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WO2021077581A1 - Levure modifiée pour la production par fermentation de sulfate de chondroïtine, et son utilisation - Google Patents

Levure modifiée pour la production par fermentation de sulfate de chondroïtine, et son utilisation Download PDF

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WO2021077581A1
WO2021077581A1 PCT/CN2019/126014 CN2019126014W WO2021077581A1 WO 2021077581 A1 WO2021077581 A1 WO 2021077581A1 CN 2019126014 W CN2019126014 W CN 2019126014W WO 2021077581 A1 WO2021077581 A1 WO 2021077581A1
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c6st
c4st
chondroitin sulfate
kfoa
kfoc
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康振
金学荣
陈坚
堵国成
李江华
张天萌
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Jiangnan University
Bloomage Biotech Co Ltd
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Bloomage Biotech Co Ltd
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    • C12Y208/02017—Chondroitin 6-sulfotransferase (2.8.2.17)

Definitions

  • 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.
  • Chondroitin sulfate 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.
  • 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).
  • 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.
  • 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.
  • the synthesis of chondroitin sulfate by microorganisms can effectively avoid the above problems.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • the gene sequences of kfoC, kfoA, tuaD, C4ST, and C6ST are shown in SEQ ID NOs. 1 to 5, respectively.
  • the yeast is Pichia pastoris or Saccharomyces cerevisiae and a mutant or mutagenic strain thereof.
  • the yeast is Pichia GS115 or Saccharomyces cerevisiae S-CA.
  • the gene is expressed by a plasmid, or is integrated and expressed on the genome of the host.
  • 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,
  • the method for constructing Pichia pastoris that produces chondroitin sulfate includes the following steps: the specific steps are as follows:
  • 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;
  • step (1) 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;
  • 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;
  • the construction method of Saccharomyces cerevisiae producing chondroitin sulfate includes the following steps: the specific steps are as follows:
  • 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 third objective of the present invention is to provide the application of the strain in the production of chondroitin sulfate or its derivative products.
  • 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.
  • the fermentation medium further contains yeast extract, peptone, potassium phosphate buffer, MnSO 4 and an amino acid mixture.
  • 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.
  • the composition includes, but is not limited to, a cytoprotective agent.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • Pichia pastoris GS115 and Saccharomyces cerevisiae CEN.PK2-1C (coded as S288C) were purchased from NTCC Type Culture Collection.
  • PrimeSTAR DNA polymerase, phosphorylase, DNA Marker, Solution I, AvrII and other enzyme reagents were purchased from TaKaRa (Dalian).
  • ClonExpress one-step directed cloning kit was purchased from Vazyme Biotech (Nanjing).
  • Glue recovery kit EcoRI, NotI, KpnI and other fast cutting enzymes were purchased from Thermo Fisher Scientific.
  • Plasmid extraction kit was purchased from Bioengineering (Shanghai) Co., Ltd.
  • 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 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 50 ⁇ g /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
  • 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 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.
  • Example 1 Construction of pGAPZB-kfoC-T2A-kfoA-T2A2-tuaD plasmid and strain GS115/CAD
  • 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.
  • 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 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).
  • SUMO Genebank accession number is NP_010798.1
  • TrxA Genebank 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.
  • 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 P2A sequence is designed on the primer, and its complete sequence is SEQ ID NO.8.
  • GS115/CADM recombinant bacteria competent cells 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°C220rpm 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.
  • glycerin in the fermentation medium 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 500 ⁇ l chondroitin sulfate sample, add 5 ⁇ l chondroitin sulfate lyase ABCI, and place it in a 37°C 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 (1.7 ⁇ m, 2.1 ⁇ 100mm, Waters, MA, USA).
  • the eluent A is acetonitrile
  • 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
  • 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.
  • Example 8 Construction of pRS305-kfoC-P2A-kfoA plasmid and construction of S. cerevisiae S-CA
  • 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.
  • Example 11 Construction of pRS304-C4ST-T2A-C6ST plasmid and S-CADMC4C6 strain
  • Example 12 Tank fermentation of chondroitin sulfate producing strain
  • the fermentation temperature was controlled to 30°C 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.
  • the acid solution for adjusting the pH is 10% phosphoric acid, and the lye is 20% ammonia water.
  • 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.

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Abstract

L'invention concerne une levure modifiée pour la production par fermentation de sulfate de chondroïtine, et son utilisation. Via l'utilisation d'une technologie de biologie synthétique et d'un moyen d'ingénierie génétique, et avec Pichia pastoris GS115 et Saccharomyces cerevisiae en tant que souches de départ, des gènes liés à une voie de synthèse de sulfate chronique sont exprimés de manière hétérologue dans des cellules : les gènes kfoC et kfoA provenant d'Escherichia coli K4, les gènes C4ST Et C6ST de chondroïtine sulfate provenant de souris, le gène de l'UDP-glucose déshydrogénase tuaD provenant de Bacillus subtilis, et le gène MET13 d'ATP sulfurylase de Saccharomyces cerevisiae, pour obtenir une souche de production synthétisant du sulfate de chondroïtine A (CSA), du sulfate de chondroïtine C (CSC) et du sulfate de chondroïtine E (CSE). Pour la première fois, une source de carbone de fermentation de micro-organismes est utilisée pour synthétiser différentes formes de sulfate de chrondroitine.
PCT/CN2019/126014 2019-10-24 2019-12-17 Levure modifiée pour la production par fermentation de sulfate de chondroïtine, et son utilisation Ceased WO2021077581A1 (fr)

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