. It can be seen from the mass spectrometry results that the synthesis of chondroitin sulfate A, chondroitin sulfate C and chondroitin sulfate E has been achieved.

It was determined that the yield of genetically engineered bacteria GS115/CADMC4 chondroitin sulfate A was 125 mg/L, the yield of genetically engineered bacteria GS115/CADMC6 chondroitin sulfate C was 54 mg/L, and the yield of genetically engineered bacteria GS115/CADMC4C6 chondroitin sulfate E was 36 mg/L. .

Example 8: Construction of pRS305-kfoC-P2A-kfoA plasmid and construction of S. cerevisiae S-CA

(1) Using the synthetic genes kfoC and kfoA as templates, using primers skfoC-F/skfoC-R, skfoA-F/skfoA-R, PCR amplification of the two genes respectively, using Gibson assembly to connect to the vector pRS305 to construct Into pRS305-kfoC-P2A-kfoA plasmid, in which the P2A sequence is designed on the primer, and its complete sequence is the same as SEQ ID NO.8; screened with ampicillin resistance plate, selected colony PCR to verify the correct strain for sequencing, and obtained pRS305 with correct sequence -kfoC-P2A-kfoA plasmid.

Prepare S.cerevisiae CEN.PK2-1C haploid Saccharomyces cerevisiae competent cells, transform the above-obtained plasmid pRS305-kfoC-P2A-kfoA into competent cells, screen positive clones through leucine-deficient plates, and get integrated The strain of genes kfoC and kfoA was named S-CA.

Example 9: Construction of pRS303-tuaD-F2A-MET13 plasmid and S-CADM strain

Using the S. cerevisiae S288C genome and the synthetic gene tuaD (shown in SEQ ID NO. 3) as templates, and primers sMET13-F/sMET13-R, stuaD-F/stuaD-R, PCR was performed to amplify the endogenous genes MET13 and The exogenous gene tuaD was assembled by Gibson and connected to the expression vector pRS303, screened with ampicillin resistance plate, selected colony PCR to verify the correct strain and sequenced, and finally constructed into pRS303-tuaD-F2A-MET13 plasmid, in which the F2A sequence was designed in the primer Above, its complete sequence is SEQ ID NO.9.

The genome of S. cerevisiae S288C strain was extracted, and the endogenous gene MET13 and exogenous gene tuaD were amplified with primers sMET13-F/sMET13-R, stuaD-F/stuaD-R, and connected to pRS303 vector using Gibson assembly to construct pRS303- tuaD-F2A-MET13 plasmid.

Prepare competent cells of strain S-CA constructed in Example 6, linearize plasmid pRS303-tuaD-F2A-MET13 and integrate it into the genome of Saccharomyces cerevisiae S-CA, and screen with leucine and tryptophan double defect plates The positive clone is the S-CADM strain.

Example 10: Construction of S-CADMC4 and S-CADMC6 strains

Using the optimized C4ST and C6ST genes (sequences such as SEQ ID NO. 4 and SEQ ID NO. 5) obtained in Example 4 as a template, primers sC4ST-F/sC4ST-R, sC6ST-F/sC6ST-R, Carry out PCR amplification of C4ST, C6ST two genes respectively, homologous recombination connected to pRS304 vector, construct pRS304-C4ST and pRS304-C6ST plasmid;

Prepare S-CADM recombinant bacteria competent cells, linearize plasmids pRS304-C4ST and pRS304-C6ST, respectively transform them into S-CADM competent cells, screen positive clones with tryptophan deficient plates to obtain S-CADMC4 and S -CADMC6 strain.

Example 11: Construction of pRS304-C4ST-T2A-C6ST plasmid and S-CADMC4C6 strain

Using the optimized C4ST and C6ST genes (sequences such as SEQ ID NO.4, SEQ ID NO.5) obtained in Example 4 as templates, primers s2C4ST-F/s2C4ST-R, s2C6ST-F/s2C6ST-R, PCR was performed to amplify the C4ST and C6ST genes, and the Gibson assembly was used to connect to the expression vector pRS304, screened with ampicillin resistance plates, and the colony PCR verified the correct strains for sequencing, and finally constructed pRS304-C4ST-T2A-C6ST plasmid (among which The T2A sequence is designed on the primer, and its complete sequence is SEQ ID NO. 6).

Prepare S-CADM recombinant bacteria competent cells, linearize pRS304-C4ST-T2A-C6ST plasmid, and transform into S-CADM recombinant bacteria competent cells, screen positive clones with uracil-deficient plates to obtain S-CADMC4C6 strain.

Example 12: Tank fermentation of chondroitin sulfate producing strain

The recombinant strains S-CADMC4, S-CADMC6 and S-CADMC4C6 were subjected to 3-L fed-batch fermentation. Firstly, a single colony is obtained by dividing and streaking. Pick a single colony and inoculate it in 50ml YPD liquid medium. Incubate at 30°C and 220rpm for 16-18h until the OD ₆₀₀ of the bacteria reaches about 6, and transfer to fresh _{with the initial OD 600 = 0.4} In the 250ml YPD liquid medium, cultured at 30℃220rpm for about 12h until the OD _{600 of the} bacteria was about 12, and then inoculated at 15% in a 3-L fermentor containing 1L fermentation medium. The fermentation temperature was controlled to 30℃ and pH It is 6, the aeration rate is 2vvm, the stirring speed is related to the dissolved oxygen, the dissolved oxygen is controlled at 30%, and the stirring speed is at 300-900rpm. Sampling is taken every 6h to determine the glucose concentration, and the glucose flow rate is adjusted in time according to the glucose consumption rate to stabilize the glucose concentration at 1-2g/L. The fermentation period is 96h. Among them, the acid solution for adjusting the pH is 10% phosphoric acid, and the lye is 20% ammonia water.

According to the method of Example 5, the chondroitin sulfate in the fermentation broth was detected. It was determined that the yield of genetically engineered strain S-CADMC4 chondroitin sulfate A was 75 mg/L after 96 hours of fermentation, the yield of genetically engineered strain S-CADMC6 chondroitin sulfate C was 48 mg/L, and the yield of genetically engineered strain S-CADMC4C6 chondroitin sulfate E was 34mg/L.

Although the present invention has been disclosed as above in preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (10)

1. A yeast producing chondroitin sulfate, characterized in that it expresses (a), (b) or (c); or contains the gene shown in (d), (e) or (f); wherein,

   (a) Chondroitin synthase, UDP-N-acetylglucosamine C4 isomerase, UDP-glucose dehydrogenase, chondroitin 4-O-sulfatase C4ST and ATP sulfurylase;

   (b) Chondroitin synthase, UDP-N-acetylglucosamine C4 isomerase, UDP-glucose dehydrogenase and chondroitin 6-O-sulfatase C6ST, ATP sulfurylase;

   (c) Chondroitin synthase, UDP-N-acetylglucosamine C4 isomerase, UDP-glucose dehydrogenase, chondroitin 4-O-sulfatase C4ST, chondroitin 6-O-sulfatase C6ST and ATP Sulfurylase

   (d) DNA sequences containing kfoC, kfoA, tuaD, C4ST and MET13;

   (e) DNA sequences containing kfoC, kfoA, tuaD, C6ST and MET13;

   (f) DNA sequences containing kfoC, kfoA, tuaD, C4ST, C6ST and MET13.
2. The yeast according to claim 1, wherein the KfoC and KfoA are derived from Escherichia coli K4; the tuaD is derived from Bacillus subtilis 168; the C4ST and C6ST are derived from Musmusculus; The ATP sulfurylase MET13 is from Saccharomyces cerevisiae.
3. The yeast according to claim 1 or 2, wherein the host cell is Pichia pastoris, Saccharomyces cerevisiae, or a mutant or mutagenic strain of Pichia pastoris or Saccharomyces cerevisiae.
4. A starter, characterized in that it contains the yeast according to any one of claims 1 to 3 ^{with a cell concentration ≥ 1×10 5} CFU/g or 1×10 ^{5 CFU/mL.}
5. A method for constructing the yeast of any one of claims 1 to 3, characterized in that (a), (b) or (c) are linked to the genome of the yeast; wherein,

   (a) DNA sequence containing: kfoC, kfoA, tuaD and C4ST;

   (b) DNA sequences containing kfoC, kfoA, tuaD and C6ST genes;

   (c) DNA sequences containing kfoC, kfoA, tuaD, C4ST and C6ST.
6. Use of the yeast according to any one of claims 1 to 3 in the production of chondroitin sulfate or its derivative products.
7. A method for producing chondroitin sulfate, characterized in that a medium containing 40-60 g/L glucose is used as a fermentation medium, and the yeast according to any one of claims 1 to 3 or the starter according to claim 4 Inoculate into the fermentation medium and ferment for 80-120h at 25-35°C.
8. The method according to claim 7, characterized in that the fermentation also controls the following conditions: 1-5 vvm ventilation, dissolved oxygen not less than 30%, and pH maintained at 5-7.
9. The method according to claim 8, characterized in that the glucose solution is added after 46 to 48 hours of fermentation to maintain the residual sugar in the fermentation process at 1-2 g/L.
10. Use of the yeast according to any one of claims 1 to 3 or the starter according to claim 4 in the preparation of foods, medicines, and health products containing chondroitin sulfate or its derivative products.

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