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
- Publication number
- CN115873733A CN115873733A CN202210854597.0A CN202210854597A CN115873733A CN 115873733 A CN115873733 A CN 115873733A CN 202210854597 A CN202210854597 A CN 202210854597A CN 115873733 A CN115873733 A CN 115873733A
- Authority
- CN
- China
- Prior art keywords
- pichia pastoris
- lysozyme
- strain
- rong1
- mutant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010014251 Muramidase Proteins 0.000 title claims abstract description 60
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 title claims abstract description 60
- 102000016943 Muramidase Human genes 0.000 title claims abstract description 50
- 235000010335 lysozyme Nutrition 0.000 title claims abstract description 48
- 229960000274 lysozyme Drugs 0.000 title claims abstract description 47
- 239000004325 lysozyme Substances 0.000 title claims abstract description 47
- 241000235058 Komagataella pastoris Species 0.000 title claims abstract description 36
- 241000894006 Bacteria Species 0.000 claims abstract description 13
- 238000002703 mutagenesis Methods 0.000 claims abstract description 13
- 231100000350 mutagenesis Toxicity 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 9
- 241000235648 Pichia Species 0.000 claims abstract description 6
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 5
- 239000013612 plasmid Substances 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 230000002018 overexpression Effects 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 14
- 230000000694 effects Effects 0.000 description 13
- 239000002609 medium Substances 0.000 description 12
- 241001465754 Metazoa Species 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000000855 fermentation Methods 0.000 description 8
- 230000004151 fermentation Effects 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000003242 anti bacterial agent Substances 0.000 description 6
- 229940088710 antibiotic agent Drugs 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 238000001976 enzyme digestion Methods 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 108010084455 Zeocin Proteins 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 3
- 244000144977 poultry Species 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229920002683 Glycosaminoglycan Polymers 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 241000282849 Ruminantia Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 108010060371 endo-N-acetylmuramidase Proteins 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000000383 hazardous chemical Substances 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008057 potassium phosphate buffer Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000004145 Endometritis Diseases 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 1
- 101710132682 Lysozyme 1 Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- MNLRQHMNZILYPY-MDMHTWEWSA-N N-acetyl-alpha-D-muramic acid Chemical compound OC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@H](O)[C@@H]1NC(C)=O MNLRQHMNZILYPY-MDMHTWEWSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 208000027954 Poultry disease Diseases 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000021050 feed intake Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 210000003701 histiocyte Anatomy 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 229940088592 immunologic factor Drugs 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 208000004396 mastitis Diseases 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229940051921 muramidase Drugs 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 235000003715 nutritional status Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210854597.0A CN115873733A (en) | 2022-07-20 | 2022-07-20 | Pichia pastoris strain for high yield of lysozyme and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210854597.0A CN115873733A (en) | 2022-07-20 | 2022-07-20 | Pichia pastoris strain for high yield of lysozyme and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115873733A true CN115873733A (en) | 2023-03-31 |
Family
ID=85769511
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210854597.0A Pending CN115873733A (en) | 2022-07-20 | 2022-07-20 | Pichia pastoris strain for high yield of lysozyme and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115873733A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2022
- 2022-07-20 CN CN202210854597.0A patent/CN115873733A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| 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 |
| 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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108285900A (en) | A kind of recombination algin catenase and its construction method and application | |
| CN107164344B (en) | Heat-resistant phytase mutant and encoding gene and application thereof | |
| CN100526458C (en) | Alpha-galactosidase gene, its coding protein, production and use | |
| TWI654304B (en) | Lysozyme having improved enzymatic activity | |
| CN110577946B (en) | Beta-mannase mutant with improved enzyme activity and heat resistance as well as encoding gene and application thereof | |
| CN102392002A (en) | Improved escherichia coli phytase HTP6M and gene and application thereof | |
| CN115873733A (en) | Pichia pastoris strain for high yield of lysozyme and application thereof | |
| CN111607582A (en) | Lysozyme with enzyme activity improving function | |
| CN112920280B (en) | Method for efficiently expressing acid protease and application thereof | |
| CN104356222B (en) | Mutant of one boar derived antimicrobial peptide PR 39 and its preparation method and application | |
| CN108841740B (en) | Pichia pastoris strain for high yield of alpha-galactosidase | |
| CN107488221B (en) | Swollenin protein from fungi and gene and application thereof | |
| CN105753958B (en) | A kind of Novel fish derived antimicrobial peptide mutant and its preparation method and application | |
| JP2021185912A (en) | Novel chitosanase chi1 as well as coding gene and application thereof | |
| CN114426571A (en) | Pinctada fucata galactose-binding lectin protein PFL-96 and coding gene and application thereof | |
| CN101368175B (en) | Novel phytase, encoding gene, cell and feedstuff additive including the enzyme | |
| CN1233826C (en) | Reformed lactase of Pichia pastoris and its application | |
| CN1302112C (en) | Production for phytase with high living rate high temp. resisting by pichia | |
| CN110591931A (en) | Pichia pastoris, generated high-temperature-resistant enzyme and application | |
| RU2388820C2 (en) | Gene abfb-2 penicillium funiculosum | |
| CN1157477C (en) | Engineering fungus containing large-scale salmon growth factor | |
| CN105524904B (en) | A kind of heat-resisting recombined xylanase and its preparation method and application | |
| CN102978182A (en) | Lipase mutant | |
| CN115094051B (en) | Recombinant lysozyme from bemyces fumosoroseus as well as preparation method and application thereof | |
| CN102229666B (en) | Preparation method and application of animal-derived cationic antimicrobial peptides |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |