CN117867005A - Saccharomyces cerevisiae engineering bacteria with high yield of T4N5 and construction method and application thereof - Google Patents
Saccharomyces cerevisiae engineering bacteria with high yield of T4N5 and construction method and application thereof Download PDFInfo
- Publication number
- CN117867005A CN117867005A CN202410059789.1A CN202410059789A CN117867005A CN 117867005 A CN117867005 A CN 117867005A CN 202410059789 A CN202410059789 A CN 202410059789A CN 117867005 A CN117867005 A CN 117867005A
- Authority
- CN
- China
- Prior art keywords
- gene
- saccharomyces cerevisiae
- xiv
- locus
- sed1
- 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
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 107
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 title claims abstract description 107
- 241000894006 Bacteria Species 0.000 title claims abstract description 34
- 238000010276 construction Methods 0.000 title claims abstract description 20
- 230000014509 gene expression Effects 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 30
- 101150006457 sed1 gene Proteins 0.000 claims abstract description 29
- 210000004027 cell Anatomy 0.000 claims abstract description 24
- 108090000623 proteins and genes Proteins 0.000 claims description 81
- 101100434663 Bacillus subtilis (strain 168) fbaA gene Proteins 0.000 claims description 69
- 101150095274 FBA1 gene Proteins 0.000 claims description 69
- 239000013598 vector Substances 0.000 claims description 42
- 108020005004 Guide RNA Proteins 0.000 claims description 30
- 108020004414 DNA Proteins 0.000 claims description 29
- 239000002773 nucleotide Substances 0.000 claims description 17
- 125000003729 nucleotide group Chemical group 0.000 claims description 17
- 210000000349 chromosome Anatomy 0.000 claims description 15
- 108091033409 CRISPR Proteins 0.000 claims description 12
- 101000877447 Enterobacteria phage T4 Endonuclease V Proteins 0.000 claims description 12
- 238000003776 cleavage reaction Methods 0.000 claims description 9
- 230000007017 scission Effects 0.000 claims description 9
- 230000010354 integration Effects 0.000 claims description 8
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 7
- 238000011144 upstream manufacturing Methods 0.000 claims description 6
- 108091005804 Peptidases Proteins 0.000 claims description 5
- 239000004365 Protease Substances 0.000 claims description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 4
- 101100010928 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) tuf gene Proteins 0.000 claims description 4
- 101150001810 TEAD1 gene Proteins 0.000 claims description 4
- 101150074253 TEF1 gene Proteins 0.000 claims description 4
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 4
- 150000007523 nucleic acids Chemical group 0.000 claims description 3
- 102100040540 60S ribosomal protein L3 Human genes 0.000 claims description 2
- 101100351264 Candida albicans (strain SC5314 / ATCC MYA-2876) PDC11 gene Proteins 0.000 claims description 2
- 101100083070 Candida albicans (strain SC5314 / ATCC MYA-2876) PGA6 gene Proteins 0.000 claims description 2
- 101000673985 Homo sapiens 60S ribosomal protein L3 Proteins 0.000 claims description 2
- 101000579123 Homo sapiens Phosphoglycerate kinase 1 Proteins 0.000 claims description 2
- 101100073891 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) nic-3 gene Proteins 0.000 claims description 2
- 101150050255 PDC1 gene Proteins 0.000 claims description 2
- KJWZYMMLVHIVSU-IYCNHOCDSA-N PGK1 Chemical compound CCCCC[C@H](O)\C=C\[C@@H]1[C@@H](CCCCCCC(O)=O)C(=O)CC1=O KJWZYMMLVHIVSU-IYCNHOCDSA-N 0.000 claims description 2
- 102100028251 Phosphoglycerate kinase 1 Human genes 0.000 claims description 2
- 101100166584 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) CCW12 gene Proteins 0.000 claims description 2
- 101150118997 bna4 gene Proteins 0.000 claims description 2
- 239000002207 metabolite Substances 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 238000000746 purification Methods 0.000 abstract description 12
- 210000002421 cell wall Anatomy 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- 108700008625 Reporter Genes Proteins 0.000 abstract description 8
- 238000001042 affinity chromatography Methods 0.000 abstract description 5
- 230000003834 intracellular effect Effects 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 4
- 231100000277 DNA damage Toxicity 0.000 abstract description 3
- 238000010612 desalination reaction Methods 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 230000001909 effect on DNA Effects 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 239000012634 fragment Substances 0.000 description 35
- 239000013612 plasmid Substances 0.000 description 35
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 32
- 102000004169 proteins and genes Human genes 0.000 description 20
- 235000018102 proteins Nutrition 0.000 description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 235000014304 histidine Nutrition 0.000 description 7
- 101710191666 Lactadherin Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 102100039648 Lactadherin Human genes 0.000 description 5
- 241001052560 Thallis Species 0.000 description 5
- 238000011033 desalting Methods 0.000 description 5
- 238000011010 flushing procedure Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 108091093078 Pyrimidine dimer Proteins 0.000 description 4
- 241000793189 Saccharomyces cerevisiae BY4741 Species 0.000 description 4
- 101150063416 add gene Proteins 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 230000009089 cytolysis Effects 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 description 3
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 3
- 101150008604 CAN1 gene Proteins 0.000 description 3
- VEQPNABPJHWNSG-UHFFFAOYSA-N Nickel(2+) Chemical compound [Ni+2] VEQPNABPJHWNSG-UHFFFAOYSA-N 0.000 description 3
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical class C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 229960000448 lactic acid Drugs 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 229910001453 nickel ion Inorganic materials 0.000 description 3
- 239000013635 pyrimidine dimer Substances 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 2
- SEHFUALWMUWDKS-UHFFFAOYSA-N 5-fluoroorotic acid Chemical compound OC(=O)C=1NC(=O)NC(=O)C=1F SEHFUALWMUWDKS-UHFFFAOYSA-N 0.000 description 2
- 230000033616 DNA repair Effects 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102100029727 Enteropeptidase Human genes 0.000 description 2
- 108010013369 Enteropeptidase Proteins 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- 108010060309 Glucuronidase Proteins 0.000 description 2
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 2
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 2
- 102100024023 Histone PARylation factor 1 Human genes 0.000 description 2
- 101001047783 Homo sapiens Histone PARylation factor 1 Proteins 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 208000000453 Skin Neoplasms Diseases 0.000 description 2
- 206010042496 Sunburn Diseases 0.000 description 2
- ASJWEHCPLGMOJE-LJMGSBPFSA-N ac1l3rvh Chemical class N1C(=O)NC(=O)[C@@]2(C)[C@@]3(C)C(=O)NC(=O)N[C@H]3[C@H]21 ASJWEHCPLGMOJE-LJMGSBPFSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- UPUOLJWYFICKJI-UHFFFAOYSA-N cyclobutane;pyrimidine Chemical class C1CCC1.C1=CN=CN=C1 UPUOLJWYFICKJI-UHFFFAOYSA-N 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- KDQPSPMLNJTZAL-UHFFFAOYSA-L disodium hydrogenphosphate dihydrate Chemical compound O.O.[Na+].[Na+].OP([O-])([O-])=O KDQPSPMLNJTZAL-UHFFFAOYSA-L 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 150000002411 histidines Chemical class 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 201000000849 skin cancer Diseases 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 238000000825 ultraviolet detection Methods 0.000 description 2
- 108010082737 zymolyase Proteins 0.000 description 2
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 1
- FYGDTMLNYKFZSV-WFYNLLPOSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,3s,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-WFYNLLPOSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 101710135546 Apurinic-apyrimidinic endonuclease Proteins 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 241000282461 Canis lupus Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 102000010719 DNA-(Apurinic or Apyrimidinic Site) Lyase Human genes 0.000 description 1
- 108010063362 DNA-(Apurinic or Apyrimidinic Site) Lyase Proteins 0.000 description 1
- 101710109420 DNA-(apurinic or apyrimidinic site) endonuclease Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229920002581 Glucomannan Polymers 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 101150009006 HIS3 gene Proteins 0.000 description 1
- 101100394989 Rhodopseudomonas palustris (strain ATCC BAA-98 / CGA009) hisI gene Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000001014 amino acid Nutrition 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
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000008004 cell lysis buffer Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000007872 degassing Methods 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002616 endonucleolytic effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000005530 etching Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229940046240 glucomannan Drugs 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- LGQLOGILCSXPEA-UHFFFAOYSA-L nickel sulfate Chemical compound [Ni+2].[O-]S([O-])(=O)=O LGQLOGILCSXPEA-UHFFFAOYSA-L 0.000 description 1
- 229910000363 nickel(II) sulfate Inorganic materials 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002818 protein evolution Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000014493 regulation of gene expression Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000004227 thermal cracking Methods 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Abstract
The invention discloses a saccharomyces cerevisiae engineering bacterium for high yield of T4N5, and a construction method and application thereof. The invention uses saccharomyces cerevisiae as chassis cells, digs sites, integrates different reporter genes (GUS or YPET) on the site XIV-68 and characterizes the reporter genes, and determines that the XIV-68 site is a high expression site in the saccharomyces cerevisiae. After the T4N5 expression cassette is knocked in to an XIV-68 locus, the high-efficiency expression of the recombinant T4N5 is realized; the mutant strain obtained by further knocking out the SED1 gene can efficiently lyse cell walls and release intracellular substances to the outside of cells, and the effective purification of the recombinant T4N5 is completed by an affinity chromatography method and desalination, so that the T4N5 produced by the method has good repairing effect on DNA damage formed by ultraviolet irradiation. The production of T4N5 by using the recombinant saccharomyces cerevisiae strain can effectively solve the medicine source problem, and has the advantages of saving material resources and protecting environment.
Description
Technical Field
The invention belongs to the field of genetic engineering, and relates to a saccharomyces cerevisiae engineering strain for high yield of T4 endonuclease V (T4N 5), and a construction method and application thereof.
Background
T4 endonuclease V (T4 endonucleolytic V, T4N 5) is an endonuclease derived from a T4 phage and has a molecular weight of about 16.1kDa, originally isolated from E.coli infected with a T4 phage by Tanaka et al in 1975. T4 endonuclease V, which is a bifunctional enzyme comprising both pyrimidine dimer-DNA glycosidase and apurinic-apyrimidinic endonuclease activities, is involved in the repair of damage to DNA caused by UV irradiation. T4 endonuclease V can specifically recognize cyclobutane pyrimidine dimers (cyclobutane pyrimidine dimer, CPD) formed by ultraviolet irradiation in DNA, and can be cut after recognizing CPD, in the process, pyrimidine dimers-DNA glycosidase can cut the glycosidic bond between 5' -pyrimidine and deoxyribose in CPD to form a pyrimidine-removing site, and apurinic endonuclease can cut the phosphodiester bond between two adjacent deoxyribose in the pyrimidine-removing site to form the nicked DNA.
Ultraviolet light is an important factor in inducing skin cancer. When exposed to sunlight for a long period of time, the ultraviolet radiation will likely induce DNA in skin cells to form pyrimidine dimers, causing DNA damage. Some experiments have shown that T4 endonuclease V can effectively reduce the incidence of skin cancer. For example, one mouse study by Wolf et al showed that T4N5 liposomes could be an effective adjunct to sunburn, reducing sunburn cell formation.
The production of recombinant T4 endonuclease V has been reported both at home and abroad, and the current mainstream production method is to use E.coli as the expression host for production. The production of T4 endonuclease V using E.coli as a host tends to cause the formation of inclusion bodies, resulting in a complicated subsequent purification process, which limits its mass production.
Saccharomyces cerevisiae (Saccharomyces cerevisiae), a widely studied single-cell eukaryotic microorganism, has been used in large numbers for the biosynthesis of various natural compounds, which have complex cell wall structures. The cell wall thickness of Saccharomyces cerevisiae is about 100-300 nm, and is similar to a sandwich structure, and mainly comprises beta-D glucan (beta-D-glucomannan), alpha-D-mannase (alpha-D-mannase) and a small amount of chitin (chitosan). The SED1 gene is present in the Saccharomyces cerevisiae genome and its translated protein Sed1p is glycosylated to form mannoprotein, which is finally anchored outside the cell wall by covalent bonding with dextran (Shimeo H, et al Sed1p is a major cell wall protein of Saccharomyces cerevisiae in the stationary phase and is involved in lytic enzyme resistance.J Bacteriol,1998, 180:3381-7.). Because mannoprotein is distributed outside the cell wall, some researchers have expressed the foreign protein in fusion with the Sed1p protein, causing the foreign protein to be displayed on the cell surface (Kuroda K, et al, enhancement of display efficiency in yeast display sys-temby vector engineering and gene display. Appl Microbiol Biotechnol,2009, 82:713-9.). The Sed1p protein maintains the stability of the cell wall and mitochondrial genome, and knock-out of the Sed1 gene does not only affect the normal growth of the cell, but also increases the tolerance of the yeast cell to lactic acid (Phadnis et al. Role of the putative structural protein Sed1p in mitochondrial genome maitenance. J Moliol,2004,342 (4): 1115-29;Toshihiro S,et al.Disruption of multiple genes whose deletion causes lactic-acid resistance improves lactic-acid resistance and productivity in Saccharomyces cerevisiae. J BIOSCI BIOENG,2013,115 (5): 467-474).
Therefore, the obtained strain capable of producing T4N5 at high yield and rapidly releasing intracellular proteins has important significance for industrial production.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a saccharomyces cerevisiae engineering strain with high yield of T4N5.
The invention further aims at providing a construction method of the saccharomyces cerevisiae engineering bacteria with high yield of T4N5.
The invention also aims to provide application of the saccharomyces cerevisiae engineering bacteria with high yield of T4N5.
The invention uses Saccharomyces cerevisiae as chassis cells to excavate sites. The XIV-68 locus was determined to be a high expression locus in Saccharomyces cerevisiae by integrating and characterizing different reporter genes (GUS or YPET) at locus XIV-68. After the T4 endonuclease V (T4N 5) expression cassette is knocked in to an XIV-68 locus, the high-efficiency expression of the recombinant T4N5 is realized; the mutant strain obtained by further knocking out the SED1 gene in the saccharomyces cerevisiae genome can efficiently lyse cell walls and release intracellular substances to the outside of cells, and the effective purification of the recombinant T4N5 is completed by an affinity chromatography and desalination, so that the T4N5 produced by the invention has good repairing effect on DNA damage (namely DNA of pyrimidine dimer) formed by ultraviolet irradiation.
The aim of the invention is achieved by the following technical scheme:
the application of the saccharomyces cerevisiae chromosome XIV-68 locus in high expression of exogenous metabolites by using saccharomyces cerevisiae as a chassis cell.
Preferably, the nucleotide sequence of the XIV-68 locus of the Saccharomyces cerevisiae chromosome comprises the gRNA target sequence of the XIV-68 locus and sequences within 500bp upstream and downstream respectively;
further preferred nucleotide sequences for the XIV-68 locus of the Saccharomyces cerevisiae chromosome include the gRNA target sequence for the XIV-68 locus and sequences within 100bp upstream and downstream, respectively.
Wherein the gRNA target sequence at the XIV-68 site is 5'-AAAGAATCATAGATCGTCAA-3'.
The higher expression intensity of the XIV-68 locus is verified by integrating a beta-glucuronidase Gene (GUS), a YPET fluorescent reporter gene or a T4N5 gene in the XIV-68 locus of the saccharomyces cerevisiae chromosome, which indicates that the XIV-68 locus is a high expression locus of the saccharomyces cerevisiae.
A saccharomyces cerevisiae engineering bacterium with high yield of T4N5 is prepared by taking saccharomyces cerevisiae as an initial strain, integrating a T4N5 gene into a saccharomyces cerevisiae chromosome XIV-68 locus, and obtaining the saccharomyces cerevisiae engineering bacterium with high yield of T4N5 integrated with the T4N5 gene.
In order to further improve the harvesting efficiency of T4N5, the cleavage efficiency of Saccharomyces cerevisiae needs to be improved, and the SED1 gene is knocked out on the basis of the Saccharomyces cerevisiae engineering bacteria.
Further, the method further comprises the following steps: and knocking out the SED1 gene in the saccharomyces cerevisiae genome to obtain the saccharomyces cerevisiae engineering bacteria integrating the T4N5 gene and knocking out the high-yield T4N5 of the SED1 gene.
Wherein, the amino acid sequence encoded by the T4N5 gene is shown as NP 049733.1.
Preferably, the nucleotide sequence of the T4N5 gene is shown as 37 to 450bp of SEQ ID No. 1.
Furthermore, for purification convenience, a histidine tag and/or a protease cleavage site is added at the 5' end of the T4N5 gene to correspond to the coding nucleic acid sequence;
preferably, the histidine tag is a histidine tag formed by connecting 4-10 histidines in series; more preferably, the nucleotide sequence of the histidine tag (6 XHis tag) formed by connecting 6 histidines in series is shown as 4 to 21bp of SEQ ID No. 1.
Preferably, the protease cleavage site is enterokinase cleavage site DDDDK, and the nucleotide sequence is shown as 22-36 bp of SEQ ID No. 1.
Further preferably, the nucleotide sequence of the T4N5 gene with histidine tag and protease cleavage site corresponding to the coding nucleic acid sequence is shown in SEQ ID No. 1.
Further, the expression cassette for the T4N5 gene is integrated into the Saccharomyces cerevisiae chromosome XIV-68.
Preferably, the expression cassette of the T4N5 gene comprises a promoter, a T4N5 gene and a terminator; in particular selected from the group consisting of strong promoters (P CCW12 、P TDH3 、P PDC1 And P TEF1 At least one of them) and a terminator (T) RPL3 、T PGK1 、T BNA4 And T FBA1 At least one of) to be expressed in combination with a gene of interest.
Further preferably, the expression cassette of the T4N5 gene is P TDH3-524 -T4N5-T FBA1 The nucleotide sequence is shown as 41 to 1428bp of SEQ ID No. 2.
A construction method of a saccharomyces cerevisiae engineering bacterium with high yield of T4N5 comprises the following steps:
1) Designing a gRNA target sequence according to the XIV-68 locus of the saccharomyces cerevisiae chromosome;
2) Constructing a recombinant vector according to the gRNA target sequence of the step 1);
3) Preparing donor DNA comprising a T4N5 gene;
4) Simultaneously transforming the recombinant vector of the step 2) and the donor DNA containing the T4N5 gene of the step 3) into the Saccharomyces cerevisiae transferred with the Cas9 gene, realizing the integration of the T4N5 gene, and obtaining the Saccharomyces cerevisiae engineering bacteria with high yield of T4N5 integrated with the T4N5 gene.
In order to further improve the harvesting efficiency of T4N5, the cleavage efficiency of Saccharomyces cerevisiae needs to be improved, and the SED1 gene is knocked out on the basis of the Saccharomyces cerevisiae engineering bacteria.
Further, the method further comprises the following steps:
5) Knocking out the SED1 gene in the saccharomyces cerevisiae engineering bacteria genome of the step 4), realizing the integration of the T4N5 gene and the knocking out of the SED1 gene, and obtaining the saccharomyces cerevisiae engineering bacteria integrating the T4N5 gene and knocking out the SED1 gene and producing high-yield T4N5.
Preferably, the gRNA target sequence in step 1) is 5'-AAAGAATCATAGATCGTCAA-3'.
Preferably, the recombinant vector in step 2) comprises a gXIV-68 expression cassette, the nucleotide sequence of which gXIV-68 expression cassette may be any sequence of the gXIV-68 expression cassette, further preferably as shown in SEQ ID No. 3.
The starting vector used for constructing the recombinant vector can be any Saccharomyces cerevisiae vector, preferably p426 (commercial plasmid p426-SNR52p-gRNA. CAN1.Y-SUP4t of Addgene company) starting vector, and the constructed recombinant vector is p426-gXIV-68.
Preferably, the donor DNA comprising the T4N5 gene in step 3) comprises a 40-100bp (preferably 40 bp) homologous sequence upstream of the gRNA target sequence, an expression cassette for the T4N5 gene or the T4N5 gene, a 40-100bp (preferably 40 bp) homologous sequence downstream of the gRNA target sequence; further preferred is as shown in SEQ ID No. 2.
Preferably, the Saccharomyces cerevisiae having been transformed with the Cas9 gene in step 4) is obtained by transferring a plasmid carrying the Cas9 gene; specifically, the gene is obtained by transferring a p414 plasmid (commercial plasmid p414-TEF1p-Cas9-CYC1t of Addgene).
Preferably, the Saccharomyces cerevisiae in step 4) is Saccharomyces cerevisiae BJ5464, saccharomyces cerevisiae BY4741 or Saccharomyces cerevisiae CEN.PK2-1Ca, but is not limited thereto.
Preferably, the saccharomyces cerevisiae engineering bacteria in the step 4) are saccharomyces cerevisiae BJ5464/XIV-68:: P TDH3-524 -T4N5-T FBA1 Saccharomyces cerevisiae BY4741/XIV-68:: P TDH3-524 -T4N5-T FBA1 Or Saccharomyces cerevisiae CEN.PK2-1Ca/XIV-68:: P TDH3-524 -T4N5-T FBA1 。
Preferably, in step 5), the SED1 gene in the saccharomyces cerevisiae engineering bacteria genome of step 4) is knocked out, and the method comprises the following steps:
a) Designing a gRNA target sequence according to the SED1 gene in the genome;
b) Constructing a recombinant vector according to the gRNA target sequence of step a);
c) Simultaneously transforming the recombinant vector and the donor DNA of the step b) into the saccharomyces cerevisiae engineering bacteria of the step 4) transferred with the Cas9 gene, so as to realize the integration of the T4N5 gene and the knockout of the SED1 gene.
Preferably, the gRNA target sequence in step a) is 5'-AGAGGAGGAAGTGACATCGG-3'.
Preferably, the recombinant vector in step b) comprises a gSED1 expression cassette, the nucleotide sequence of which gSED1 expression cassette may be any sequence of the gXIV-68 expression cassette, further preferably as shown in SEQ ID No. 4.
Preferably, the nucleotide sequence of the donor DNA in step c) may be any sequence of the donor DNA, more preferably as shown in SEQ ID No. 5.
The starting vector used for constructing the recombinant vector can be any Saccharomyces cerevisiae vector, preferably p426 (commercial plasmid p426-SNR52p-gRNA. CAN1.Y-SUP4t of Addgene company) starting vector, and the constructed recombinant vector is p426-gSED1.
Preferably, the saccharomyces cerevisiae engineering bacteria of step 4) in step c) into which the Cas9 gene has been transferred are obtained by transferring a plasmid carrying the Cas9 gene; specifically, the gene is obtained by transferring a p414 plasmid (commercial plasmid p414-TEF1p-Cas9-CYC1t of Addgene).
Preferably, the saccharomyces cerevisiae engineering bacteria in the step 5) are saccharomyces cerevisiae BJ5464/XIV-68:: P TDH3-524 -T4N5-T FBA1 Delta sed1, saccharomyces cerevisiae BY4741/XIV-68:: P TDH3-524 -T4N5-T FBA1 P is:. DELTA.sed 1 or Saccharomyces cerevisiae CEN.PK2-1Ca/XIV-68 TDH3-524 -T4N5-T FBA1 /Δsed1。
The saccharomyces cerevisiae engineering bacteria are applied to high-yield T4N5.
Further, a method for producing T4N5, activating the saccharomyces cerevisiae engineering bacteria; inoculating the activated strain to a fermentation medium for fermentation culture, and collecting bacterial cells after fermentation culture to extract T4N5.
Preferably, the method for producing T4N5 comprises inoculating Saccharomyces cerevisiae engineering bacteria into a seed culture medium, activating at 30+ -2deg.C and 220+ -20 rpm for 12+ -2 h, transferring to a fresh seed culture medium, fermenting at 30+ -2deg.C and 220+ -20 rpm for 30+ -2 h, collecting thallus, and performing steps of breaking and cracking, ni affinity chromatography, desalting, etc. to obtain recombinant T4N5 protein with higher purity; wherein the seed culture medium is YPD.
Compared with the prior art, the invention has the following advantages and effects:
the invention realizes the biosynthesis of T4N5 by taking glucose as a carbon source in the saccharomyces cerevisiae strain, and integrates an optimized T4N5 gene expression cassette into a genome XIV-68 site in the recombinant saccharomyces cerevisiae strain to realize the purpose of producing T4N5. The SED1 gene in the saccharomyces cerevisiae genome is further knocked out, and the obtained mutant strain can efficiently lyse cell walls and release intracellular substances to the outside of cells. The production of T4N5 by using the recombinant saccharomyces cerevisiae strain can effectively solve the medicine source problem, and has the advantages of saving material resources and protecting environment.
Drawings
FIG. 1 shows the results of fluorescence detection of strains in example 4 in which YPET expression cassettes were integrated into the XIV-68 and LEU sites, respectively.
FIG. 2 shows the results of the enzyme activity assays of the strains in example 4 in which GUS expression cassettes were integrated into the XIV-68 locus and the LEU locus, respectively.
FIG. 3 shows the SDS-PAGE results of the strains of example 5 in which the T4N5 expression cassette was integrated into the XIV-68 locus and the LEU locus, respectively; wherein LEU:: T4N5 means BJ5464/LEU:: P TDH3-524 -T4N5-T FBA1 XIV-68:: T4N5 means BJ5464/XIV-68:: P TDH3-524 -T4N5-T FBA1 。
FIG. 4 is the effect on the amount of T4N5 protein obtained after knock-out of the SED1 gene in example 6; wherein XIV-68:: T4N5 means BJ5464/XIV-68:: P TDH3-524 -T4N5-T FBA1 XIV-68:: T4N5. DELTA. SED1 means BJ5464/XIV-68:: P TDH3-524 -T4N5-T FBA1 /Δsed1。
FIG. 5 shows the purification and desalting results of recombinant T4 endonuclease V (T4N 5) of example 7.
FIG. 6 is a result of verifying the activity of recombinant T4 endonuclease V (T4N 5) in example 8.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention. It should be noted that variations and modifications, e.g., changing the type of expression vector, changing the method of construction of the expression vector, changing the type of host cell, etc., can be made on the basis of the above-described variations and modifications, without departing from the spirit of the invention. These are all within the scope of the present invention.
In the examples, the YEp181 plasmid refers to the YEplac181 plasmid, the plasmid p426 refers to the plasmid p426-SNR52p-gRNA. CAN1.Y-SUP4t, and the plasmid p414 refers to the plasmid p414-TEF1p-Cas9-CYC1t.
Example 1: construction of Gene integration expression cassettes
In this example, the codon-optimized nucleotide sequence of T4N5 (NP-049733.1) fused with a 6 XHis tag and enterokinase site (HHHHHHDDDDK) at the amino terminus is shown as SEQ ID NO.1, which is synthesized by whole gene artificial synthesis, embedded into Puc57 plasmid, and the synthesized sequence is consistent with the designed sequence by DNA detection analysis, and is used for constructing the following recombinant plasmid, named Puc57-T4N5.
The vector YEp181-P used in the examples TDH3-524 -T4N5-T FBA1 The construction process is as follows:
in order to amplify the T4N5 gene, 1 pair of primer pairs P1 and P2 were designed and synthesized, and the primer sequences were as follows:
P1：5′-AACAAACAAActcgagATGCATCATCATCATCATCATGAC-3′；
P2：5′-AATTTGAATTAACTCTGCAGTTAAGCGTAGATAGCTTTAC-3′；
note that: the underlined sequence is the base homologous to the sequence of the fragment to be ligated, and the lower case letter is the XhoI cleavage site.
The plasmid Puc57-T4N5 is used as a template, and a primer P1/P2 is used for carrying out PCR reaction to obtain a T4N5 gene fragment.
PCR reactions were performed using the YEp181 plasmid from Invitrogen as a template, using the primers YEp181-F1/YEp181-R1 to obtain a linearized fragment of the YEp181 plasmid, the primer sequences being as follows:
YEp181-F1：5′-AAGCTTGGCGTAATCATGGT-3′；
YEp181-R1：5′-GGATCCCCGGGTACCGAGCT-3′；
the PCR reaction system and conditions are shown in Table 1.
TABLE 1 Prime STAR PCR System and conditions
After the PCR reaction, whether the amplified gene fragment was correct in size or not was detected by agarose gel electrophoresis, and if the band was correct in size, the amplified fragment was purified by an oligonucleotide purification kit (purchased from Bio-engineering (Shanghai) Inc.), and the concentration of the gene fragment was detected by a micro-spectrophotometer and recorded.
The genome of Saccharomyces cerevisiae BJ5464 was extracted using Yeast DNA Kit. Using the genome as a template, the TDH3 promoter fragment was amplified using the PCR enzyme KOD FX (available from TOYOBO, japan) using the primer 181-TDH3-F/TDH3-R, and the FBA1 terminator fragment was amplified using the primer FBA1t-F/181-FBA1 t-R.
Wherein the specific primer sequences are as follows (underlined indicates that the sequences are bases homologous to the vector sequences):
181-TDH3-F：5′-AGCTCGGTACCCGGGGATCCATAACATCGTAGGTGTCTGG-3′；
TDH3-R：5′-CATCTCGAGTTTGTTTGTTTA-3′；
FBA1t-F：5′-CTGCAGAGTTAATTCAAATTAAT-3′；
181-FBA1t-R：5′-ACCATGATTACGCCAAGCTTAGTAAGCTACTATGAAAGA-3′；
the PCR reaction system and conditions are shown in Table 2.
TABLE 2 KOD-FX PCR System and conditions
After the PCR reaction is finished, whether the size of the amplified gene fragment is correct or not is detected by agarose gel electrophoresis, if the size of the band is correct, the amplified fragment is purified by an oligonucleotide purification kit, and the concentration of the gene fragment is detected by a micro spectrophotometer and recorded.
By ClonExpress R MultiS One Step Cloning Kit recombinant cloning kit (purchased from Nanjinofuzan Co.) the obtained TDH3 promoter fragment, FBA1 terminator fragment, YEp181 plasmid vector fragment and T4N5 fragment were subjected to multi-fragment homologous recombination ligation to obtain vector YEp181-P TDH3-524 -T4N5-T FBA1 。
Then the connection product is transformed into E.coli DH5 alpha competent cells by a chemical method, the E.coli DH5 alpha competent cells are coated on LB/Amp (10 g/L Tryptone,5g/L Yeast Extract,5g/L NaCl, 100 mu g/mL Ampicillin) plates, single clone is picked up, the single clone is scratched on LB/Amp plates, cultured for 5 to 6 hours, the single clone is positively screened by colony PCR technology, 3 to 5 single colonies are picked up in an ultra clean workbench and respectively dissolved in 20 mu L ddH 2 In O, the supernatant obtained after thermal cracking for 10min at 95 ℃ and centrifugation for 5min at 14000rpm can be used as a template for colony PCR. The reaction system and conditions for colony PCR are shown in Table 3.
TABLE 3 PCR reaction System and conditions for E.coli colonies
Primers for colony PCR are shown below:
M13fwd：5′-GTAAAACGACGGCCAGT-3′；
Check-R：5′-TTTCACACAGGAAACAGCTA-3′；
after colony PCR reaction, agarose gel electrophoresis was performed on the PCR product to verify whether the fragment size was the expected one. According to electrophoresis detection result, selecting strain with corresponding band size, inoculating into LB/Amp+ liquid culture medium, culturing at 37deg.C and 220rpm overnight, extracting recombinant plasmid in thallus with rapid plasmid small extraction kit (purchased from Tiangen (Beijing) Co., ltd.), and sending to biological engineering (Shanghai) Co., ltd for gene sequencing. Analyzing the sequencing result by utilizing Snapgene software to finally obtain the recombinant plasmid YEp181-P containing the T4N5 gene TDH3-524 -T4N5-T FBA1 。
The E.coli DH 5. Alpha. Genomic DNA was used as a template and GUS-F/GUS-R as primers to amplify the beta-glucuronidase Gene (GUS) fragment.
The primer sequences are as follows: (underlined indicates that the sequence is a base homologous to the sequence of the fragment to be ligated)
GUS-F：5′-AACAAACAAActcgagATGTTACGTCCTGTAGAAAC-3′；
GUS-R：5′-AATTTGAATTAACTCTGCAGTCATTGTTTGCCTCCCTGCT-3′；
By YEp181-P TEF1 -YPET-T FBA1 The YPET fluorescent reporter gene fragment was amplified using the plasmid as a template and YPET-F/YPET-R as primers. Wherein Yep181-P TEF1 -YPET-T FBA1 Plasmids are disclosed in "CN202110962577.0, a method for fine regulation of gene expression based on genomic loci".
The primer sequences are as follows: (underlined indicates that the sequence is a base homologous to the sequence of the fragment to be ligated)
YPet-F：5′-AACAAACAAActcgagATGTCTAAAGGTGAAGAATTAT-3′；
YPet-R：5′-AATTTGAATTAACTCTGCAGTTATTTGTACAATTCATCAAT-3′；
Recombinant plasmids as shown in Table 4 were obtained by the method of multi-fragment homologous recombination according to the above-described method.
TABLE 4 recombinant plasmid
| Plasmid name |
| YEp181-P TDH3-524 -YPet-T FBA1 |
| YEp181-P TDH3-524 -GUS-T FBA1 |
EXAMPLE 2 construction of Saccharomyces cerevisiae recombinant vector and donor DNA
Construction of Saccharomyces cerevisiae recombinant vectors p426-gXIV-68, p426-gLEU (leucine (LEU) site in genome is used as control site) and corresponding donor DNA, the specific construction method is as follows:
1. construction of recombinant vector p426-gXIV-68
Based on the CRISPR-Direct website prediction, the gRNA target sequence of the XIV-68 site (S.cerevisiae/NC_ 001146.8) is determined as follows: 5'-AAAGAATCATAGATCGTCAA-3';
PCR amplification was performed using commercial plasmid P426 as a template and P3/P5 and P4/P6 as primer pairs, respectively, to obtain gXIV-68-1 and gXIV-68-2 fragments. Using ClonExpress R And II, recombining the gXIV-68-1 fragment and the gXIV-68-2 fragment to obtain a recombinant vector p426-gXIV-68 carrying the guide RNA sequence of the XIV-68 site, wherein the nucleotide sequence of the gXIV-68 expression frame is shown as SEQ ID No. 3.
Wherein the specific primer sequences are as follows (underlined indicates that the sequences are bases homologous to the vector sequences):
P3：5′-TTGACGATCTATGATTCTTTGATCATTTATCTTTCACTGC-3′；
P4：5′-AAAGAATCATAGATCGTCAAGTTTTAGAGCTAGAAATAGC-3′；
P5：5′-TAATAATGGTTTCTTAGTATGA-3′；
P6：5′-ACTAAGAAACCATTATTATCAT-3′；
2. construction of recombinant vector p426-gLEU
Based on the predicted result of the CRISPR-Direct website, determining the gRNA target sequence of the LEU locus is as follows: 5'-AATATAATCAGATGGTTGCG-3';
the fragments gLEU-1 and gLEU-2 were amplified using the commercial plasmid P426 as a template and P5/P11 and P6/P12 as primer pairs, respectively. The rest method is the same as the above, and a recombinant vector p426-gLEU carrying LEU site guide RNA sequence is obtained.
Wherein, the specific amplification primers are as follows (underlined indicates the sequence is a base homologous to the vector sequence):
P11：5′-CGCAACCATCTGATTATATTGATCATTTATCTTTCACTGC-3′；
P12：5′-AATATAATCAGATGGTTGCGGTTTTAGAGCTAGAAATAGC-3′；
3. amplification of donor DNA
1)Donor-LEU-YPet
By YEp181-P TDH3-524 -YPet-T FBA1 As templates, the Donor-LEU-YPET fragment was amplified using P13/P14 as primers, and the specific amplification primers were as follows (underlined indicates the sequence is a base homologous to the genomic sequence):
P13：5′-CAGCCACAGGTTGGTCATCAATACCACTGGGGGAGCATGCATAACATCGTAGGTGTCTGG-3′；
P14：5′-ATCTTAACTAATGTGTTCAGTTTTAGCTCTACTAGCTATTAGTAAGCTACTATGAAAGAC-3′；
2)Donor-LEU-GUS
by YEp181-P TDH3-524 -GUS-T FBA1 As a template, the Donor-LEU-GUS fragment was amplified using P13/P14 as a primer as described above.
3)Donor-LEU-T4N5
By YEp181-P TDH3-524 -T4N5-T FBA1 As a template, the Donor-LEU-T4N5 fragment was amplified using P13/P14 as a primer as described above.
4)Donor-XIV-68-YPet
By YEp181-P TDH3-524 -YPet-T FBA1 As templates, the Donor-XIV-68-YPET fragment was amplified using P15/P16 as primers, and the specific amplification primers were as follows (underlined indicates the sequence is a base homologous to the genomic sequence):
P15：5′-TATAAAAGAATAAATAATAGCGGTATTACGTGTGTTGGAAATAACATCGTAGGTGTCTGG-3′；
P16：5′-CGATCCATATTATAATAATACTGGTAATACAAATACTAGTAGTAAGCTACTATGAAAGAC-3′；
5)Donor-XIV-68-GUS
by YEp181-P TDH3-524 -GUS-T FBA1 As a template, the Donor-XIV-68-GUS fragment was amplified using P15/P16 as a primer as described above.
6)Donor-XIV-68-T4N5
By YEp181-P TDH3-524 -T4N5-T FBA1 As a template, the Donor-XIV-68-T4N5 fragment (the nucleotide sequence of which is shown as SEQ ID No. 2) was amplified using P15/P16 as a primer as described above.
Example 3 construction of Gene-integrated Yeast Strain Using CRISPR/Cas9 System
Competent cells of S.cerevisiae BJ5464 (MAT. Alpha. Ura3-52 trp1 leu2Δ1his3 Δ200pep4:: HIS3 prb1 Δ 1.6R can1 GAL,ATCC 208288) were prepared using the yeast transformation kit S.c. easy Comp Transformation Kit (available from Invitrogen, USA). P426-gLEU and the corresponding Donor-LEU-YPET were simultaneously transformed into competent cells transformed into commercial plasmid p414 at the same time, 500ng of plasmid, 1000ng of Donor and 200. Mu.L of Solution III (Transformation Solution) were added per 25. Mu.L of competent cells, the mixture was homogenized by vortexing, placed in a 30℃incubator for 15min, and after removal, homogenized again by shaking for 15min, and the procedure was repeated twice, and then the whole bacterial Solution was blotted on an auxotroph plate SD/. DELTA.Trp. DELTA.Ura (6.7 g/L yeast nitrogen source, 0.62g/L defective amino acid mixture, 20g/L glucose, 60mg/L leucine), incubated at 30℃for 2-4 days, and the transcribed gRNA target sequence of the gene was located with the aid of Cas9 protein, while the Donor DNA was integrated into the editing site of the chromosome by homologous recombination, and the knock-in of the reporter gene was achieved.
To verify that the fluorescent reporter gene was integrated into the genome, 3 Saccharomyces cerevisiae transformants were randomly selected from each transformation plate, patched for 12h, positive selection was performed on the monoclonal by colony PCR technique, and part of the thalli was selected in an ultra clean bench and dissolved in 20. Mu.L NaOH (0.025 mol/L), thermally cracked at 98℃for 20min, and centrifuged at 14000rpm for 5min to obtain a supernatant that was used as a template for colony PCR. Colony PCR amplification was performed using LEU-check-F/LEU-check-R as primers, the specific reaction conditions are shown in Table 2, and the PCR products were sent to the assay. Sequencing results showed that all 3 transformants were mutated at the LEU site, i.e.YEp 181-P was successfully integrated TDH3-524 -YPet-T FBA1 The gene is obtained into mutant BJ5464/LEU:: P TDH3-524 -YPet-T FBA1 。
LEU-check-F：5′-CAACAGGTGTGTATCCAGAA-3′；
LEU-check-R：5′-CTCTTCCGTAATCTCGAGCC-3′。
The p426-gLEU/gXIV-68 and the corresponding Donor fragment were simultaneously transformed into competent cells of S.cerevisiae BJ5464, which had been transformed into commercial plasmid p414, according to the procedure described above, wherein the XIV-68 locus was subjected to colony PCR amplification using XIV-68-check-F/XIV-68-check-R as primer.
XIV-68-check-F：5′-AGAACATATTTGCATATGTG-3′；
XIV-68-check-R：5′-TGAATGTTAGAATTGTGCAA-3′。
Finally obtaining mutant BJ5464/LEU:: P TDH3-524 -YPet-T FBA1 、BJ5464/LEU::P TDH3-524 -GUS-T FBA1 、BJ5464/LEU::P TDH3-524 -T4N5-T FBA1 、BJ5464/XIV-68::P TDH3-524 -YPet-T FBA1 、BJ5464/XIV-68::P TDH3-524 -GUS-T FBA1 And BJ5464/XIV-68:: P TDH3-524 -T4N5-T FBA1 。
Similarly, with reference to the above method, S.cerevisiae BJ5464 is replaced with S.cerevisiae CEN.PK2-1Ca or S.cerevisiae BY4741. The following mutants were finally obtained:
CEN.PK2-1Ca/LEU::P TDH3-524 -YPet-T FBA1 、CEN.PK2-1Ca/LEU::P TDH3-524 -GUS-T FBA1 、CEN.PK2-1Ca/LEU::P TDH3-524 -T4N5-T FBA1 、CEN.PK2-1Ca/XIV-68::P TDH3-524 -YPet-T FBA1 、CEN.PK2-1Ca/XIV-68::P TDH3-524 -GUS-T FBA1 and CEN.PK2-1Ca/XIV-68:: P TDH3-524 -T4N5-T FBA1 。
BY4741/LEU::P TDH3-524 -YPet-T FBA1 、BY4741/LEU::P TDH3-524 -GUS-T FBA1 、BY4741/LEU::P TDH3-524 -T4N5-T FBA1 、BY4741/XIV-68::P TDH3-524 -YPet-T FBA1 、BY4741/XIV-68::P TDH3-524 -GUS-T FBA1 And BY4741/XIV-68:: P TDH3-524 -T4N5-T FBA1 。
Example 4 verification of Yeast Strain reporter Gene integration
(1) Detection of YPet fluorescence
The correct BJ5464/LEU:: P will be verified TDH3-524 -YPet-T FBA1 And BJ5464/XIV-68:: P TDH3-524 -YPet-T FBA1 The mutants were streaked onto YPD plates (glucose 20g/L, yeast extract 10g/L, peptone 20g/L, agar 20 g/L) and cultured at 30℃for 72 hours. Picking single colony, respectively inoculating into YPD (glucose 20g/L, yeast extract 10g/L, peptone 20 g/L) liquid culture medium, culturing at 30deg.C to stationary phase, adding 10mL YPD liquid culture medium into 50mL conical flask, adding bacterial liquid into conical flask, and controlling initial OD of bacterial liquid in flask 600 0.05, and incubated at 30℃and 220rpm for 12h. 200. Mu.L of the bacterial suspension was centrifuged at 4000g for 2min, resuspended in PBS (phosphate-buffered saline) and plated in 96-well plates. OD is performed separately 600 And fluorescence measurement, the excitation wavelength of the yellow fluorescent protein (YPET) is 528nm, and the emission wavelength is 485nm. From the measured fluorescence intensity of the sample/OD of the sample 600 Obtaining the relative fluorescence intensity of the sample to characterize the site expressionThe strength is shown in FIG. 1, and BJ5464/XIV-68:: P TDH3-524 -YPet-T FBA1 The mutant strain was used as an experimental group, BJ5464/LEU:: P TDH3-524 -YPet-T FBA1 The mutant strain is used as a control group, 3 parallel samples are arranged in the control group and the experimental group, and the difference multiple of the mutant strain of the XIV-68 relative to the LEU site is 52.42 times under different positions measured by a fluorescence enzyme-linked immunosorbent assay, so that the XIV-68 site has higher expression intensity.
By the same token, the verification is carried out by CEN.PK2-1Ca/XIV-68:: P TDH3-524 -YPet-T FBA1 Mutant strains were used as experimental groups, CEN.PK2-1Ca/LEU:: P TDH3-524 -YPet-T FBA1 The mutant strain is used as a control group, 3 parallel samples are arranged in the control group and the experimental group, and the fluorescence intensity of the XIV-68 relative to the mutant strain of the LEU site is higher under different positions measured by a fluorescence enzyme-labeled instrument, so that the XIV-68 site has higher expression intensity.
BY4741/XIV-68:: P TDH3-524 -YPet-T FBA1 Mutant strains were used as experimental groups, BY4741/LEU:: P TDH3-524 -YPet-T FBA1 The mutant strain is used as a control group, 3 parallel samples are arranged in the control group and the experimental group, and the fluorescence intensity of the XIV-68 relative to the mutant strain of the LEU site is higher under different positions measured by a fluorescence enzyme-labeled instrument, so that the XIV-68 site has higher expression intensity.
(2) Detection of Gus enzyme Activity
The relative expression intensities of the LEU and XIV-68 sites were further verified by exchanging the YPET reporter protein for the beta-glucosidase Gene (GUS). The BJ5464/LEU:: P was activated and cultured according to the method described in the above step (1) TDH3-524 -GUS-T FBA1 And BJ5464/XIV-68:: P TDH3-524 -GUS-T FBA1 Mutant strain to 24h.
Cell lysis:
1) The cultured bacterial liquid was centrifuged at 3000 Xg for 5min.
2) The obtained cells were washed 2 times with Tris-HCl buffer (pH 7.5).
3) Resuspension with buffer Tris-HCl (pH 7.5) to make its OD 600 And (3) obtaining Saccharomyces cerevisiae suspension after the reaction time reaches 0.6-0.7.
4) 200 mu L of Saccharomyces cerevisiae suspension is respectively added into a 96-well plate, 10U/mL Zymolyase is added, and shaking culture is carried out at 30 ℃ and 250rpm for 6 hours, thus obtaining the yeast lysate.
Detection of enzyme activity:
1) mu.L of PBS (pH 7.0) buffer was added to another 96-well plate, followed by 1g/L of pnitrophenyl-. Beta.substrate, D-galactopyranoside (pNPG). Mu.L.
2) 10 mu L of yeast lysate was added to the above identification system and reacted at 37℃for 10min.
3) The reaction was quenched by the addition of 20. Mu.L of 1M NaOH.
4) OD determination with an ELISA apparatus 405 Values.
5) Determination of OD of different concentrations (0, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500. Mu.M) of product p-nitrophenol (pNP) 405 Values, a standard curve is drawn.
6) The Gus enzyme activity, i.e.the amount of enzyme required to release 1. Mu. Mol of p-nitrophenol per hour of substrate at pH 7.0 and 37℃was calculated from the standard curve and was designated U.
BJ5464/LEU:: P under incubation of 10U/mL Zymolyase for 6h TDH3-524 -GUS-T FBA1 And BJ5464/XIV-68:: P TDH3-524 -GUS-T FBA1 As shown in FIG. 2, the mutant strain has 16.03 times of site difference, which means that the expression difference of two sites is still larger under the same promoter and terminator, so that the XIV-68 site has higher expression specificity.
Example 5 expression and preparation method of T4N5
BJ5464/LEU:: P was activated and cultured as described in example 4 TDH3-524 -T4N5-T FBA1 And BJ5464/XIV-68:: P TDH3-524 -T4N5-T FBA1 Mutant strain was grown to 16h and then at initial OD 600 0.05 was transferred to a 100mL Erlenmeyer flask containing 20mL of YPD medium and incubated at 30℃for 30h. Collecting bacterial liquid, centrifuging at 8000rpm for 1min, discarding supernatant, collecting bacterial cells, and storing in a refrigerator at-20deg.C.
The thawed thalli is ice-bathed for 10min, cell lysis buffer (50mM HEPES pH 7.3,1%TritonX-100, 10mM imidozole) is added to resuspend thalli, a 1.5mL cell disruption tube is taken, 1g of 0.5mM glass beads are added, 1.5mL of fungus liquid is added, the thalli is lysed by using a cell disruption instrument, the lysis intensity is 6m/s, the single lysis time is 20s, the total lysis time is 3min, the interval ice-bath time is 2min, and the thalli is centrifuged at 12000rpm for 5min after complete microscopic examination, and the sediment and supernatant are separated. The supernatant was used for SDS-PAGE detection.
As shown in FIG. 3, BJ5464/XIV-68:: P TDH3-524 -T4N5-T FBA1 The mutant strain has a brighter target band around 15-20kD, which indicates that the target protein T4N5 is expressed in a large quantity at the XIV-68 site.
Example 6 improving the efficiency of mutant Strain to lyse T4N5 and method of constructing the same
The construction method of the saccharomyces cerevisiae recombinant vector p426-gSED1 and donor DNA is as follows:
1. construction of recombinant vector p426-gSED1
Based on the predicted result of the CRISPR-Direct website, the gRNA target sequence of the SED1 gene is determined as follows: 5'-AGAGGAGGAAGTGACATCGG-3';
and respectively amplifying by taking the P426 carrier as a template and respectively taking P5/P17 and P6/P18 as primer pairs to obtain SED1-1 and SED1-2 fragments. By ClonExpress R II, recombining the SED1-1 and SED1-2 fragments to obtain a recombinant vector p426-gSED1 carrying the SED1 guide RNA sequence, wherein the nucleotide sequence of the gSED1 expression frame is shown as SEQ ID No. 4.
Wherein the specific primer sequences are as follows (underlined indicates that the sequences are bases homologous to the vector sequences):
P17：5′-CCGATGTCACTTCCTCCTCTGATCATTTATCTTTCACTGC-3′；
P18：5′-AGAGGAGGAAGTGACATCGGGTTTTAGAGCTAGAAATA-3′；
2. synthesis of donor DNA which can block SED1 expression
The primers were as follows:
P25：5′-TACTTTGGCCCAATTTTCCAACAGTACATCTGCTTCTTAAACCGATGTCACTTCCTCCT-3′；
p26:5'-TGTGATAGTTACTGAGCCAGAGGAAGTGGAGATGGAAGAGGAGGAAGTGACATCGGTTT-3'; p25 and P26 are mutually used as templates to amplify and obtain donor DNA, and the sequence of the donor DNA is shown as SEQ ID No. 5.
3. Yeast genome SED1 site editing verification
The activated BJ5464/XIV-68:: P TDH3-524 -T4N5-T FBA1 Single colonies of the mutant strain were plated on SD/. DELTA.Trp+5-FOA (5-fluoroorotic acid, 1 g/L) plates, and single colonies grown on the plates were picked and inoculated on SD/. DELTA.Trp liquid medium to prepare competent cells, for specific procedures, see example 4.
P was transformed simultaneously with P426-gSED1 and donor DNA into BJ5464/XIV-68 transformed with the commercial plasmid P414 TDH3-524 -T4N5-T FBA1 In competent cells of (1) under the assistance of Cas9 protein, the gRNA sequence transcribed by SED1 gene is positioned, and simultaneously donor DNA is recombined to an editing site to block the normal expression of SED1 gene, so as to realize the knockout of SED1 gene in genome and obtain mutant BJ5464/XIV-68:: P TDH3-524 -T4N5-T FBA1 /Δsed1。
3 Saccharomyces cerevisiae transformants were randomly selected, colony PCR amplification was performed using SED1-check-F/SED1-check-R as primers, and the PCR products were sent to detection. Sequencing results show that the SED1 loci of the target genes of the 3 transformants are mutated, namely the SED1 genes are knocked out successfully.
SED1-check-F：5′-CCCTCTTTTGAACTGTCATA-3′；
SED1-check-R：5′-GTAGTTGGTGGGAAAGCTGA-3′。
The obtained mutant was subjected to protein expression, and the specific procedure is as in example 5, and the result is shown in FIG. 4, and after the SED1 gene is knocked out, the target band is brighter, which indicates that deletion of the SED1 gene is more favorable for cell lysis, so that more target protein is released.
Similarly, with reference to the above method, a mutant strain was obtained: CEN.PK2-1Ca/XIV-68:: P TDH3-524 -T4N5-T FBA1 /Δsed1、BY4741/XIV-68::P TDH3-524 -T4N5-T FBA1 /Δsed1。
EXAMPLE 7 affinity chromatography and desalting of recombinant T4N5
The procedure of example 5 was followed for mutant BJ 5464/XIV-68:P TDH3-524 -T4N5-T FBA1 Culture and lysis were performed with/. DELTA.sed1, and 50mL of the lysed supernatant was collected as a subsequent protein purification sample. Purification of T4N5 was performed using a Bio-rad chromatography system, and all reagents required degassing prior to purification.
Flushing the system with ultrapure water at a maximum flow rate (6.5 mL/min) for 5min to remove 20% ethanol from the system tubing; reducing the flow rate to 1mL/min, connecting a Hi-TrapTM chelating HP column, then adjusting the flow rate to 3mL/min in a grading manner, flushing until the conductivity indicating line and the ultraviolet detection baseline are stably kept at about 0; adding 0.5M sodium hydroxide solution, removing residual impurities in the column, and flushing to a baseline for stability; the ultrapure water is accessed to remove the sodium hydroxide solution in the flow path of the purification system until the baseline value is stable; the nickel sulfate solution is connected to flow to the base line stably, so that the chelating column is fully combined with the nickel ions; buffer A (17 mM disodium hydrogen phosphate dihydrate, 3mM sodium dihydrogen phosphate dihydrate, 500mM sodium chloride, 20mM imidazole) was accessed, nickel ions not chelated to the column were removed, and the column was rinsed to baseline plateau; accessing a protein sample, adjusting the flow rate to 1.5mL/min, after the protein sample completely enters a purification system, changing the protein sample into a Buffer A3 mL/min to wash until an ultraviolet detection baseline is stable, and collecting flow-through liquid; adjusting the proportion of Buffer B (17 mM disodium hydrogen phosphate, 3mM sodium dihydrogen phosphate dihydrate, 500mM sodium chloride, 500mM imidazole), performing gradient elution and collecting; respectively introducing ultrapure water, stripping Buffer (17 mM disodium hydrogen phosphate dihydrate, 3mM sodium dihydrogen phosphate dihydrate, 500mM sodium chloride, 55mM disodium ethylenediamine tetraacetate) and flushing the system with 0.5M sodium hydroxide solution to remove nickel ions and impurities in the column; accessing 20% ethanol, flushing 10 column volumes, unloading the column, and closing the chromatographic system; partially collecting the protein sample, preparing SDS-PAGE sample by the method and performing SDS-PAGE verification; and (3) placing the collected protein sample into a 3kDa ultrafiltration centrifuge tube, centrifuging at 4 ℃ at 8000rpm for 30min for desalting and concentrating, adding 10mL of PBS solution 3 times during the desalting and concentrating, collecting the T4N5 solution which is desalted in the ultrafiltration tube, quick-freezing the solution by liquid nitrogen, and storing the solution in a refrigerator at-80 ℃. The purification results are shown in FIG. 5, and the results show that the T4N5 protein with higher purity is successfully obtained through nickel column affinity chromatography and desalination.
EXAMPLE 8 determination of in vitro DNA repair Activity of recombinant T4N5
And (3) performing in vitro activity verification on T4N5 by adopting a DNA etching method. 20. Mu.L of plasmid YEp181 at a concentration of 500 ng/. Mu.L was placed at an intensity of 21. Mu.W/cm 2 Irradiation under ultraviolet lamp at λ=254 nm for 30min to form pyrimidine dimers; the T4N5 protein sample obtained by concentration in example 7 was diluted 5-fold, 10-fold, 20-fold and 100-fold, 1. Mu.L of each was aspirated, and 1. Mu.L of the stock solution was aspirated and added to 1x T4N5 Reaction Buffer (25 mM sodium phosphate, 100mM sodium chloride, 1mM ethylenediamine tetraacetic acid, 1mM dithiothreitol, 100. Mu.g/mL bovine serum albumin) together with 500ng of plasmid YEp181 after ultraviolet irradiation, to prepare a 20. Mu.L system, and reacted at 37℃for 30 minutes; 1. Mu.L was taken for agarose gel electrophoresis and gray-scale scanning was performed by imageJ to examine the repair effect of recombinant T4 endonuclease V. The repair effect results are shown in FIG. 6, which shows that the purified T4N5 sample has good DNA repair efficiency.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The application of the saccharomyces cerevisiae chromosome XIV-68 locus in the high expression of exogenous metabolites by using saccharomyces cerevisiae as a chassis cell is characterized in that: the nucleotide sequence of the XIV-68 locus of the saccharomyces cerevisiae chromosome comprises a gRNA target sequence of the XIV-68 locus and sequences within 500bp upstream and downstream respectively; further, the nucleotide sequence of the XIV-68 locus of the saccharomyces cerevisiae chromosome comprises a gRNA target sequence of the XIV-68 locus and sequences within 100bp upstream and downstream respectively;
the gRNA target sequence of the XIV-68 locus is 5'-AAAGAATCATAGATCGTCAA-3'.
2. A saccharomyces cerevisiae engineering bacterium for high yield of T4N5 is characterized in that:
integrating a T4N5 gene into a Saccharomyces cerevisiae chromosome XIV-68 locus in claim 1 by taking Saccharomyces cerevisiae as an original strain to obtain a high-yield T4N5 Saccharomyces cerevisiae engineering strain integrated with the T4N5 gene; or,
taking saccharomyces cerevisiae as an initial strain, integrating a T4N5 gene into a saccharomyces cerevisiae chromosome XIV-68 locus in claim 1, and knocking out an SED1 gene in a saccharomyces cerevisiae genome to obtain a high-yield T4N5 saccharomyces cerevisiae engineering bacterium integrated with the T4N5 gene and knocking out the SED1 gene;
wherein T4N5 refers to T4 endonuclease V.
3. The saccharomyces cerevisiae engineering bacteria of claim 2 wherein:
the amino acid sequence coded by the T4N5 gene is shown as NP 049733.1;
or, adding a histidine tag and/or a protease cleavage site at the amino terminus of the amino acid sequence encoded by the T4N5 gene.
4. A saccharomyces cerevisiae engineering bacterium according to claim 3 wherein:
the nucleotide sequence of the T4N5 gene is shown as 37 to 450bp of SEQ ID No. 1;
the nucleotide sequence of the T4N5 gene with histidine tag and protease cleavage site corresponding to the coding nucleic acid sequence is shown in SEQ ID No. 1.
5. The saccharomyces cerevisiae engineering bacteria of claim 2 wherein:
integrating the expression cassette of the T4N5 gene into the Saccharomyces cerevisiae chromosome XIV-68 site;
the expression cassette of the T4N5 gene comprises a promoter, a T4N5 gene and a terminator; the promoter is selected from constitutive strong promoters, P CCW12 、P TDH3 、P PDC1 And P TEF1 At least one of (a) and (b); the terminator is selected from T RPL3 、T PGK1 、T BNA4 And T FBA1 At least one of them.
6. A method for constructing saccharomyces cerevisiae engineering bacteria with high yield of T4N5 according to any one of claims 2-5, which is characterized in that: the method comprises the following steps:
1) Designing a gRNA target sequence according to the XIV-68 locus of the saccharomyces cerevisiae chromosome;
2) Constructing a recombinant vector according to the gRNA target sequence of the step 1);
3) Preparing donor DNA comprising a T4N5 gene;
4) Simultaneously transforming the recombinant vector of the step 2) and the donor DNA containing the T4N5 gene of the step 3) into the Saccharomyces cerevisiae transferred with the Cas9 gene to realize the integration of the T4N5 gene and obtain the Saccharomyces cerevisiae engineering bacteria with high yield of T4N5 integrated with the T4N5 gene;
alternatively, the method further comprises:
5) Knocking out the SED1 gene in the saccharomyces cerevisiae engineering bacteria genome of the step 4), realizing the integration of the T4N5 gene and the knocking out of the SED1 gene, and obtaining the saccharomyces cerevisiae engineering bacteria integrating the T4N5 gene and knocking out the SED1 gene and producing high-yield T4N5.
7. The construction method according to claim 6, wherein:
the gRNA target sequence in step 1) is 5'-AAAGAATCATAGATCGTCAA-3';
in the step 2), the starting vector used for constructing the recombinant vector is any one Saccharomyces cerevisiae vector;
the donor DNA comprising the T4N5 gene in step 3) comprises a homologous sequence of 40-100bp upstream of the gRNA target sequence, an expression cassette of the T4N5 gene or the T4N5 gene, and a homologous sequence of 40-100bp downstream of the gRNA target sequence.
8. The construction method according to claim 6 or 7, characterized in that:
in the step 5), the SED1 gene in the saccharomyces cerevisiae engineering bacteria genome of the step 4) is knocked out, and the method comprises the following steps:
a) Designing a gRNA target sequence according to the SED1 gene in the genome;
b) Constructing a recombinant vector according to the gRNA target sequence of step a);
c) Simultaneously transforming the recombinant vector and the donor DNA of the step b) into the saccharomyces cerevisiae engineering bacteria of the step 4) transferred with the Cas9 gene, so as to realize the integration of the T4N5 gene and the knockout of the SED1 gene.
9. The method of construction according to claim 8, wherein:
the gRNA target sequence in step a) is 5'-AGAGGAGGAAGTGACATCGG-3';
in the step b), the starting vector used for constructing the recombinant vector is any one of Saccharomyces cerevisiae vectors.
10. The use of the saccharomyces cerevisiae engineering bacteria according to any one of claims 2-5 in high yield of T4N5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410059789.1A CN117867005A (en) | 2024-01-15 | 2024-01-15 | Saccharomyces cerevisiae engineering bacteria with high yield of T4N5 and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410059789.1A CN117867005A (en) | 2024-01-15 | 2024-01-15 | Saccharomyces cerevisiae engineering bacteria with high yield of T4N5 and construction method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117867005A true CN117867005A (en) | 2024-04-12 |
Family
ID=90580790
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410059789.1A Pending CN117867005A (en) | 2024-01-15 | 2024-01-15 | Saccharomyces cerevisiae engineering bacteria with high yield of T4N5 and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117867005A (en) |
-
2024
- 2024-01-15 CN CN202410059789.1A patent/CN117867005A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0292609B1 (en) | Process and signal sequence for the extracellular production of proteinaceous material | |
| US4959317A (en) | Site-specific recombination of DNA in eukaryotic cells | |
| JPH0515379A (en) | Yeast hybrid vector and method for producing polypeptide using said vector | |
| Filho et al. | Stable Yeast Transformants that Secrete Functional α–Amylase Encoded by Cloned Mouse Pancreatic cDNA | |
| US20110281362A1 (en) | Electrotransformation of Gram-Positive, Anaerobic, Thermophilic Bacteria | |
| WO2016029802A1 (en) | Identification and use of synthetic gene cluster of cordycepin | |
| CN113106114A (en) | Factor for regulating and controlling trichoderma reesei protein expression efficiency, regulating and controlling method and application | |
| JPS61257189A (en) | Novel plasmid vector | |
| CN104232611A (en) | Recombinant beauveria brongniartii proteinase K as well as industrial production and purification method thereof | |
| JP2016538865A (en) | New genome modification system for microorganisms | |
| US4464471A (en) | Biologically engineered plasmid coding for production of β-glucosidase, organisms modified by this plasmid and methods of use | |
| CN117867005A (en) | Saccharomyces cerevisiae engineering bacteria with high yield of T4N5 and construction method and application thereof | |
| JPH0576375A (en) | Improved yeast vector | |
| CN115141763A (en) | Yeast engineering bacterium capable of efficiently secreting protein and construction method and application thereof | |
| CA2017176A1 (en) | Albumin gene-containing plasmid, transformant carrying same, production of such transformant and production of albumin | |
| CN112626102B (en) | Pichia pastoris fixed-point DSB system and application | |
| CN109337921B (en) | Recombinant vector for constructing saccharomyces cerevisiae cracking engineering bacteria and application thereof | |
| WO2014030644A1 (en) | Transformant of schizosaccharomyces pombe mutant, and cloning vector | |
| CN114107360A (en) | Method for improving cellulase expression of trichoderma reesei by interfering phosphatase gene | |
| JP7388194B2 (en) | Mutant strain of Trichoderma reesei and method for producing protein using the same | |
| CN108949579B (en) | Thermoascus thermophilus gene expression system | |
| CN111378674B (en) | Myceliophthora isopterans glucoamylase MhglaA, coding gene thereof and application thereof in glucose production | |
| CZ279427B6 (en) | Micro-organism of escherichia coli or pseudomonas putida strain and recombinant dna | |
| CN114634883B (en) | Recombinant engineering bacterium for producing 2' -fucosyllactose and construction method and application thereof | |
| JPS58501406A (en) | Increased expression of protein synthesis in Escherichia coli |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |