Co-oxygen protein VHb and its recombinant strain X33-pPICZαA-102C300C-Vgb and applications

Abstract

The present invention relates to a co-oxygen protein VHb, its recombinant bacterium X33-pPICZαA-102C300C-Vgb and applications, belonging to the technical field of genetic engineering. The nucleotide sequence of the gene of the co-oxygen protein VHb is as shown in SEQ ID N0.1, and the gene of the co-oxygen protein VHb can be used to improve the efficiency of the secretion of alginate lyase by the Pichia pastoris engineering bacterium. The present invention also provides that the amino acid sequence of the co-oxygen protein VHb is as shown in SEQ ID N0.2. The present invention also provides a recombinant expression vector and a recombinant bacterium X33-pPICZαA-102C300C-Vgb comprising an alginate lyase gene and the gene of the co-oxygen protein VHb. The recombinant bacterium improves the utilization rate of oxygen by the bacterial cells in a low-oxygen environment in the later stage of fermentation, and finally improves the yield of alginate lyase.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a co-oxygen protein VHb, its recombinant strain X33-pPICZαA-102C300C-Vgb, and applications thereof. Background Art

[0002] Pichia pastoris is the most commonly used protein expression system after Escherichia coli, and it is currently widely used for laboratory-scale protein preparation, characterization, and structural analysis. In the field of biopharmaceuticals, various proteins can be expressed and produced by Pichia pastoris, such as insulin, hepatitis B surface antigen, human serum albumin, and epidermal growth factor. It has many advantages compared with Escherichia coli and Bacillus subtilis expression systems. For example, it can achieve effective secretory expression, reduce the workload required for the subsequent separation and purification of the target protein, the stable expression of foreign genes integrated into the genome, and simple and inexpensive high-density biomass accumulation.

[0003] Pichia pastoris X33 belongs to a wild-type strain, which can rapidly utilize methanol, integrate the recombinant vector into the strain genome, and combine with the alcohol oxidase promoter (AOX) on the vector to control the expression of foreign proteins in the host. Currently, during the fermentation process, since Pichia pastoris is a highly aerobic microorganism, the limited oxygen supply capacity of the equipment often restricts the cell density and the rate of methanol oxidative degradation, thereby limiting the expression level of its target protein.

[0004] Vitreoscilla hemoglobin (VHb) is an intracellular soluble oxygen substance similar to human hemoglobin, with a molecular weight of about 15.47 kDa. After being transformed into yeast, it can improve the ability of VHb engineering bacteria to utilize low oxygen during high-density fermentation at the molecular level, thereby increasing the expression level of the target protein.

[0005] Aiming at the problem of oxygen utilization rate during the high-density fermentation of Pichia pastoris, the present invention cloned the gene Vgb of Vitreoscilla hemoglobin, constructed a high-expression strain producing alginate lyase, improved the oxygen utilization rate of the cells in the low-oxygen environment during the late fermentation stage, and finally increased the yield of alginate lyase. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a co-oxygen protein VHb, its recombinant strain X33-pPICZαA-102C300C-Vgb, and applications thereof, and the co-oxygen protein can improve the efficiency of secreting alginate lyase by Pichia pastoris engineering bacteria.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A gene of co-oxygen protein VHb, whose nucleotide sequence is shown in SEQ ID N0.1.

[0009] The amino acid sequence of the co-oxygen protein VHb is shown in SEQ ID N0.2.

[0010] A recombinant expression vector, which contains the alginate lyase gene and the gene of co-oxygen protein VHb, and the nucleotide sequence of the alginate lyase gene is shown in SEQ ID N0.3.

[0011] A recombinant bacterium, which contains the recombinant expression vector, and the gene of co-oxygen protein VHb is linked to the end of the alginate lyase gene to form a tandem expression cassette.

[0012] The application of the co-oxygen protein in improving the secreted expression of alginate lyase, and the secreted expression of the alginate lyase is the secreted expression in a eukaryotic expression system;

[0013] Preferably, it is the secreted expression in Pichia pastoris X33.

[0014] The nucleotide sequence of the gene of co-oxygen protein VHb:

[0015] atgttggaccagcagaccatcaacatcatcaaggctaccgttccagtcttgaaagaacacggtgttactatcaccaccacct

[0016] tctacaagaacctgttcgctaagcacccagaggttagaccactgttcgatatgggtagacaagagtctttggagcagcctaa

[0017] ggctttggctatgactgttttggctgctgctcagaacatcgagaacttgccagctattttgccagccgttaagaagatcgccgt

[0018] taagcactgtcaagctggtgttgctgctgcacattacccaatcgttggtcaagagttgctgggtgccatcaaagaagttttag

[0019] gcgacgctgctactgacgacattttggatgcttggggtaaagcctacggtgttatcgctgacgttttcatccaagttgaggctgacttgtacgctcaggctgttgagtaa.

[0020] Protein encoded by the gene of co-oxygen protein VHb:

[0021] MLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQES LEQPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELL GAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE.

[0022] Alginase gene:

[0023] actgaatctggttctggttcttcttctggtggttcttcctccggatcttcttcttcctcatcctcttccggtggatcatcctctggtg

[0024] gatcaggtggtagtagttcaggtggatctttggacccaaacttgccaccatcttccaacttcgatttgtccgcttggtacttgtc

[0025] cgttccaactgataacaacggtgacggtaaggccgactccatcaaagaaaacgatttgaacgctggttacgccgacggta

[0026] cttacttttacactgctgctgatggtggtatggtgttcagatgtccaatctgtggttacaagacttctaccaacacctcctacac

[0027] cagaaccgagttgagagaaatgctgagaagaggtgacacctccattgctactcaaggtgtcaacggtaacaactgggtttt

[0028] cggttctgctccagcttccgctagagaagctgctggtggtgtagatggtgttttgagagctactttggccgttaaccacgttac

[0029] tactactggtgactctggtcaggttggtagagttatcgttggtcagatccacgctaacaacgacgaaccattgagactgtact

[0030] acagaaagttgccaggtcactccaagggttccgtttacattgctcatgaacctaacggtggttccgactcttggtacgacatg

[0031] attggttctagatcctcctctgcttctgacccatctgacggtattgctttggacgaagtttggtcctacgaggttaaggttgtcg

[0032] gtaacactttgaccgtgaccatcttcagagctggtaaggacgacgttgttcaggttgttgacatgggtaactccggttacgat

[0033] gttgctgaccagtaccagtacttcaaggccggtgtttacaaccagaacaatactggtaactgttccgactacgttcaggttactttctacgctttggagcaatctcacgatcatcatcaccatcaccactaa.

[0034] Advantages of the present invention over the prior art:

[0035] 1. By constructing an expression vector in which the gene of the oxygen-binding protein VHb is combined with the alginate lyase-encoding gene at the end of the nucleotide sequence SEQ ID NO. 3, i.e., the original alginate lyase gene, the present invention improves the secretion expression of the alginate lyase-producing engineering bacteria in a hypoxic environment.

[0036] 2. The present invention provides a gene of the oxygen-binding protein VHb, which can improve the utilization rate of oxygen by the bacterial cells in a hypoxic environment and achieve high expression of alginate lyase. In particular, it provides an effective element for expressing the alginate lyase gene in Pichia pastoris. Brief Description of the Drawings

[0037] Figure 1 It is the gene electrophoresis map of amplifying co-oxygen protein VHb in Example 1; among them, lane M: marker DNA; lane 1 is the gene fragment of co-oxygen protein VHb;

[0038] Figure 2 It is a schematic diagram of constructing the pPICZαA-102C300C-Vgb recombinant plasmid in Example 1;

[0039] Figure 3 It is the comparison of the dry weights of the genetically engineered bacteria for constructing co-oxygen protein VHb in Example 2 and the control group before transformation;

[0040] Figure 4 It is the comparison of the alginate lyase activities of the genetically engineered bacteria for constructing co-oxygen protein VHb in Example 2 and the control group before transformation. Detailed implementation manners

[0041] The present invention will be further described in detail below in conjunction with examples, but the implementation manners of the present invention are not limited thereto.

[0042] The molecular biology experimental techniques adopted in the following examples, including PCR amplification, plasmid extraction, DNA fragment ligation, gel electrophoresis, etc., are specifically referred to "Molecular Cloning: A Laboratory Manual" (Third Edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Translated by Huang Peitang et al., 2002, Beijing: Science Press).

[0043] Example 1 Construction and screening of co-oxygen protein recombinant engineering bacteria

[0044] By means of one-step cloning, an expression vector combining the gene of co-oxygen protein VHb and the alginate lyase gene is constructed, and by combining a high-throughput screening method, a positive clone with the highest alginate lyase activity is obtained from the clones, which specifically includes the following steps:

[0045] (1) Retrieval and purification of the gene of co-oxygen protein VHb: The original sequence of co-oxygen protein is downloaded from the National Center for Biotechnology Information website. The nucleotide sequence of the original sequence is as shown in SEQ ID N0.6. After codon optimization, it is artificially synthesized. The optimized nucleotide sequence is shown in SEQ ID N0.1 and the amino acid sequence is shown in SEQ ID N0.2. The optimized nucleotide sequence is ligated to the pUC57 plasmid to construct the pUC57-Vgb strain, and the plasmid of the pUC57-Vgb strain is extracted ( Plasmid DNA Mini Kit (Kit D6942), the gene of the oxygen-binding protein VHb was amplified by PCR from the pUC57-Vgb plasmid genome using two synthetic primer fragments SEQ ID N0.4 and SEQ ID N0.5; verified by nucleic acid electrophoresis (see Figure 1 ) and purified ( Gel Extraction Kit) to recover the target fragment for later use.

[0046] Forward primer: 5'-caccatcaccactaagcggccgccaagcttgatgttggaccagc-3';

[0047] Reverse primer: 5'-tgttctagaaagctggcggccgcttactcaacagcctgagcgtaca-3'.

[0048] Original sequence of the gene of the oxygen-binding protein VHb:

[0049] atgttggatcaacagactatcaacatcatcaaggctactgttccagtcttgaaggaacatggtgttactatcactactactttct

[0050] acaagaacttgtttgctaagcatccagaagttagaccattgtttgatatgggtagacaagaatctttggaacaaccaaaggct

[0051] ttggctatgactgtcttggctgctgctcagaacattgagaacttgccagctatcttgccagctgttaagaagattgctgttaag

[0052] cattgtcaagctggtgttgctgctgctcattacccaattgttggtcaagaattgttgggtgctatcaaggaagtcttgggtgatg

[0053] ctgctactgatgatatcttggatgcttggggtaaggcttacggtgttattgctgatgtcttcattcaagttgaagctgatttgtacgctcaagctgttgaataa.

[0054] (2) Digest the pPICZαA-102C300C plasmid linearly with restriction enzymes: Digest the pPICZαA-102C300C plasmid with Not I (TaKaRa) alone to obtain a linearized vector fragment. The linearized vector is digested with Fast Digest Dpn I, and the treated fragment is purified using a purification kit ( Cycle Pure Kit D6492) and recovered for later use.

[0055] (3) Construction and verification of the pPICZαA-102C300C-Vgb plasmid: The construction process of the pPICZαA-102C300C-Vgb plasmid is as Figure 2 shown. Using DNA seamless cloning (Clon Express II One Step Cloning Kit), according to the DNA concentrations of the recombinant gene and the linearized pPICZαA-102C300C fragment, appropriate dilutions are made and then recombinant ligation is carried out. The ligated system is transformed into E. coli DH5α by heat shock method, and then spread on an LB (Zeocin) plate for screening of positive recombinants. The constructed vector is verified by monoclonal sequencing using the universal primers of the pPICZαA vector.

[0056] (4) Construction of the Pichia pastoris strain X33-pPICZαA-102C300C-Vgb: After activating the correctly sequenced transformant, extract the plasmid ( Plasmid DNA Mini Kit D6942), and obtain a cloning vector containing the gene of the co-oxygen protein VHb. The cloning vector is electrotransformed into the Pichia pastoris X33 strain to construct the Pichia pastoris X33-pPICZαA-102C300C-Vgb strain.

[0057] (5) High-throughput screening of recombinant strains: Pick the positive transformants into a 48-well deep-well plate for cultivation, and add 1 mL of BMGY liquid medium to each well. Incubate at 30 °C and 200 rpm, add 1% methanol every 24 h, and continuously induce for 3 days. Centrifuge at 4000 rpm for 5 min, take the supernatant for alginate lyase activity detection, and screen out the strains with high enzyme activity.

[0058] Example 2 Evaluation of the growth and enzyme production levels of strains containing co-oxygen protein

[0059] (1) Inoculate the strains constructed in Example 1 and the control group strains constructed without the gene of the co-oxygen protein VHb into 20 mL of liquid YPD medium respectively, and shake culture at 37 °C and 200 r / min for 24 h as seed solutions.

[0060] (2) Absorb 1 mL of the seed liquid into 20 mL of BMGY medium, add 1% methanol every 24 h for enzyme induction, and perform induction for 5 times in total. Incubate with shaking at 30 °C and 200 r / min for the first four days, and then at 30 °C and 50 r / min for the next two days. Take 2 mL of the bacterial liquid every 24 h, centrifuge at 5000 r / min for 5 min, measure the dry weight of the precipitate, and measure the enzyme activity of the supernatant.

[0061] (3) Take a clean flat weighing bottle made of glass, place it in a drying oven at 105 °C, prop the bottle cap obliquely on the edge of the bottle, heat for 2 h, take it out and cover it, place it in a desiccator to cool for 0.5 h, weigh it, and repeat the drying until the mass difference between the previous and the next time does not exceed 2 mg, which is the constant weight.

[0062] (4) The enzyme activity of alginate lyase is determined by the DNS method. The definition of enzyme activity unit: 1 enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 μmol of reducing sugar per 1 min.

[0063] (5) Determination of the dry weight of Pichia pastoris cells and the enzyme activity of alginate lyase: Compared with the control group (13.05 g / L), the dry weight of the Pichia pastoris engineering bacteria producing alginate lyase containing co-oxygen protein was 14.675 g / L at 144 h of fermentation, showing an upward trend, while the cell mass of the control group showed a downward trend, as Figure 3 shown. The gene of co-oxygen protein VHb can increase the production of alginate lyase by Pichia pastoris in a low-oxygen environment from 7.76 U / mL to 10.86 U / mL, an increase of 39.9% (see Figure 4 ).

Claims

1. A recombinant expression vector, characterized in that, The recombinant expression vector contains an alginate lyase gene and a gene of the oxygen-binding protein VHb. The nucleotide sequence of the gene of the oxygen-binding protein VHb is shown as SEQ ID NO.1, and the nucleotide sequence of the alginate lyase gene is shown as SEQ ID NO.

3.

2. A recombinant bacterium X33-pPICZαA- 102C300C - Vgb , characterized in that The recombinant strain X33-pPICZαA- 102C300C - Vgb comprises the recombinant expression vector described in claim 1, and connects the gene of the co-oxygen protein VHb to the end of the alginate lyase gene to form a tandem expression cassette.

3. Use of the recombinant expression vector according to claim 1 in enhancing the secreted expression of alginate lyase, characterized in that, The secretion expression of the alginate lyase is the secretion expression in a eukaryotic expression system, and the eukaryotic expression system is Pichia pastoris X33.

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