CN115873733B

CN115873733B - Pichia pastoris strain for high yield of lysozyme and application thereof

Info

Publication number: CN115873733B
Application number: CN202210854597.0A
Authority: CN
Inventors: 程斯达; 鲍锴; 康丽华; 张静静; 刘文瑶; 葛菁华
Original assignee: Qingdao Vland Biotech Group Co Ltd
Current assignee: Qingdao Vland Biotech Group Co Ltd
Priority date: 2022-07-20
Filing date: 2022-07-20
Publication date: 2024-06-25
Anticipated expiration: 2042-07-20
Also published as: CN115873733A

Abstract

The invention relates to the technical field of genetic engineering, in particular to a pichia pastoris strain for high yield of lysozyme and application thereof. Firstly, carrying out over-expression on lysozyme genes in a Pichia pastoris (Pichia pastoris) host, and constructing a recombinant strain; then, mutant bacteria with obviously improved lysozyme yield are obtained by an ultraviolet mutagenesis method, which are named pichia pastoris RONG-14 (Pichia pastoris RONG 1-14) and are preserved in China center for type culture collection (CCTCC NO) of university of Wuhan in China, wherein the preservation number is CCTCC NO: m2022977.

Description

Pichia pastoris strain for high yield of lysozyme and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a pichia pastoris strain for high yield of lysozyme and application thereof.

Background

Lysozyme (lysozyme), also known as muramidase (muramidase) or N-acetylmuramidase hydrolase (N-acetylmuramide glycanohydrlase), is an alkaline protein capable of hydrolyzing mucopolysaccharides in bacteria. Lysozyme is widely distributed in animals, plants and microorganisms, and is expressed in the plasma, trachea, intestinal tract, stomach, kidney, liver and other tissue cells of mammals. The action mechanism is mainly to break beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in cell wall to decompose insoluble mucopolysaccharide in cell wall into soluble glycopeptide, which leads to overflow of cell wall rupture content to dissolve bacteria.

The abused quantity of antibiotics and a series of problems such as drug residue, pesticide pollution and the like make people feel uneasy to eat. The use of antibiotics has long been aimed at improving the nutritional status and antimicrobial growth promotion of animals, and there has been a great interest in the study of feed antibiotic substitutes that are effective in antimicrobial, growth promotion, applicable, residue-free, and pollution-free. Lysozyme is used as a non-specific immune factor of animals, and attracts the vision of a plurality of researchers by the characteristics of broad-spectrum efficient sterilization, no residue, no pollution, safety to human bodies, no toxicity and no harm. The research shows that the lysozyme can be used as a potential antibiotic substitute for the fields of livestock and poultry feed, food, biological engineering, medical treatment, aquatic products and the like.

The lysozyme is a natural protein, is a feed additive with high safety due to safety, no residue and sterilization effect on pathogenic bacteria, and has wide application prospect in feed application. (1) The lysozyme is used in piglet feed, can provide daily gain and feed intake, reduce feed conversion rate and diarrhea rate, and improve the health level of piglets. (2) The lysozyme is used in poultry feed, can improve the growth performance of poultry, reduce the morbidity, improve the survival rate, and simultaneously can improve the lysozyme content and the immune function in serum, thereby having positive effect on the resistance of poultry to diseases. (3) The lysozyme is used in ruminant feed, can effectively improve the milk quality level, reduce the use amount of medicines, reduce the medicine residue in milk and reduce the formation of drug resistance, and simultaneously, the lysozyme is also used as a medicine for treating recessive mastitis and endometritis of cows, so that the effect is remarkable. Meanwhile, the lysozyme can be applied to disease control of aquatic animals, and chemical medicines used in aquaculture are reduced or replaced. Therefore, lysozyme as a safe and efficient green additive plays an increasingly important role in livestock production under the background that food safety is increasingly important at present.

With the increasing trend of market competition of products, the development of green, safe, high-quality and efficient livestock products is a necessary trend of the development of animal husbandry, and the production of the green livestock products is concerned by society and favored by people. In view of the hazards posed by feeding antibiotics to human health and the environment, many countries, particularly those with developed economies, have or are ready to prohibit the use of feeding antibiotics. The problem of antibiotic replacement has been forced and lysozyme is certainly a good choice. Many researches and applications prove that the feeding lysozyme can effectively improve the growth performance of animals, meet the requirements of people on animal products, improve the economic benefit of animal husbandry and reduce the harm to the environment and human health. However, the lysozyme products in the current market have the problems of low enzyme activity, low yield, unstable properties and the like, so that the lysozyme products are difficult to meet the development requirements of the feed industry.

Disclosure of Invention

The invention provides a pichia pastoris strain for high yield of lysozyme and application thereof, aiming at solving the problems in the prior art. The applicant firstly carries out over-expression on lysozyme genes in a Pichia pastoris (Pichia pastoris) host, and constructs a recombinant strain; then, mutant bacteria with obviously improved lysozyme yield are obtained by an ultraviolet mutagenesis method, so that the production cost of the lysozyme is greatly reduced, and the wide application of the lysozyme is promoted.

One aspect of the invention relates to a Pichia pastoris engineered strain carrying a recombinant plasmid expressing a lysozyme gene.

The nucleotide sequence of the lysozyme gene is SEQ ID NO:1, the encoded amino acid sequence is SEQ ID NO:2.

The invention also relates to a pichia pastoris mutant strain, which is obtained by taking the pichia pastoris engineering strain as a starting strain through an ultraviolet mutagenesis method.

The mutant strain is pichia pastoris RONG-14 (Pichia pastoris RONG 1-14), and is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan in China at the year 2022, month 6 and day 27: m2022977.

The invention also relates to application of the pichia pastoris strain in lysozyme production.

The mutant strain Pichia pastoris RONG-14 provided by the invention can greatly improve the expression quantity of lysozyme, the enzyme activity of lysozyme in the shake flask fermentation supernatant is as high as 9305U/ml, and the mutant strain Pichia pastoris is improved by 74.5% compared with the starting strain, so that unexpected technical effects are achieved.

The mutant strain can be widely applied to the production of lysozyme, thereby being beneficial to reducing the production cost of the lysozyme and promoting the popularization and application of the lysozyme in the industrial field.

Detailed Description

The present invention uses conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as those described in MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art may adopt other methods, experimental schemes and reagents which are conventional in the art on the basis of the technical scheme described in the present invention, and are not limited to the specific embodiments of the present invention.

Strains and vectors: coli DH 5. Alpha. Host deposited with Pichia pastoris GS115, vector pPIC9k, pPICZA, amp, G418, zeocin was purchased from Invitrogen.

Enzyme and kit: the DNA polymerase was purchased from Takara, T4 ligase, restriction enzyme from Fermentas, plasmid extraction kit and gel purification recovery kit from Omega, geneMorph II random mutagenesis kit from Beijing Bomais Biotechnology Co.

The formula of the culture medium comprises:

Coli medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCl, pH7.0;

LB+Amp Medium: 100 mug/mL ampicillin was added to LB medium;

Yeast Medium (YPD Medium): 1% yeast extract, 2% peptone, 2% glucose;

Ypd+zeocin medium: YPD medium plus 100. Mu.g/ml Zeocin;

Yeast screening medium (MD medium): 1.34% YNB, 4X 10 ^-5% biotin, 1% glycerol, 2% agarose;

BMGY medium: 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4X 10 ^-5% biotin, 1% glycerol;

BMMY medium: 2% peptone, 1% yeast extract, 100 mM% potassium phosphate buffer (pH 6.0), 1.34% YNB, 4X 10 ^-5% biotin, 0.5% methanol.

The invention is further described in conjunction with the following detailed description.

EXAMPLE 1 cloning of lysozyme Gene

Based on the amino acid sequence of lysozyme gene (GenBank is NP-001001470.1), the amino acid sequence of the lysozyme is analyzed, the signal peptide of the lysozyme is removed, and then the codon optimization is carried out on the lysozyme according to the codon preference of Pichia pastoris, so that the complete gene synthesis is carried out by Huada gene company. The lysozyme gene is named RONG to 1, and the nucleotide sequence of the lysozyme gene is SEQ ID NO:1, the encoded amino acid sequence is SEQ ID NO:2.

The gene fragment of lysozyme RONG1 was cloned by PCR reaction, and the primer sequences and reaction conditions were as follows:

primer 1 (F): GCGCGAATTCGGCACCGGCTGCTACGGCTCTGTTT (restriction site underlined as EcoR I);

primer 1 (R): TAAAGCGGCCGCGTAGCCGTGCTGCTTGAAGTACTGA (restriction site underlined as Not I).

The PCR conditions were: denaturation at 94℃for 5min; then denaturation at 94℃for 30s, renaturation at 56℃for 30s, extension at 72℃for 1min,35 cycles, and incubation at 72℃for 10min. The full length of RONG gene is 555bp.

EXAMPLE 2 construction of Pichia pastoris engineering bacteria expressing recombinant lysozyme RONG1

1. Construction of recombinant plasmids

The cloned lysozyme RONG gene was subjected to double cleavage with restriction enzymes EcoR I and Not I, and 100. Mu.l of the cleavage system was as follows: the PCR products of lysozyme 1 gene RONG.mu.l, 10 XH buffer 10. Mu.l, 10 XBSA 10. Mu.l, ecoR I5. Mu.l, not I5. Mu.l, ddH ₂ O30. Mu.l were used. After digestion at 37℃of 4 h, agarose gel electrophoresis was recovered.

The expression vector pPIC9K was first subjected to a single cleavage with the restriction enzyme EcoR I, and 100. Mu.l of the cleavage system was as follows: expression vector pPIC9K 20. Mu.l, 10 XH buffer 10. Mu.l, ecoR I5. Mu.l, ddH ₂ O65. Mu.l. After digestion at 37℃of 4 h, agarose gel electrophoresis was recovered. The recovered fragment was subjected to single cleavage with restriction enzyme Not I, and 100. Mu.l of the cleavage system was as follows: pPIC9K was used to collect 20. Mu.l of fragment, 10 XH buffer 10. Mu.l, 10 XBSA 10. Mu.l, 10 XTriton 10. Mu.l, not I5. Mu.l, ddH ₂ O45. Mu.l. After digestion at 37℃of 4 h, agarose gel electrophoresis was recovered.

The RONG gene fragment which is subjected to double digestion by EcoR I and Not I is connected with an expression vector pPIC9K to construct an expression vector pPIC9K-RONG1. The connection system is as follows: expression vector pPIC9K double cleavage product 5. Mu.l, RONG1 gene double cleavage product 3. Mu.l, 10 XT ₄ ligase buffer 1 μl、T₄ ligase 1. Mu.l. 22. Overnight ligation was performed at C.and transformed into E.coli DH 5. Alpha. And transformants were picked and sequenced for verification. Sequencing verifies that the correct transformant is transferred into LB+Amp liquid medium, cultured overnight at 37 ℃, and the plasmid is recombinant yeast expression plasmid pPIC9K-RONG1.

2. Transformation and screening

The recombinant yeast expression plasmid pPIC9K-RONG1 was linearized with Sal I, and after purification of the linearized product with a column purification kit, pichia pastoris GS115 was transformed by electroporation and coated on MD plates. The colonies grown on the MD plates are Pichia pastoris engineering strains, and then YPD plates containing different concentrations of geneticin G418 are coated to screen for multiple copies of transformants.

3. Shake flask fermentation verification

The single multicopy transformants were picked and inoculated into BMGY medium, shake-cultured at 30℃and 220rpm for 24 hours, and then transferred into BMMY medium, shake-cultured at 30℃and 220rpm, and 0.5% methanol was added every 24 hours. After 4d of induction expression, the cells were removed by centrifugation and the lysozyme activity in the fermentation supernatant was examined as described in GB/T25879-2010.

The result shows that under the shake flask condition, the fermentation enzyme activity of the pichia pastoris engineering bacteria of the recombinant expression lysozyme RONG1 constructed by the invention reaches 5330U/ml at most. The transformant was designated as Pichia pastoris RONG1 (Pichia pastorisRONG 1).

EXAMPLE 3 UV mutagenesis screening

Mutation caused by ultraviolet mutagenesis is very random, and the effect of mutation is also random and difficult to predict. Therefore, in order to obtain effective positive mutation, the skilled person is usually required to perform multiple rounds of ultraviolet mutagenesis, the screening effort is large, and there is a possibility that effective positive mutation cannot be obtained. However, since the equipment required for ultraviolet mutagenesis is simple and low in cost, and a large number of mutants can be obtained in a short time, it is still a commonly used mutagenesis breeding method.

The applicant uses pichia pastoris RONG1 as an original strain, and genetically modifies the pichia pastoris by an ultraviolet mutagenesis method to further improve the yield of lysozyme.

Inoculating Pichia pastoris RONG to YPD plate, culturing at 30deg.C for 2-3 days, washing thallus with sterile water to obtain suspension, diluting to 1× ⁶ pieces/mL, irradiating with ultraviolet lamp (40W) for 2-10min for about 22cm until mortality reaches 90%, coating plate, and culturing at 30deg.C for 48 hr.

A total of about 200 individual colonies of mutant bacteria were obtained by the first round of UV mutagenesis. Each single colony was inoculated into a 96-well plate containing 200ul of BMGY liquid medium, and after shaking culture at 30℃and 250rpm for 1 day, the upper medium was centrifuged off, and then 200ul of BMMY medium was added thereto, and shaking culture at 30℃and 250rpm was carried out for 2 days, with 0.5% methanol added each day. After induced expression for 2 days, removing thalli by centrifugation to obtain supernatant containing lysozyme, measuring the activity of the lysozyme, and screening mutant strains with obviously improved lysozyme activity by taking starting bacteria as a control.

The results show that in the mutant bacteria obtained by the first round of ultraviolet mutation screening, the lysozyme activity in the fermentation supernatant of none of the mutant bacteria is higher than that of the starting bacteria. The applicant continues to carry out 55 rounds of mutagenesis screening according to the method, and finally obtains 1 mutant strain with the yield of lysozyme remarkably higher than that of the original strain, and names pichia pastoris RONG-14 (Pichia pastorisRONG 1-14).

Under the shake flask fermentation condition, the lysozyme activity in the fermentation supernatant of the mutant pichia pastoris RONG-14 is as high as 9305U/ml, which is improved by 74.5% compared with that of the original bacteria, and unexpected technical effects are achieved.

The applicant has preserved pichia pastoris RONG-14 (Pichia pastoris RONG 1-14) at the China center for type culture collection, with the preservation number CCTCC NO: m2022977.

The mutant pichia pastoris RONG-14 can be used for fermentation production of lysozyme, is beneficial to reducing the production cost of the enzyme and promotes wide application in the field of feed.

Claims

1. A pichia pastoris mutant, wherein the mutant is pichia pastoris RONG-14 (Pichia pastoris RONG 1-14) and has been deposited at the China center for type culture collection (CCTCC NO: m2022977.

2. The use of the pichia pastoris mutant strain according to claim 1 in lysozyme production.