Method for improving II type full-length collagen production capacity of pichia pastoris

Abstract

The invention discloses a method for improving the production capacity of II type full-length collagen of pichia pastoris. The method comprises the following steps: in a methanol-induced pichia pastoris fermentation production system, adding three stabilizers, namely dimethyl sulfoxide (DMSO), 4-phenylbutyric acid (4-PBA) and tetramethylurea (TMU), into a fermentation culture medium in a combined manner. According to the method, the yield of the complete recombinant human II type full-length collagen is remarkably improved, and compared with the sum of target protein yield improvement effects of independently using the three stabilizers, the combined use of the three stabilizers achieves a synergistic target protein yield improvement effect. The method also has the advantages of simple process, low cost, no need of gene modification and easiness in large-scale production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of synthetic biology. Specifically relates to a method for improving the production capacity of Pichia pastoris for fermentative production of type II full-length collagen. BACKGROUND

[0002] Type II collagen is the main structural protein of articular cartilage tissue, and has a broad application prospect in the fields of tissue engineering, medical repair and cosmetics due to its good biocompatibility and biological activity. Using genetic engineering means, especially Pichia pastoris expression system, to produce recombinant human type II full-length collagen has become a research hotspot to solve the problems of limited source and high risk of pathogen in traditional extraction method.

[0003] However, there are still major technical challenges in using Pichia pastoris system to express full-length type II collagen with high yield, mainly in the following two aspects: Aspect one: Type II collagen has a large molecular weight (about 110 kDa) and a complex structure, and its functional unit is a triple helix superhelix structure formed by three alpha chains winding around each other. The correct formation of this structure depends on a large number of proline and hydroxyproline residues and accurate disulfide bond pairing between chains. Although Pichia pastoris has strong protein secretion expression ability, its intracellular oxidative folding environment and chaperone network are still insufficient for such a complex macromolecular protein, resulting in protein misfolding during expression, retention in the endoplasmic reticulum, triggering the unfolded protein response (UPR), and ultimately being recognized and removed by the endoplasmic reticulum-associated degradation pathway (ERAD), resulting in low expression efficiency.

[0004] Aspect two: Even if a small amount of protein is successfully expressed and secreted into the extracellular, it is extremely unstable in high-density fermentation environment. During fermentation, the autolysis of the bacterial cells releases a large amount of endogenous proteases (such as alkaline protease, metalloprotease, etc.), and the loose structure of collagen and its rich Gly-X-Y repeat sequence make it an easy target for protease attack, resulting in severe degradation of the product and the generation of a large amount of inactive small molecule fragments, which not only greatly reduces the final yield of the target protein, but also brings great difficulty to the downstream separation and purification.

[0005] In view of the above technical problems, the skilled person in the art uses technical means such as screening suitable host strains, adding molecular chaperones or epigenetic regulators in the culture medium to help the correct expression of recombinant human type II full-length collagen and avoid its degradation. However, in fact, there are still technical problems such as limited effect of single means and difficulty in achieving synergistic effect of combined means. SUMMARY

[0006] The present application aims to overcome the deficiencies of the prior art and provide a new method for synergistically and efficiently expressing recombinant human type II full-length collagen in a Pichia pastoris system.

[0007] The inventors have conducted in-depth research to solve the above technical problems, and have found that by selecting a specific Pichia pastoris strain as a production strain and selecting specific chaperones and epigenetic regulators as technical means to promote the correct expression of recombinant human type II full-length collagen and avoid its degradation, synergistic effects between the technical means can be achieved, thereby efficiently expressing recombinant human type II full-length collagen.

[0008] Specifically, the present application comprises: 1. A method for fermentatively producing type II full-length collagen, the method comprising: Step A: culturing a Pichia pastoris strain capable of secreting and expressing recombinant human type II full-length collagen in a fermentation medium; Step B: after the carbon source in the fermentation medium is depleted, adding glycerol for fed-batch culture; and Step C: after the glycerol in the fermentation medium is depleted, adding methanol for induction culture, and adding dimethyl sulfoxide (DMSO), 4-phenylbutyric acid (4-PBA), and tetramethylurea (TMU) in the fermentation medium.

[0009] The depletion of the carbon source or the depletion of the glycerol can be indicated by a sharp increase in the dissolved oxygen value of the system, for example, a rapid increase from 20%-30% to 80%-90% within 1 min.

[0010] 2. The method of item 1, wherein dimethyl sulfoxide is added to a final concentration of 0.5%-1.5% (v / v) in the fermentation medium, for example, 1.2% (v / v).

[0011] 3. The method of item 1, wherein 4-phenylbutyric acid is added to a final concentration of 1-10 mM in the fermentation medium, for example, 5 mM can be added.

[0012] 4. The method of item 1, wherein tetramethylurea is added to a final concentration of 10-100 mM in the fermentation medium, for example, 50 mM can be added.

[0013] 5. The method of item 1, wherein the Pichia pastoris strain is Pichia pastoris GS115 or X33.

[0014] In the step A, the Pichia pastoris strain can be Pichia pastoris GS115 or X33. The Pichia pastoris strain secreting and expressing the recombinant type II full-length collagen can be constructed by using the methods commonly used by those skilled in the art. For example, the codon optimization of the yeast expression system can be performed according to the amino acid sequence of the recombinant type II full-length collagen to obtain the target gene sequence, the obtained target gene sequence is subjected to gene synthesis, the synthesized gene is connected into the pPic9K plasmid to obtain the pPic9K-II plasmid. Then the pPic9K-II is linearized by using the restriction enzyme SacII and XhoI, and the linearized pPic9K-II is transformed into the competent cells of Pichia pastoris GS115 or X33, and the transformants are screened by using G418 resistance as a screening marker, so as to obtain the yeast expression strain. Sac I linearized and transformed into the competent cells of Pichia pastoris GS115 or X33, and the transformants are screened by using G418 resistance as a screening marker, so as to obtain the yeast expression strain.

[0015] 6. The method according to item 1, wherein the glycerol, methanol, dimethyl sulfoxide, 4-phenylbutyric acid and / or tetramethylurea are added in one portion or as a flow addition.

[0016] 7. The method according to item 1, wherein the amino acid sequence of the recombinant human type II full-length collagen is shown in SEQ ID NO: 1.

[0017] SEQ ID NO: 1 In step B, the fermentation medium can be those commonly used by those skilled in the art when using Pichia pastoris to ferment to produce target proteins, for example, it can be a basic medium, and the formula of the basic medium can be H3PO4 (85%), 13.4 ml / L; CaSO4·2H2O, 0.46 g / L; K2SO4, 9.1 g / L; MgSO4·7H2O, 7.5 g / L, KOH, 2.1 g / L; glycerol, 40 g / L; trace elements, 4.36 ml / L.

[0018] In step B, the culture conditions can be, for example, a temperature of 28-32℃, a pH of 4.5-6.5, and a dissolved oxygen control of 15-30%.

[0019] In step B, the depletion of the carbon source can be indicated by, for example, a sharp rise in the dissolved oxygen value. In step C, the depletion of the glycerol feed can be indicated by, for example, a sharp rise in the dissolved oxygen value, for example, a rapid rise from 20%-30% to 80%-90% within 1 min. In step B, if the glycerol is added by flow addition, the addition of glycerol can be stopped when, for example, OD 600 is 250-350.

[0020] In step B, the culture conditions for methanol induction can be, for example, an induction temperature of 25-28℃ and an induction time of 35-45 h.

[0021] Inventive Effects Compared with the prior art, the present application has the following significant technical effects: 1. The present application combines two chemical chaperones (DMSO / TMU) and one epigenetic regulator (4-PBA), a total of three stabilizers, significantly improves the yield of intact recombinant human type II full-length collagen, and compared with the additive effect of the yield of target proteins when each of the three stabilizers is used alone, the combination of the three stabilizers achieves a synergistic effect of improving the yield of target proteins.

[0022] 2. The recombinant human type II full-length collagen production method of the present application has the advantages of simple process, low cost, no need for genetic modification, and easy scaling. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The SDS-PAGE analysis results of the target protein yield of each example.

[0024] Figure 2 The SDS-PAGE analysis results of the target protein yield of each example. DETAILED DESCRIPTION

[0025] The application will be further described in conjunction with the examples. It should be understood that the examples are only for further illustrating and explaining the application, and are not intended to limit the application.

[0026] Example 1 According to the amino acid sequence of recombinant type II full-length collagen (SEQ ID NO: 1), the codon optimization of the yeast expression system was carried out to obtain the target gene sequence (SEQ ID NO: 3) as follows: CAAATGGCTG GAGGTTTTGA CGAAAAGGCC GGCGGAGCTC AATTAGGAGT TATGCAGGGTCCAATGGGAC 71 CAATGGGACC AAGAGGTCCT CCCGGTCCTG CTGGTGCACC AGGACCACAAGGTTTTCAAG GTAACCCAGG 141 TGAACCAGGT GAACCCGGAG TTAGTGGTCC AATGGGACCTAGAGGTCCTC CTGGTCCACC TGGTAAGCCA 211 GGAGACGATG GAGAGGCCGG TAAACCTGGAAAGGCTGGCG AAAGAGGACC TCCTGGACCA CAGGGCGCCA 281 GGGGTTTTCC CGGAACCCCAGGACTTCCTG GTGTTAAGGG ACATCGTGGT TATCCTGGCT TGGATGGTGC 351 TAAAGGTGAGGCCGGTGCCC CAGGAGTTAA AGGCGAATCC GGATCACCTG GAGAAAATGG TAGTCCTGGT 421 CCAATGGGAC CTCGAGGTCT GCCAGGCGAG AGGGGTCGTA CAGGTCCAGC AGGTGCTGCC GGTGCCAGAG 491 GTAATGATGG TCAGCCAGGT CCTGCTGGCC CTCCAGGTCC CGTTGGTCCT GCCGGAGGTCCAGGATTTCC 561 CGGCGCCCCC GGTGCTAAGG GCGAGGCTGG ACCTACTGGA GCTAGAGGACCAGAAGGTGC ACAAGGTCCT 631 AGAGGCGAGC CAGGAACACC TGGTTCTCCC GGTCCAGCTGGCGCATCAGG TAACCCTGGT ACCGATGGTA 701 TTCCTGGTGC AAAAGGTTCT GCAGGAGCTCCTGGAATCGC CGGTGCACCT GGTTTTCCTG GCCCTAGAGG 771 ACCACCAGGA CCCCAAGGTGCTACTGGACC ATTGGGTCCT AAAGGTCAGA CTGGTGAACC TGGCATCGCT 841 GGTTTCAAAGGTGAGCAGGG CCCAAAAGGT GAGCCAGGTCCAGCCGGACC ACAAGGAGCT CCCGGTCCCG 911 CCGGTGAAGA AGGTAAGAGA GGCGCTAGAG GAGAACCTGG CGGAGTGGGT CCAATCGGAC CTCCTGGTGA981 AAGAGGAGCA CCCGGAAACC GTGGTTTCCC AGGTCAAGAC GGTCTGGCAG GTCCTAAGGGCGCTCCTGGT 1051 GAAAGAGGAC CTTCAGGATT AGCTGGTCCA AAAGGAGCTA ACGGTGACCCTGGACGTCCT GGAGAACCAG 1121 GATTGCCAGG AGCCAGAGGA CTTACCGGAC GTCCTGGCGATGCTGGTCCT CAAGGCAAAG TCGGTCCTAG 1191 TGGTGCTCCT GGAGAAGATG GTAGACCCGGTCCACCCGGT CCACAGGGTG CAAGAGGTCA ACCAGGAGTT 1261 ATGGGATTCC CAGGCCCTAAGGGTGCAAAC GGTGAACCAG GTAAGGCCGG AGAAAAAGGT TTGCCAGGCG 1331 CACCAGGATTACGTGGATTG CCCGGTAAAG ACGGAGAAAC AGGTGCTGCC GGACCACCTG GACCAGCTGG 1401 CCCC GCAGGA GAGAGAGGTG AACAGGGTGC TCCCGGACCC TCTGGTTTCC AAGGTCTTCC AGGTCCTCCT 1471 GGACCTCCAG GAGAAGGAGG AAAGCCTGGT GATCAGGGAG TTCCCGGAGA GGCCGGTGCCCCTGGATTGG 1541 TCGGACCCCG AGGAGAACGA GGATTCCCAG GTGAGCGTGG TAGTCCCGGAGCCCAGGGTT TGCAAGGTCC 1611 TCGTGGCTTA CCAGGTACTC CTGGTACGGA CGGACCAAAGGGCGCTTCTG GTCCTGCTGG ACCACCAGGT 1681 GCTCAAGGTC CACCAGGATT GCAAGGTATGCCTGGCGAAA GAGGCGCAGC CGGAATAGCT GGTCCAAAAG1751 GTGATAGGGG TGACGTTGGAGAGAAGGGTC CAGAGGGTGC ACCTGGTAAG GACGGCGGTA GAGGTCTGAC 1821 CGGCCCAATCGGTCCTCCAG GACCCGCCGG AGCAAACGGA GAAAAGGGTG AAGTTGGCCC TCCCGGACCC 1891GCTGGCAGTG CAGGTGCTAG AGGAGCACCC GGAGAACGAG GCGAGACTGG TCCACCCGGC CCTGCAGGTT1961 TCGCCGGCCC TCCTGGTGCC GATGGACAAC CAGGAGCAAA AGGTGAACAA GGAGAGGCTGGTCAAAAGGG 2031 CGACGCCGGA GCTCCAGGTC CCCAAGGTCC TTCCGGTGCT CCTGGTCCTCAAGGACCAAC TGGTGTCACA 2101 GGACCAAAAG GTGCTAGAGG TGCACAGGGA CCCCCCGGTGCTACAGGTTT TCCTGGAGCC GCTGGTAGAG 2171 TAGGCCCTCC AGGTTCCAAC GGTAACCCAGGCCCTCCTGG TCCACCTGGA CCATCCGGCA AAGATGGTCC 2241 TAAAGGTGCC AGAGGAGACTCAGGACCACC TGGAAGAGCT GGTGAACCCG GACTTCAAGG TCCCGCTGGT 2311 CCTCCAGGAGAAAAGGGAGA GCCAGGCGAT GATGGACCCT CCGGAGCTGA AGGCCCCCCA GGACCTCAAG 2381GCCTTGCTGG TCAACGTGGA ATTGTAGGTT TGCCTGGACA ACGTGGTGAA AGAGGTTTCC CCGGCCTACC2451 AGGACCTTCC GGTGAGCCCG GTAAACAAGG TGCTCCAGGT GCTTCAGGAG ACAGAGGCCCACCCGGTCCA 2521 GTTGGACCAC CTGGTTTGAC AGGACCAGCA GGTGAACCAG GTAGAGAAGGTTCCCCTGGC GCAGATGGTC 2591 CCCCAGGCAG AGATGGTGCCGCTGGTGTTA AGGGAGATAGGGGCGAAACA GGAGCTGTTG GCGCTCCTGG 2661 TGCCCCTGGA CCTCCAGGTA GTCCTGGACCCGCTGGTCCC ACTGGTAAGC AAGGAGATAG GGGAGAGGCT 2731 GGTGCTCAAG GTCCTATGGGTCCTTCTGGT CCCGCTGGTG CTAGGGGAAT TCAGGGTCCA CAAGGACCAA 2801 GAGGTGATAAGGGAGAAGCT GGTGAGCCTG GAGAGAGGATTGAAGGGT CATAGGGGTT TCACCGGTTT 2871 GCAAGGTTTG CCTGGTCCCC CTGGTCCTTC AGGAGACCAA GGAGCCTCTG GTCCCGCTGG TCCAAGTGGA 2941 CCTCGTGGCCC ACCAGGACCAGTCGGACCT TCCGGTAAAG ACGGAGCTAACGGTATACCAGGCCCTATTG 3011 GACCACCCGG TCCAAGAGGT AGATCTGGAG AGACTGGACCTGCCGGACCACCCGGAAATC CAGGTCCTCC 3081 AGGTCCACCA GGTCCCCCTG GCCCAGGAAT TGACATGTCCGCTTTTGCTG GATTGGGACC ACGAGAAAAA 3151 GGTCCAGATC CACTTCAATA CATGAGAGCT。

[0027] The obtained target gene sequence is entrusted to GenScript Biotech Corporation for gene synthesis, and the synthesized gene is connected to pPic9K plasmid to obtain pPic9K-II plasmid. After linearization of pPic9K-II with Sac I, the linearized pPic9K-II is transformed into competent Pichia pastoris GS115, and G418 resistance is used as a screening marker to screen transformants, thereby obtaining a yeast expression strain.

[0028] The recombinant Pichia pastoris expressing human type II full-length collagen is used to produce human type II full-length collagen by fermentation, and the amino acid sequence of the human type II full-length collagen is shown as SEQ ID NO: 1.

[0029] The fermentation medium is: H3PO4(85%), 13.4 ml / L; CaSO4.2H2O, 0.46 g / L; K2SO4, 9.1 g / L; MgSO4.7H2O, 7.5 g / L, KOH, 2.1 g / L; glycerol, 40 g / L; trace elements, 4.36 ml / L.

[0030] The seed liquid is inoculated into a 125 L fermenter containing 50 L of fermentation medium at an inoculation amount of 10%. The initial culture parameters are stirring speed 100 rpm, tank pressure 0.05 MPa, the dissolved oxygen (DO) is maintained above 30% by adjusting the air flow and speed, the culture temperature is 30°C, and the pH is controlled at 5.0 by automatic ammonia water flow.

[0031] After the base carbon source is depleted (indicated by a sharp rise in dissolved oxygen value), the feeding culture is started by flowing 50% glycerol solution, and the OD 600 is 300.

[0032] After the glycerol is depleted (indicated by a sharp rise in dissolved oxygen value), the induction is started by flowing methanol, and no stabilizer is added. The fermentation condition parameters are controlled during the induction stage: temperature 23°C, pH 6.5, and the dissolved oxygen is controlled above 15%, and the induction time is 40 h.

[0033] After the fermentation broth is centrifuged, the fermentation broth supernatant is subjected to gray scale scanning quantification by protein electrophoresis (SDS-PAGE analysis), and it is found that the yield of the human type II full-length collagen protein is 4.01 g / L.

[0034] Example 2 The fermentation production of human type II full-length collagen protein is carried out as in Example 1 above, except that DMSO is added to a final concentration of 1.2% (v / v) at once while flowing methanol for induction. Gray scale scanning quantification by protein electrophoresis (SDS-PAGE analysis) shows that the yield of the human type II full-length collagen protein is 4.81 g / L.

[0035] Example 3 The fermentation production of human type II full-length collagen protein is carried out as in Example 1 above, except that TMU is added to a final concentration of 50 mM at once while flowing methanol for induction. Gray scale scanning quantification by protein electrophoresis (SDS-PAGE analysis) shows that the yield of the human type II full-length collagen protein is 5.41 g / L.

[0036] Example 4 The fermentation production of human type II full-length collagen was carried out as in Example 1 above, except that 4-PBA was added to a final concentration of 5 mM at the same time as the methanol was fed for induction. The yield of the human type II full-length collagen was 4.85 g / L, as quantified by gray scale scanning of protein electrophoresis (SDS-PAGE analysis).

[0037] Example 5 The fermentation production of human type II full-length collagen was carried out as in Example 1 above, except that DMSO was added to a final concentration of 1.2% (v / v), TMU was added to a final concentration of 50 mM, and 4-PBA was added to a final concentration of 5 mM at the same time as the methanol was fed for induction. The yield of the human type II full-length collagen was 9.51 g / L, as quantified by gray scale scanning of protein electrophoresis (SDS-PAGE analysis).

[0038] Comparative Example 1 The preparation of the engineered bacteria and the protein production capacity test were carried out as in Example 1 above, except that the target gene in the recombinant expression vector pPIC9K-II was replaced with a recombinant type III collagen gene optimized according to the preferences of Pichia pastoris, and the amino acid sequence of the recombinant type III collagen is shown as SEQ ID NO: 2.

[0039] SEQ ID NO: 2: The yield of the recombinant collagen type III was 3.67 g / L, as determined by gray scale scanning quantification of protein electrophoresis (SDS-PAGE analysis).

[0040] Comparative Example 2 The same procedure as in Example 2 was used, except that the fermentation strain and target protein were as described in Comparative Example 1. The yield of the recombinant collagen type III was 4.59 g / L, as determined by gray scale scanning quantification of protein electrophoresis (SDS-PAGE analysis).

[0041] Comparative Example 3 The same procedure as in Example 3 was used, except that the fermentation strain and target protein were as described in Comparative Example 1. The yield of the recombinant collagen type III was 5.20 g / L, as determined by gray scale scanning quantification of protein electrophoresis (SDS-PAGE analysis).

[0042] Comparative Example 4 The same procedure as in Example 4 was used, except that the fermentation strain and target protein were as described in Comparative Example 1. The yield of the recombinant collagen type III was 4.63 g / L, as determined by gray scale scanning quantification of protein electrophoresis (SDS-PAGE analysis).

[0043] Comparative Example 5 The same procedure as in Example 5 was used, except that the fermentation strain and target protein were as described in Comparative Example 1. The yield of the recombinant collagen type III was 7.57 g / L, as determined by gray scale scanning quantification of protein electrophoresis (SDS-PAGE analysis).

[0044] The stabilizers used in each of the examples and comparative examples and the results of target protein yield determination are shown in Table 1. In addition, the synergistic index (CI) of the combination of the three stabilizers DMSO, TMU and 4-PBA was calculated and is also shown in Table 1.

[0045] wherein CI = P ABC / P A x P B x P C P A , P B , P C respectively represent the fold of target protein yield relative to the case where no stabilizer was used (Example 1) when DMSO, TMU and 4-PBA were used alone; P ABC represents the fold of target protein yield relative to the case where no stabilizer was used (Example 1) when DMSO, TMU and 4-PBA were used in combination.

[0046] CI > 1 indicates that the effect of the combination of the three on the yield of the target protein is greater than the sum of the effects of the three used alone, i.e., the combination of the three has a synergistic effect. CI > 1.1 indicates that the synergistic effect is significant.

[0047] Table 1

Claims

1. A method for fermenting and producing type II full-length collagen, the method comprising: Step A: Cultivate Pichia pastoris strains capable of secreting and expressing recombinant human type II full-length collagen in fermentation medium; Step B: After the carbon source in the fermentation medium is depleted, add glycerol for fed culture. and Step C: After the glycerol in the fermentation medium is depleted, methanol is added for induction culture, and dimethyl sulfoxide (DMSO), 4-phenylbutyric acid (4-PBA) and tetramethylurea (TMU) are added to the fermentation medium.

2. The method according to claim 1, wherein, Dimethyl sulfoxide is added to bring the final concentration in the fermentation medium to 0.5%-1.5% (v / v), for example, 1.2% (v / v).

3. The method according to claim 1, wherein, Add 4-phenylbutyric acid to bring its final concentration in the fermentation medium to 1-10 mM, for example, 5 mM can be added.

4. The method according to claim 1, wherein, Add tetramethylurea to bring the final concentration in the fermentation medium to 10-100 mM, for example, 50 mM can be added.

5. The method according to claim 1, wherein, The Pichia pastoris strain is Pichia pastoris GS115 or X33.

6. The method according to claim 1, wherein, The glycerol, methanol, dimethyl sulfoxide, 4-phenylbutyric acid and / or tetramethylurea are added either in a single addition or in a continuous flow.

7. The method according to claim 1, wherein, The amino acid sequence of the recombinant human type II full-length collagen is shown in SEQ ID NO: 1.

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