CN115873733A - Pichia pastoris strain for high yield of lysozyme and application thereof - Google Patents
Pichia pastoris strain for high yield of lysozyme and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, and particularly relates to a pichia pastoris strain for high yield of lysozyme and application thereof. The present invention firstly puts the lysozyme gene in pichia pastoris (A), (B)Pichia pastoris) Carrying out overexpression in a host to construct a recombinant strain; then obtaining mutant bacteria with obviously improved lysozyme yield by an ultraviolet mutagenesis method, which is named as pichia pastoris RONG1-14 (Pichia pastorisRing 1-14) of the ring, has been preserved in China Wuwu in 2022 at 6 months and 27 daysChina center for type culture Collection of the university of Han, the preservation number is CCTCC NO: m2022977.
Description
Technical Field
The invention relates to the technical field of genetic engineering, and particularly relates to a pichia pastoris strain for high yield of lysozyme and application thereof.
Background
Lysozyme (also called muramidase) or N-acetylmuramidase (N-acetylmuramidase glycohydrolase) is a basic protein that hydrolyzes mucopolysaccharides in bacteria. The lysozyme is widely distributed in animals, plants and microorganisms, and is expressed in histiocytes such as plasma, trachea, intestinal tract, stomach, kidney and liver of mammals. The action mechanism is mainly that beta-1, 4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in the cell wall are broken, so that insoluble mucopolysaccharide of the cell wall is decomposed into soluble glycopeptide, and the content of broken cell wall overflows to dissolve the bacteria.
A series of problems of massive abuse of antibiotics, drug residues, pesticide pollution and the like cause difficulty in sleeping and eating. The use of antibiotics has long been the goal of improving the nutritional status of animals and antimicrobial growth promotion, and thus research on alternatives to antibiotics for feeding, which are effective in antimicrobial, growth promotion, applicable, residue-free, and pollution-free, has been attracting attention. The lysozyme is used as a non-specific immune factor of animals, and attracts the sight 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. Researches find that the lysozyme can be used as a potential antibiotic substitute in the fields of livestock and poultry feed, food, bioengineering, medical treatment, aquatic products and the like.
The lysozyme is a natural protein, is a feed additive with high safety due to the safety, no residue and sterilization effect on pathogenic bacteria, and has wide application prospect in feed application. (1) The lysozyme is used in the piglet feed, can provide daily gain and feed intake, reduce feed conversion ratio 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, simultaneously improve the content of lysozyme in serum and the immune function, and has positive effect on poultry disease resistance. (3) The lysozyme is used in the feed for ruminants, can effectively improve the milk quality level of the ruminants, reduces the dosage of the medicament, reduces the medicament residue in milk, reduces the formation of medicament resistance, is used as a medicament for treating recessive mastitis and endometritis of cows, and has remarkable effect. Meanwhile, the lysozyme can be applied to disease control of aquatic animals, and chemical drugs used in aquaculture can be reduced or replaced. Therefore, the lysozyme as a safe and efficient green additive plays an increasingly important role in animal production under the background that food safety is more and more emphasized at present.
With the increasingly fierce market competition of products, the development of green, safe, high-quality and high-efficiency animal products is a necessary trend of animal husbandry development, and the production of green animal products is greatly concerned by the society and favored by people. In view of the health and environmental hazards posed by feed antibiotics, many countries, particularly economically developed countries, have or are prepared to ban the use of feed antibiotics. The problem of replacing antibiotics is imminent, and lysozyme is undoubtedly a good choice. A plurality of 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, 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 cannot meet the development requirements of the feed industry.
Disclosure of Invention
The invention provides a pichia pastoris strain for high yield lysozyme and application thereof, aiming at solving the problems of the prior art. The applicants first applied the lysozyme gene to Pichia pastoris (A), (B)Pichia pastoris) Carrying out overexpression in a host to construct a recombinant strain; then mutant bacteria with obviously improved lysozyme yield are obtained by an ultraviolet mutagenesis method, so that the production cost of lysozyme is greatly reduced, and the wide application of the lysozyme is promoted.
One aspect of the invention relates to a pichia pastoris engineering bacterium, which carries a recombinant plasmid for expressing a lysozyme gene.
The nucleotide sequence of the lysozyme gene is SEQ ID NO:1, the coded 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 and adopting an ultraviolet mutagenesis method.
The mutant strain is pichia pastoris RONG1-14 (Pichia pastorisRONG 1-14), has been preserved in China center for type culture Collection of Wuhan university in China at 27.6 months 2022, with the preservation number of CCTCC NO: m2022977.
The invention also relates to application of the pichia pastoris strain in lysozyme production.
The mutant strain pichia pastoris RONG1-14 provided by the invention can greatly improve the expression quantity of lysozyme, the enzyme activity of the lysozyme in the shake flask fermentation supernatant is as high as 9305U/ml, and is improved by 74.5 percent compared with the original strain, and unexpected technical effects are obtained.
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 the methods 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 can adopt other conventional methods, experimental schemes and reagents in the field on the basis of the technical scheme described in the invention, and the invention is not limited to the specific embodiment of the invention.
Strain and carrier: coli DH 5. Alpha. Deposited from Invitrogen, pichia pastoris GS115, vectors pPIC9k, pPICZA, amp, G418, zeocin.
Enzyme and kit: 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 Ltd.
The formula of the culture medium is as follows:
coli medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCl, pH7.0;
LB + Amp medium: adding 100 mu g/mL ampicillin into LB culture medium;
yeast medium (YPD medium): 1% yeast extract, 2% peptone, 2% glucose;
YPD + Zeocin medium: adding 100 mu g/ml Zeocin into YPD culture medium;
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 present invention will be further described with reference to the following detailed description.
EXAMPLE 1 cloning of the lysozyme Gene
Based on the amino acid sequence of lysozyme gene (GenBank number is NP-001001470.1), the amino acid sequence of the lysozyme is analyzed, the signal peptide of the lysozyme is removed, codon optimization is carried out on the lysozyme according to the codon preference of pichia pastoris, and the whole gene synthesis is carried out by Huada gene company. The lysozyme gene is named as RONG1, and the nucleotide sequence of the lysozyme gene is SEQ ID NO:1, the coded amino acid sequence is SEQ ID NO:2.
the gene fragment of lysozyme RONG1 was cloned by PCR, and the primer sequence and reaction conditions were as follows:
primer 1 (F): GCGCGAATTCGGCACCGGCTGCTGCTACGGCTCTGTTT (underlined)EcoThe cleavage site of R I);
primer 1 (R): TAAAGCGGCCGCGTAGCCGTGCTGCTTGAAGTACTGA (underlined)NotCleavage site of I).
The PCR conditions were: denaturation at 94 deg.C for 5min; then, the mixture is denatured at 94 ℃ for 30s, renatured at 56 ℃ for 30s, extended at 72 ℃ for 1min, and after 35 cycles, the mixture is kept at 72 ℃ for 10min. The whole length of the RONG1 gene is 555bp.
Example 2 construction of Pichia pastoris engineering bacteria expressing recombinant lysozyme RONG1
1. Construction of recombinant plasmid
The cloned lysozyme RONG1 gene is treated with restriction enzymeEcoR I andNoti, carrying out double digestion, wherein 100 mu l of digestion system is as follows: PCR product of lysozyme 1 gene RONG1 40. Mu.l, 10 XH buffer 10. Mu.l, 10 XBSA 10. Mu.l,EcoR I 5 μl、Not I 5 μl、ddH 2 O30. Mu.l. After digestion at 37 ℃ for 4h, the product was recovered by agarose gel electrophoresis.
The expression vector pPIC9K is firstly treated with restriction endonucleaseEcoR I is subjected to single enzyme digestion, and a 100-microliter digestion system is as follows: 20 ul of expression vector pPIC9K, 10 uL of 10 XHbuffer,EcoR I 5 μl、ddH 2 O65. Mu.l. After digestion at 37 ℃ for 4h, the product was recovered by agarose gel electrophoresis. Reuse of the recovered fragments with restriction enzymesNotI, performing single enzyme digestion, wherein 100 mu l of enzyme digestion system is as follows: pPIC9K fragments 20. Mu.l, 10 XH buffer 10. Mu.l, 10 XBSA 10. Mu.l, 10 XTUTON 10. Mu.l,Not I 5 μl、ddH 2 And O45 mu.l. After digestion at 37 ℃ for 4h, the product was recovered by agarose gel electrophoresis.
Will be passedEcoR I andNotthe RONG1 gene fragment subjected to double enzyme digestion is connected with an expression vector pPIC9K to construct an expression vector pPIC9K-RONG1. The linking system is as follows: 5 mul of expression vector pPIC9K double enzyme digestion product, 3 mul of RONG1 gene double enzyme digestion product and 10 XT 4 ligase buffer 1 μl、T 4 1 μ l of ligase. 22. The mixture is connected at the temperature of overnight, transformed into escherichia coli DH5 alpha, and transformants are picked and sequenced for verification. And transferring the transformant which is verified to be correct by sequencing into an LB + Amp liquid culture medium, carrying out overnight culture at 37 ℃, and obtaining the quality-improved particles, namely the recombinant yeast expression plasmid pPIC9K-RONG1.
2. Transformation and screening
The recombinant yeast expression plasmid pPIC9K-RONG1 is usedSalI, linearization, purifying the linearization product by using a column purification kit, and converting pichia pastoris by an electroporation methodGS115, MD plate coated. The colony grown on the MD plate is the pichia pastoris engineering strain, and then YPD plates containing different concentrations of geneticin G418 are coated to screen multi-copy transformants.
3. Shake flask fermentation validation
Selecting single multi-copy transformants, respectively inoculating into BMGY culture medium, performing shake culture at 30 ℃ and 220rpm for 24h, then transferring into BMMY culture medium, performing shake culture at 30 ℃ and 220rpm, and adding 0.5% methanol every 24 h. After the induction expression is carried out for 4d, thalli are removed by centrifugation, and the lysozyme activity in the fermentation supernatant is detected by referring to the method of GB/T25879-2010.
The result shows that the highest fermentation enzyme activity of the Pichia pastoris engineering bacteria for recombinant expression of lysozyme RONG1 constructed by the invention reaches 5330U/ml under the condition of shaking the flask. The transformant was named Pichia pastoris RONG1 (Pichia pastorisRONG1）。
Example 3 UV mutagenesis screening
The mutation caused by ultraviolet mutagenesis has strong randomness, and the effect generated by mutation is random and difficult to predict. Therefore, in order to obtain effective positive mutations, technicians usually need to perform multiple rounds of ultraviolet mutagenesis, the screening workload is large, and the possibility that effective positive mutations cannot be obtained exists. However, ultraviolet mutagenesis requires simple equipment and low cost, and can obtain a large number of mutants in a short time, so that it is still a common mutagenesis breeding method.
The applicant takes pichia pastoris RONG1 as an original strain, and carries out genetic modification on the strain by an ultraviolet mutagenesis method, so that the yield of lysozyme is further improved.
Inoculating Pichia pastoris RONG1 on YPD plate, culturing at 30 deg.C for 2-3 days, washing thallus with sterile water to obtain suspension, and diluting to 1 × 10 6 Irradiating with ultraviolet lamp (40W) for 2-10min at a distance of about 22cm, coating the plate, and culturing at 30 deg.C for 48 hr.
A total of about 200 single colonies of mutant bacteria were obtained in the first round of UV mutagenesis. Each single colony was inoculated to a 96-well plate containing 200. Mu.l of BMGY liquid medium, subjected to shaking culture at 30 ℃ and 250rpm for 1 day, centrifuged to remove the upper layer medium, added with 200. Mu.l of BMMY medium, subjected to shaking culture at 30 ℃ and 250rpm for 2 days, and added with 0.5% methanol every day. After 2 days of induced expression, the thalli are removed by centrifugation to obtain supernatant containing lysozyme, the activity of the lysozyme is measured, and a mutant strain with the significantly improved lysozyme activity is screened out by taking spawn running as a control.
The result shows that the lysozyme enzyme activity in the fermentation supernatant of no mutant strain is higher than that of the original strain in the mutant strains obtained by the first round of ultraviolet mutagenesis screening. The applicant continues 55 rounds of mutagenesis and screening according to the method, finally obtains 1 mutant strain with lysozyme yield obviously higher than that of the original strain, and names Pichia pastoris RONG1-14 (Pichia pastorisRONG1-14）。
Under the condition of shake flask fermentation, the lysozyme enzyme activity in the fermentation supernatant of the mutant pichia pastoris RONG1-14 is up to 9305U/ml, which is improved by 74.5 percent compared with the original strain, and unexpected technical effects are obtained.
The applicant has already introduced Pichia pastoris RONG1-14 (at 27.6.2022: (R))Pichia pastorisRONG 1-14) is preserved in China center for type culture Collection of Wuhan university in China, and the preservation number is CCTCC NO: m2022977.
The mutant pichia pastoris RONG1-14 can be used for fermentation production of lysozyme, is favorable for reducing the production cost of the lysozyme, and promotes wide application of the lysozyme in the field of feeds.
Claims (5)
1. A pichia pastoris engineering bacterium is characterized in that the pichia pastoris engineering bacterium carries a recombinant plasmid for expressing a lysozyme gene.
2. The pichia pastoris engineered bacterium of claim 1, wherein the nucleotide sequence of the lysozyme gene is SEQ ID NO:1, the coded amino acid sequence is SEQ ID NO:2.
3. a Pichia pastoris mutant strain, which is characterized in that the mutant strain is obtained by taking the Pichia pastoris engineering strain of claim 1 or 2 as a starting strain through an ultraviolet mutagenesis method.
4. The Pichia pastoris mutant bacterium according to claim 3, wherein the mutant bacterium is Pichia pastoris RONG1-14 (R-RONG 1-14)Pichia pastorisRONG 1-14), has been preserved in China center for type culture Collection of Wuhan university in Wuhan, china at 27.6 months in 2022, with the preservation number of CCTCC NO: m2022977.
5. The use of the Pichia pastoris mutant strain of claim 3 or 4 in the production of lysozyme.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116103178A (en) * | 2023-04-13 | 2023-05-12 | 中国科学院天津工业生物技术研究所 | Copper-resistant pichia pastoris strain with high copper enrichment and application thereof |
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| WO1989004320A1 (en) * | 1987-11-02 | 1989-05-18 | The Salk Institute Biotechnology/Industrial Associ | PRODUCTION OF ANIMAL LYSOZYME c VIA SECRETION FROM PICHIA PASTORIS AND COMPOSITION THEREFOR |
| CN102061291A (en) * | 2010-11-05 | 2011-05-18 | 中国科学院海洋研究所 | Lysozyme as well as preparation and application thereof |
| US20120232007A1 (en) * | 2009-10-16 | 2012-09-13 | Merck Sharp & Dohme Corp | Method for producing proteins in pichia pastoris that lack detectable cross binding activity to antibodies against host cell antigens |
| CN111607582A (en) * | 2020-05-15 | 2020-09-01 | 天津科技大学 | Lysozyme with enzyme activity improving function |
| CN113637598A (en) * | 2021-03-18 | 2021-11-12 | 上海复华兴生物技术有限公司 | Recombinant pichia pastoris engineering bacteria integrating high-copy human lysozyme gene and construction method |
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| US20120232007A1 (en) * | 2009-10-16 | 2012-09-13 | Merck Sharp & Dohme Corp | Method for producing proteins in pichia pastoris that lack detectable cross binding activity to antibodies against host cell antigens |
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| CN113637598A (en) * | 2021-03-18 | 2021-11-12 | 上海复华兴生物技术有限公司 | Recombinant pichia pastoris engineering bacteria integrating high-copy human lysozyme gene and construction method |
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| CN116103178A (en) * | 2023-04-13 | 2023-05-12 | 中国科学院天津工业生物技术研究所 | Copper-resistant pichia pastoris strain with high copper enrichment and application thereof |
| CN116103178B (en) * | 2023-04-13 | 2023-06-09 | 中国科学院天津工业生物技术研究所 | Copper-resistant pichia pastoris strain with high copper enrichment and application thereof |
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