阅读说明：本技术 利用分子伴侣构建的吡喃糖氧化酶基因、蛋白、毕赤酵母菌及制备和应用 (Pyranose oxidase gene, protein and pichia pastoris constructed by utilizing molecular chaperones, and preparation and application thereof ) 是由 董聪 王玥 高庆华 王庆庆 罗同阳 马清河 马金国 于 2019-10-28 设计创作，主要内容包括：本发明属于新基因的制备及工程菌的构建,特别是指一种利用分子伴侣构建的吡喃糖氧化酶基因、蛋白、毕赤酵母菌及制备和应用。本发明是将毕赤酵母中EroⅠ、PDI、CNE1、SEC53等基因分别克隆连接到毕赤酵母表达载体pPICZ等系列衍生质粒连接,并与P2O共表达转化入Pichia pastoris GS115菌株中,涂布于不同博来霉素浓度梯度YPD平板上考察其效果,通过分子伴侣共表达和发酵条件优化,经筛选鉴定得到一株较原有菌株增强分泌表达吡喃糖氧化酶的菌株,该菌株表达的吡喃糖氧化酶在10L发酵罐上酶活达405U/mL,比没有分子伴侣共表达菌株酶活提高1倍,为吡喃糖氧化酶规模化生产奠定了良好基础。(The invention belongs to the preparation of new genes and the construction of engineering bacteria, and particularly relates to a pyranose oxidase gene, a pyranose protein and pichia pastoris constructed by utilizing a molecular chaperone, and preparation and application thereof. The invention clones and connects Ero I, PDI, CNE1, SEC53 and other genes in Pichia pastoris to a Pichia pastoris expression vector pPICZ and other series derivative plasmids respectively, and co-expresses and converts the Ero I, PDI, CNE1, SEC53 and other genes into a Pichia pastoris GS115 strain with P2O, coats the Pichia pastoris GS115 strain on YPD plates with different bleomycin concentration gradients to investigate the effect of the Pichia pastoris strain, obtains a strain for enhancing secretion and expression of pyranose oxidase compared with the original strain through molecular chaperone co-expression and fermentation condition optimization through screening and identification, improves the enzyme activity of pyranose oxidase expressed by 405U/mL on a 10L fermentation tank by 1 time compared with the enzyme activity of the strain without the molecular chaperone co-expression, and lays a good foundation for scale production.)

1. The pyranose oxidase gene constructed by utilizing the molecular chaperone is characterized in that the gene sequence is SEQ ID No. 1.

2. The preparation method of the pyranose oxidase gene constructed by utilizing the molecular chaperone is characterized by comprising the following process steps:

A. construction of plasmids and strains

Preparing Pichia pastoris Phchia pastoris CGMCC No.16552 into competence;

B. molecular chaperone gene cloning

Designing oligonucleotide chains according to chaperonin Ero1, PDI, CNE1 and SEC53 gene sequences, synthesizing primers, and respectively amplifying Ero1, PDI, CNE1 and SEC53 gene sequences by taking a genome of pichia pastoris CGMCC No.16552 as a template and utilizing the primers and the genome of the pichia pastoris as the template through a PCR technology, wherein the primers are specifically as follows:

ERoI-F：5'-ctatttcgaaacgatgaggatagtaaggagcgtagctatc-3', wherein the BstBI cleavage site is underlined;

ERoI-R：5'-ataagaatgcggccgcttacaagtctactctatatgtggtatc-3', wherein the underline indicates a NotI cleavage site;

PDI-F：5'-ctatttcgaaacgatgcaattcaactgggatattaaaactg-3', wherein the BstBI cleavage site is underlined;

PDI-R：5'-ataagaatgcggccgctaaagctcgtcgtgagcgtctgcctc-3', wherein the underline indicates a NotI cleavage site;

CNE1-F：5'-ctatttcgaaacgatgaagatctctaccattgcaag-3', wherein the BstBI cleavage site is underlined;

CNE1-R：5'-ataagaatgcggccgctaggttctctttgtagctttagtc-3', wherein the underline indicates a NotI cleavage site;

SEC53-F：5'-ctatttcgaaacgatgtcgttttctaataaagaagatc-3', wherein the BstBI cleavage site is underlined;

SEC53-R：5'-ataagaatgcggccgcttacagggaaaagagctcct-3', wherein the underline indicates a NotI cleavage site;

the PCR reaction is carried out for 30 cycles, and the PCR reaction system is as follows: q5 polymerase 1 μ l, 5 XBuffer 20 μ l, 5 XGC enhance 20 μ l, upstream and downstream primers 4 μ l each, dNTP 2 μ l with concentration of l0mM, Pichia pastoris genome template 4 μ l, ddH₂O to 100 μ L;

after double enzyme digestion purification, mature fragments of Ero1, PDI, CNE1 and SEC53 are connected with pPICZ vectors which are subjected to the same double enzyme digestion respectively, cloning vectors pPICZ-Ero I, pPICZ-PDI, pPICZ-CNE1 and pPICZ-SEC53 are constructed, E.coliT1 competent cells are transformed, and a single clone is selected for PCR, enzyme digestion identification and gene sequencing to obtain a correct fusion vector;

C. construction of recombinant plasmid

The construction method of the recombinant plasmid pPICZ-PDI comprises the following steps: a double-chain molecule of a PDI gene is inserted between BstB I and Not I enzyme cutting sites of the pPICZ vector; the construction method of the recombinant plasmid pPICZ-Ero I comprises the following steps: double-chain molecules of EroI genes are inserted between BstB I enzyme cutting sites and Not I enzyme cutting sites of the pPICZ vector; the construction method of the recombinant plasmid pPICZ-CNE1 comprises the following steps: double-chain molecules of CNE1 gene are inserted between BstB I and Not I enzyme cutting sites of the pPICZ vector; the construction method of the recombinant plasmid pPICZ-SEC53 comprises the following steps: a double-stranded molecule of SEC53 gene is inserted between BstB I and Not I cleavage sites of the pPICZ vector.

3. The protein coded by the pyranose oxidase gene is characterized in that the sequence is SEQ ID No. 2.

4. Pichia pastoris transformed by pyranose oxidase gene, which is characterized in that the Latin classification is named as Phchia pastoris, and the preservation number is CGMCC No. 18464.

5. The use of Pichia pastoris according to claim 4, for the preparation of pyranose oxidase.

6. The use according to claim 5, characterized by comprising the following process steps:

selecting Pichia pastoris with preservation number of CGMCC No.18464, inoculating into 5mL YPD liquid culture medium, culturing for 10 hr, inoculating into 150mL BMGY culture medium to OD₆₀₀Inoculating about 6 to a fermentation tank, inoculating 10L of BSM culture medium with the liquid loading capacity of 5.4L in the fermentation tank, sterilizing, inoculating at 10% fermentation volume, controlling pH to 5.0 with concentrated ammonia water, culturing at 30 deg.C, feeding 50% glycerol for 4 hr until OD is reached₆₀₀When the concentration is approximately equal to 180 ℃, stopping the glycerol feeding; when the glycerol in the culture medium is exhausted, beginning to feed methanol for induction, wherein the volume percentage concentration of the methanol solution is 1%, adding the Vc aqueous solution every 24 hours to the final concentration of 80 mug/L, sampling every 12 hours to determine the enzyme activity of the fermentation liquid, and stopping fermentation when the enzyme activity is not increased any more.

Technical Field

The invention belongs to the preparation of new genes and the construction of engineering bacteria, and particularly relates to a pyranose oxidase gene, a pyranose protein and pichia pastoris constructed by utilizing a molecular chaperone, and preparation and application thereof.

Background

Pyranose oxidase (PROD, abbreviated as P2O) also known as pyranose-2-oxidase or glucose-2-oxidase (pyranose-2-oxidase), system number EC 1.1.3.10, is an oxidoreductase using flavoprotein as a coenzyme and a six-membered ring pyranose monosaccharide compound as a substrate, and has multi-substrate catalytic characteristics. The enzyme belongs to glucose-methanol-choline oxido-reductase (GMC) family, can catalyze C-2 site oxidation of glucose and other pyranoses to generate corresponding 2-ketose, and simultaneously oxidize O₂Reduction to H₂O₂. The pyranose oxidase is widely applied in the industries of sugar chemistry, analytical chemistry, clinical chemistry and the like.

Researches show that the heterologous expression of the protein by using the genetic engineering strain has more advantages. The engineering bacteria have clear genetic background, mature culture condition, fast growth rate and easy operation of protein expression regulation. Currently, more heterologous expression is used in prokaryotic and eukaryotic expression systems. According to the literature examination, the cloning and expression of pyranose oxidase gene have been successfully realized only in Escherichia coli, and the pyranose oxidase gene was expressed by Escherichia coli as a host bacterium such as Zhanguo at the university of Hunan, but the activity (0.11U/mL at the maximum) was detected, but the expression level was extremely low. When escherichia coli is used as a heterologous expression host, the problems of cell disruption and centrifugation are often encountered, the denaturation process of the inclusion body is easy, the renaturation is complex, and the operation is inconvenient.

The pichia pastoris expression system, as an eukaryotic expression system, has the following advantages: the genetic modification is convenient, the expression level of the foreign protein is high, the eukaryotic protein is modified after translation, the large-scale fermentation is convenient, and the secreted recombinant protein is easy to purify. Pichia expression systems have been successfully used to express a variety of recombinant foreign proteins. However, with the expression level of some foreign proteins being too high, the maximum load that the host cell can bear in post-translational processing capacity is exceeded, resulting in misfolding, non-processing or mislocalization of the protein, which in turn leads to the formation of multimers of the protein of interest in the lumen of the endoplasmic reticulum and the blocking of proteins in the endoplasmic reticulum that impede its function or are secreted extracellularly inactive. These problems are becoming more and more bottleneck problems that prevent the pichia pastoris expression system from being applied to high-level expression.

Molecular chaperones are a large group of protein molecules that play an important synergistic role in the folding, assembly and degradation of biological macromolecules, but do not undergo any change in themselves. Different cellular localized chaperones have different functions. The molecular chaperones in the cytoplasm have the main functions of assisting the transfer of protein ribosomes to endoplasmic reticulum membranes, and simultaneously, the molecular chaperones interact with each other to promote the depolymerization and the degradation of misfolded protein polymers and maintain the normal physiological metabolic state of cells. The molecular chaperone protein on the endoplasmic reticulum membrane mainly mediates the binding of protein ribosome to the endoplasmic reticulum membrane and constitutes a protein polypeptide continuous transport channel, for example, the transport channel composed of Sec61, Sec62 and Sec63 can transport polypeptide into the endoplasmic reticulum cavity. Chaperones in the lumen of the endoplasmic reticulum play an important role in ensuring correct folding of proteins.

Disulfide Isomerase (PDI) and endoplasmic reticulum oxidoreductase (Ero i) are widely present in fungi, plants, animals and humans and can aid protein secretion. Disulfide Isomerase (PDI) is one of the most abundant proteins in endoplasmic reticulum, mainly catalyzes oxidation reaction to help the proteins to form a correct folding structure and be secreted to the outside of cells, and can obviously improve the expression of heterologous proteins in Pichia pastoris. The majority exists in the yeast endoplasmic reticulum in an oxidized state, meaning that PDI mainly displays oxidative activity to help proteins form the correct folded structure for secretion outside the cell. Ero i oxidizes the reduced PDI state to an oxidized state in the endoplasmic reticulum, indirectly aiding protein secretion. EroI-deficient cells are unable to oxidize newly synthesized proteins, while cells lacking PDI are unable to survive. Zhang et al co-express PDI in Pichia pastoris to increase the specific activity of glucosidase by 1.5 times; wu et al, co-express PDI and Ero I in Pichia pastoris to increase the expression level of fusion protein of human serum albumin and human growth hormone to 304g/L in a 5L fermentor.

A significant bottleneck in increasing the production of foreign proteins in p. The process of protein folding and secretion is very complex, involving a series of interrelated intracellular mechanisms and stress responses. The applicant divides the metabolic pathways for protein production into 4 major modules, respectively: a protein folding module, a vesicle trafficking module, an endoplasmic reticulum-associated degradation mechanism (ERAD) module, and a stress response module. The functions are mutually influenced, and the function of adjusting one area directly influences the functions of other areas.

In the research, molecular chaperones having a great influence on protein synthesis and transport, namely a protein folding related gene CNE1 and a vesicle transport related gene SEC53, are selected for research and investigation. The folding module is mainly present in ER, and the endoplasmic reticulum agglutinin (calnexin) encoded by CNE1 gene belongs to another important control protein in the quality control mechanism of ER, and is mainly involved in folding and glycosylation modification of glycoprotein. The lectin (calnexin) is capable of recognizing and binding to glycoprotein intermediates that are to undergo folding, and is present in the folding machinery of the ER as a signal for sensing the folding state of the protein. Meanwhile, if the protein is continuously misfolded, the calnexin protein can slow down the release of the bound protein intermediate, so that the calnexin protein participates in the next folding step. The vesicle trafficking module plays an important role in foreign protein secretion. Studies have shown that in yeast expression systems, secretion of proteins spans several organelles, including the cytosolic space, endoplasmic reticulum, golgi apparatus, vacuole, and cell membrane. Sec53p protein encoded by SEC53 gene is a function involved in glycosylation modification of quasi-transported nascent peptide chain on ER membrane, and is related to important functional protein recognized by resident protein on ER membrane. The two molecular chaperone genes and glucose oxidase are co-expressed in pichia pastoris by Leili university in south Jiangnan, so that the expression quantity of the glucose oxidase is improved.

A resistance marker gene is introduced into a series of expression vectors commonly used by pichia pastoris. After the transformation is completed, the exogenous gene and the resistance gene are simultaneously integrated on the pichia pastoris chromosome in a homologous recombination mode. By improving the resistance, exogenous genes with multiple copies of inserted genes can be screened, and the higher the copy number of the pichia pastoris transformant is, the stronger the resistance is.

Disclosure of Invention

An object of the present invention is to provide a pyranose oxidase gene constructed using a molecular chaperone.

The second purpose of the invention is to provide a preparation method of pyranose oxidase gene constructed by using molecular chaperone.

It is a further object of the present invention to provide a protein encoded by using a pyranose oxidase gene.

The fourth purpose of the invention is to provide Pichia pastoris CGMCC No.18464 transformed by pyranose oxidase gene.

The fifth purpose of the invention is to provide the application of Pichia pastoris CGMCC No.18464 in the preparation of pyranose oxidase.

The overall technical concept of the invention is as follows:

the gene sequence of the pyranose oxidase gene constructed by utilizing the molecular chaperone is SEQ ID No. 1.

The preparation method of the pyranose oxidase gene constructed by utilizing the molecular chaperone comprises the following process steps:

A. construction of plasmids and strains

Preparing Pichia pastoris Phchia pastoris CGMCC No.16552 into competence;

B. molecular chaperone gene cloning

Designing oligonucleotide chains according to chaperonin Ero1, PDI, CNE1 and SEC53 gene sequences, synthesizing primers, and respectively amplifying Ero1, PDI, CNE1 and SEC53 gene sequences by taking a genome of pichia pastoris CGMCC No.16552 as a template and utilizing the primers and the genome of the pichia pastoris as the template through a PCR technology, wherein the primers are specifically as follows:

ERoI-F：5′-ctatttcgaaacgatgaggatagtaaggagcgtagctatc-3', wherein the BstBI cleavage site is underlined;

ERoI-R：5′-ataagaatgcggccgcttacaagtctactctatatgtggtatc-3', wherein the underline indicates a NotI cleavage site;

PDI-F：5′-ctatttcgaaacgatgcaattcaactgggatattaaaactg-3', wherein the BstBI cleavage site is underlined;

PDI-R：5′-ataagaatgcggccgctaaagctcgtcgtgagcgtctgcctc-3', wherein the underline indicates a NotI cleavage site;

CNE1-F：5′-ctatttcgaaacgatgaagatctctaccattgcaag-3', wherein the BstBI cleavage site is underlined;

CNE1-R：5′-ataagaatgcggccgctaggttctctttgtagctttagtc-3', wherein the underline indicates a NotI cleavage site;

SEC53-F：5′-ctatttcgaaacgatgtcgttttctaataaagaagatc-3', wherein the BstBI cleavage site is underlined;

SEC53-R：5′-ataagaatgcggccgcttacagggaaaagagctcct-3', wherein the underline indicates a NotI cleavage site;

the PCR reaction is carried out for 30 cycles, and the PCR reaction system is as follows: q5 polymerase 1 μ l, 5 XBuffer 20 μ l, 5 XGC enhance 20 μ l, upstream and downstream primers 4 μ l each, dNTP 2 μ l with concentration of l0mM, Pichia pastoris genome template 4 μ l, ddH₂O to 100 μ L;

after double enzyme digestion purification, mature fragments of Ero1, PDI, CNE1 and SEC53 are connected with pPICZ vectors which are subjected to the same double enzyme digestion respectively, cloning vectors pPICZ-Ero I, pPICZ-PDI, pPICZ-CNE1 and pPICZ-SEC53 are constructed, E.coliT1 competent cells are transformed, and a single clone is selected for PCR, enzyme digestion identification and gene sequencing to obtain a correct fusion vector;

C. construction of recombinant plasmid

The construction method of the recombinant plasmid pPICZ-PDI comprises the following steps: a double-chain molecule of a PDI gene is inserted between BstB I and Not I enzyme cutting sites of the pPICZ vector; the construction method of the recombinant plasmid pPICZ-Ero I comprises the following steps: double-chain molecules of EroI genes are inserted between BstB I and NotI enzyme cutting sites of the pPICZ vector; the construction method of the recombinant plasmid pPICZ-CNE1 comprises the following steps: double-chain molecules of CNE1 gene are inserted between BstB I and Not I enzyme cutting sites of the pPICZ vector; the construction method of the recombinant plasmid pPICZ-SEC53 comprises the following steps: a double-stranded molecule of SEC53 gene is inserted between BstB I and Not I cleavage sites of the pPICZ vector.

The sequence of the protein coded by the pyranose oxidase gene is SEQ ID No. 2.

The Pichia pastoris transformed by the pyranose oxidase gene is named Phchia pastoris by Latin classification, and the preservation number is CGMCC No. 18464.

The applicant submits the strains to the common microorganism center of China general microbiological culture Collection center for preservation in 2019, 9 and 4, the classification of the strains is named as Pichia pastoris, with the preservation number of CGMCC No.18464, the preservation organization is located in No. 3 of Xilu No.1 of Beijing republic of rising area, and the preservation organization is abbreviated as CGMCC.

Application of Pichia pastoris in preparing pyranose oxidase is provided.

The specific technical concept of the invention is as follows:

the application of Pichia pastoris CGMCC No.18464 in preparing pyranose oxidase includes the following steps:

selecting Pichia pastoris with preservation number of CGMCC No.18464, inoculating into 5mL YPD liquid culture medium, culturing for 10 hr, inoculating into 150mL BMGY culture medium to OD₆₀₀Inoculating about 6 to a fermentation tank, inoculating 10L of BSM culture medium with the liquid loading capacity of 5.4L in the fermentation tank, sterilizing, inoculating at 10% fermentation volume, controlling pH to 5.0 with concentrated ammonia water, culturing at 30 deg.C, feeding 50% glycerol for 4 hr until OD is reached₆₀₀When the concentration is approximately equal to 180 ℃, stopping the glycerol feeding; when the glycerol in the culture medium is exhausted, the methanol is fed for induction,the volume percentage concentration of the methanol solution is 1%, the Vc aqueous solution is added to the final concentration of 80 mu g/L every 24h, samples are taken every 12h to determine the enzyme activity of the fermentation liquor, and the fermentation is stopped when the enzyme activity is not increased any more.

The invention achieves the substantive characteristics and obvious technical progress that:

1. the invention constructs a recombinant vector containing molecular chaperones PDI, Ero I, CNE1 and SEC53 on the basis of Pichia pastoris CGMCC No.16552, and further improves the expression level of P2O by integrating molecular chaperone co-expression.

2. The constructed recombinant plasmid with correct sequencing is subjected to Sfo I linearization and then is respectively subjected to electric transformation to prepare competence, the bacteria are constructed, molecular chaperone high-copy-number strains are obtained through concentration gradient screening of different bleomycin, genes with high copy numbers are obtained through concentration gradient screening of antibiotics and are integrated onto chromosome DNA, expression quantity is improved, and meanwhile genetic stability of offspring is guaranteed.

3. The high-enzyme-activity strain is primarily fermented and screened by a fermentation medium YPCS, and the strain which is enhanced to secrete and express pyranose oxidase compared with the original strain is obtained through screening and identification, wherein the enzyme activity of the pyranose oxidase expressed by the strain on a 10L fermentation tank is 405U/mL, and is 1 time higher than that of the strain without molecular chaperone co-expression.

Detailed Description

The following examples further illustrate the invention but are not intended to limit it. The protection scope of the present invention is subject to the content of the claims, and any equivalent technical means made according to the specification can be substituted without departing from the protection scope of the present invention. The methods of the present invention, in which the specific conditions are not specified, are performed according to the techniques commonly used in the art or the conditions suggested by the manufacturers.