CN116334115A - Genetically engineered saccharomycete for intracellular expression of tetracycline degrading enzyme Tet (X) and application thereof - Google Patents
Genetically engineered saccharomycete for intracellular expression of tetracycline degrading enzyme Tet (X) and application thereof Download PDFInfo
- Publication number
- CN116334115A CN116334115A CN202310255638.9A CN202310255638A CN116334115A CN 116334115 A CN116334115 A CN 116334115A CN 202310255638 A CN202310255638 A CN 202310255638A CN 116334115 A CN116334115 A CN 116334115A
- Authority
- CN
- China
- Prior art keywords
- tetracycline
- genetically engineered
- tet
- degrading enzyme
- opt
- 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
- 239000004098 Tetracycline Substances 0.000 title claims abstract description 80
- 235000019364 tetracycline Nutrition 0.000 title claims abstract description 80
- 150000003522 tetracyclines Chemical class 0.000 title claims abstract description 80
- 229960002180 tetracycline Drugs 0.000 title claims abstract description 73
- 229930101283 tetracycline Natural products 0.000 title claims abstract description 73
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 68
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 68
- 101100480958 Sphingobacterium sp. (strain PM2-P1-29) tet(X) gene Proteins 0.000 title claims abstract description 34
- 230000000593 degrading effect Effects 0.000 title claims abstract description 32
- 230000003834 intracellular effect Effects 0.000 title claims abstract description 16
- 241000235342 Saccharomycetes Species 0.000 title claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 42
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 34
- 239000013612 plasmid Substances 0.000 claims abstract description 20
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 6
- 230000028327 secretion Effects 0.000 claims abstract description 5
- 241001506991 Komagataella phaffii GS115 Species 0.000 claims abstract description 4
- 101100010928 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) tuf gene Proteins 0.000 claims abstract 2
- 101150001810 TEAD1 gene Proteins 0.000 claims abstract 2
- 101150074253 TEF1 gene Proteins 0.000 claims abstract 2
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 claims abstract 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 229940072172 tetracycline antibiotic Drugs 0.000 claims description 19
- 244000144972 livestock Species 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 14
- 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 claims description 11
- 239000008103 glucose Substances 0.000 claims description 11
- 238000009395 breeding Methods 0.000 claims description 9
- 230000001488 breeding effect Effects 0.000 claims description 9
- 239000002351 wastewater Substances 0.000 claims description 8
- 238000010276 construction Methods 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 239000007222 ypd medium Substances 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 239000008057 potassium phosphate buffer Substances 0.000 claims description 3
- 239000012137 tryptone Substances 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 239000003124 biologic agent Substances 0.000 claims description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 abstract description 39
- 108090000623 proteins and genes Proteins 0.000 abstract description 21
- 241000894006 Bacteria Species 0.000 abstract description 15
- 239000006166 lysate Substances 0.000 abstract description 11
- 238000006065 biodegradation reaction Methods 0.000 abstract description 7
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 abstract description 5
- 239000005515 coenzyme Substances 0.000 abstract description 5
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 abstract description 5
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 238000003860 storage Methods 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 238000006731 degradation reaction Methods 0.000 description 34
- 230000015556 catabolic process Effects 0.000 description 33
- 239000010865 sewage Substances 0.000 description 17
- 238000012408 PCR amplification Methods 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 14
- 230000001580 bacterial effect Effects 0.000 description 13
- 239000000047 product Substances 0.000 description 11
- 239000003814 drug Substances 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 238000000855 fermentation Methods 0.000 description 8
- 230000004151 fermentation Effects 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 244000144977 poultry Species 0.000 description 7
- 235000013594 poultry meat Nutrition 0.000 description 7
- 239000008399 tap water Substances 0.000 description 7
- 235000020679 tap water Nutrition 0.000 description 7
- 229940040944 tetracyclines Drugs 0.000 description 7
- 238000011144 upstream manufacturing Methods 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 description 6
- 238000000137 annealing Methods 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000004925 denaturation Methods 0.000 description 6
- 230000036425 denaturation Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000012257 pre-denaturation Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 230000009466 transformation Effects 0.000 description 6
- 206010059866 Drug resistance Diseases 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 229930182555 Penicillin Natural products 0.000 description 5
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000007791 liquid phase Substances 0.000 description 5
- 229940049954 penicillin Drugs 0.000 description 5
- 230000003115 biocidal effect Effects 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- SOVUOXKZCCAWOJ-HJYUBDRYSA-N (4s,4as,5ar,12ar)-9-[[2-(tert-butylamino)acetyl]amino]-4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1C2=C(N(C)C)C=C(NC(=O)CNC(C)(C)C)C(O)=C2C(O)=C2[C@@H]1C[C@H]1[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]1(O)C2=O SOVUOXKZCCAWOJ-HJYUBDRYSA-N 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- 102100036826 Aldehyde oxidase Human genes 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 2
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000009304 pastoral farming Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000009374 poultry farming Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000010802 sludge Substances 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 229960004089 tigecycline Drugs 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- 108010006654 Bleomycin Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 102000010292 Peptide Elongation Factor 1 Human genes 0.000 description 1
- 108010077524 Peptide Elongation Factor 1 Proteins 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 241000589970 Spirochaetales Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 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(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[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 OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 238000003776 cleavage reaction Methods 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
- 239000002131 composite material Substances 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007247 enzymatic mechanism Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000004880 explosion Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- -1 glucan and the like Chemical class 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229940127554 medical product Drugs 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000006919 modified lb Substances 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000001699 photocatalysis Effects 0.000 description 1
- 238000006303 photolysis reaction Methods 0.000 description 1
- 230000015843 photosynthesis, light reaction Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Business, Economics & Management (AREA)
- Emergency Management (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of bioengineering, and particularly discloses genetically engineered saccharomycetes for intracellular expression of tetracycline degrading enzyme Tet (X) and application thereof. The invention uses pichia pastoris GS115 as an expression host bacterium, uses GAP or TEF1 as a promoter to replace a methanol-induced promoter of pPIC9K-opt (X4) plasmid, deletes secretion signal peptide of the plasmid, and constructs the genetic engineering bacterium capable of autonomously expressing tetracycline degrading enzyme Tet (X) in cells. The gene engineering yeast lysate can efficiently degrade tetracycline residues in samples in different environments on the premise of not adding coenzyme NADPH. The enzyme preparation prepared by the genetically engineered bacteria has good storage stability and good tolerance to the pH and temperature of the environment. The gene engineering yeast biodegradation strategy has the advantages of low cost, high efficiency, low risk and wide application range.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to genetically engineered saccharomycetes for intracellular expression of tetracycline degrading enzyme Tet (X) and application thereof.
Background
The first antibiotic, penicillin, was discovered since 1928, alexander Fleming, and it became an irreplaceable medical product for humans and animals. One survey predicts that global antibiotic consumption will increase by 67.0% from 63151 ±1560 tons (2010) to 105596 ±3605 tons (2030). Tetracyclines are among the most common antibiotics, known for their broad-spectrum antibacterial activity, and are effective against both gram-negative and gram-positive bacteria, spirochetes, mycoplasma and parasites. Broad spectrum, low cost, convenient production and use, and the like, facilitates the wide application of tetracycline antibiotics in preventive medicine and clinical treatment for human medicine, veterinary medicine, animal growth promoters and the like, and the tetracyclines enter human and animal organisms mostly in an oral administration way. A large number of tetracycline antibiotics are produced and used each year, particularly in livestock and poultry farming. The consumption of tetracycline in livestock and poultry farming was highest in all investigated continents, 41.2% in europe, 40.8% in america, 31.7% in africa and 31.2% in asia, respectively. In 2013, the consumption of the tetracycline antibiotics in China is about 6950 tons, and the veterinary antibiotics account for 74.6 percent (47.5 percent of pigs, 16.2 percent of chickens and 10.9 percent of other animals). According to the animal antibiotic usage publications 2020 and 2021 issued by the agricultural rural area, the tetracyclines consume 11297.65 tons and 10002.733 tons in 2019 and 2020, accounting for 36.56% and 30.51% of the total annual antibacterial drugs respectively.
However, with the widespread use of tetracycline antibiotics, some troublesome problems are increasingly manifested. Because of the low bioavailability of the tetracycline antibiotics by human and animals, a considerable proportion (70.0% -90.0%) of the tetracycline antibiotics flow into the surrounding environment along with excrement, urine and other excretions. Tetracyclines are frequently detected in feces, soil, sewage, sludge, surface water, wastewater, and even drinking water. On one hand, the residual antibiotics in the environment enter the human body through a food chain, and on the other hand, bacteria in the environment are promoted to accelerate the formation of antibiotic drug resistance genes, and enrichment and diffusion of the drug resistance genes threaten the effect of clinical treatment.
In view of the hazards of tetracycline residues, previous work has developed a series of methods for removing tetracycline. Common methods for removing tetracyclines include biodegradation and non-biodegradation. Bacteria, white rot fungi, activated sludge, sediment and constructed wetland are commonly used in the biodegradation process. For another non-biological degradation process, there are many reports on membrane technology, physical adsorption, photolysis, electrochemical degradation, photocatalytic and photoelectrocatalytic treatments, and the like. Each strategy has advantages and disadvantages and ideally, a widely accepted degradation strategy is cost effective, efficient in removal, environmentally friendly and scalable to industry levels. Non-biodegradation is expensive, energy-consuming, and environmentally unfriendly, and the biodegradation process is an economical, efficient and most suitable method of removing tetracycline from the environment at the expense of microbial enzyme mechanisms. There is no doubt that there is a risk of gene transmission in the use of bacteria, so that such methods have not been widely popularized so far.
The discovery of a new subtype of the tetracycline degradation gene Tet (X) provides a plurality of new ideas for solving the problem of tetracycline residue. Literature [ Qian He, et al reducing tetracycline antibiotics residues in aqueous environments using Tet (X) degrading enzymes expressed in Pichiapastoris [ J)],Science ofthe Total Environment,799(2021)149360]A pichia pastoris for stably expressing glycosylated tetracycline degrading enzyme Tet (X) is constructed, and the protein expressed by its exogenous gene needs to be expressed in NADPH and Mg 2+ 、O 2 Can exhibit biological functions only when participating in the process, and has degradation effect on tetracycline antibiotics. This method requires protein extraction of the fermentation broth and additional supplementation of NADPH, an expensive coenzyme, resulting in increased production and application costs. In addition, the extracted enzyme has poor degradation effect on the tetracyclic in livestock and poultry breeding sewage, and has poor tolerance to temperature and pH, and cannot meet the clinical application at the present stage. The promoter used in the document is a methanol-inducible AOX1 promoter, and can express high-level protein under the strict induction of methanol, however, the promoter used for expressing a plurality of problems (1) in the actual fermentation amplifying process needs the induction of methanol, the methanol is toxic and flammable, and special anti-riot design is needed in large-scale industrial fermentation; (2) The methanol fermentation consumes oxygen strongly, and the amount of oxygen required for methanol metabolism is three to four times the amount of oxygen required for glucose as a carbon source. The more methanol is consumed, the more pure oxygen is required, which causes great trouble to the actual industrial production. In addition, the more the consumed methanol is, the more heat is generated, and the higher the cooling capacity requirement of the required equipment is; (3) Methanol is used as a petrochemical product, is not suitable for the production of additives in some food fields, and the production cost of methanol is increased along with the explosion of petroleum crisis; (4) Methanol metabolism produces H 2 O 2 Hydrolysis of the expressed polypeptide may result.
In order to realize safe, efficient and environment-friendly tetracycline removal, developing a novel strategy for removing tetracycline residues based on tetracycline degrading enzyme Tet (X) is a primary challenge.
Disclosure of Invention
Aiming at the defects and shortcomings of the prior art, the invention provides a novel economic, efficient and safe strategy for degrading tetracycline residues in water environment, and the primary aim of the invention is to provide a construction method of a genetic engineering plasmid for intracellular expression of tetracycline degrading enzyme Tet (X), wherein the promoter of pPIC9K-opt (X4) plasmid is replaced by GAP or TEF1 promoter, and simultaneously the sequence of secretory signal peptide expressed in the plasmid is deleted.
The second object of the invention is to provide a genetically engineered yeast for intracellular expression of tetracycline degrading enzyme Tet (X), which is constructed by adopting the method to obtain genetically engineered plasmid, and the host is Pichia pastoris GS115, so as to obtain the genetically engineered strain capable of autonomously expressing the tetracycline degrading enzyme Tet (X) in cells.
In order to improve the autonomous expression of degrading enzyme to the greatest extent, the invention modifies the promoter of the existing plasmid pPIC9K-opt (X4), replaces a methanol inducible promoter with a GAP (3-glyceraldehyde phosphate dehydrogenase) promoter or a TEF1 (translation elongation factor-1) promoter which can be autonomously started, and simultaneously realizes the intracellular expression by modifying the plasmid to delete the secretion signal peptide of the plasmid for low-cost application. Finally, the low-cost application is realized by releasing the coenzyme and degrading enzyme Tet (X) of the engineering yeast cell by the cell lysis.
The third object of the present invention is to provide the application of the genetically engineered yeast and/or the biological agent prepared from the genetically engineered yeast in degrading tetracycline antibiotic residues.
The genetic engineering yeast lysate can efficiently degrade tetracycline in different environment matrixes such as tap water, lake water and livestock breeding sewage without adding coenzyme NADPH, and has wide applicability.
Preferably, the genetically engineered yeast is subjected to expansion culture, bacterial cells are collected, and a sample containing the tetracycline antibiotics is put into the culture medium after the bacterial cells are lysed.
Preferably, the sample is livestock breeding sewage.
The fourth object of the invention is to provide a tetracycline antibiotic degrading enzyme Tet (X) preparation, which is prepared from the genetically engineered yeast.
The invention also provides a method for preparing the tetracycline antibiotic degrading enzyme Tet (X) preparation by using the genetically engineered yeast, which comprises the steps of amplifying and culturing the genetically engineered yeast, collecting thalli, and then carrying out cracking, crushing and freeze drying.
Preferably, in the expansion culture of the genetically engineered yeast, the YPD medium is prepared by adding 1g of yeast extract and 2g of tryptone to 80ml of pure water, autoclaving at 121 ℃ for 20min, cooling to room temperature, and adding 10ml of 2.0% glucose solution and 10ml of 1M potassium phosphate buffer with pH of 6.8.
Preferably, in the case of the expansion culture of the genetically engineered yeast, the culture conditions are continuous culture in a shaker at 30℃and 220rpm for 4 days.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the invention, the autonomous expression of the tetracycline degrading enzyme Tet (X) in yeast cells is realized, and the maximum expression of the degrading enzyme is realized by comparing different promoters and optimizing expression parameters.
(2) Compared with the prior art, the gene engineering yeast lysate can efficiently degrade tetracycline residues in samples in different environments on the premise of not adding coenzyme NADPH.
(3) The biological safety evaluation also proves the safety, and the engineering yeast biodegradation strategy constructed based on the invention has the characteristics of low cost, high efficiency, low risk and wide application range.
(4) Compared with the freeze-drying of the traditional enzyme preparation, the enzyme preparation prepared by the genetically engineered yeast does not need to add any freeze-drying protective agent, greatly reduces the production cost, has good stability, has no obvious difference in degradation efficiency when stored for 14 days at different temperatures, still keeps the high efficiency of tetracycline degradation, and has good tolerance to both pH and temperature of the environment. In the existing tetracycline treatment strategy, the preparation is convenient to store, transport and apply, and has the potential of being capable of being upgraded to industrial production.
Drawings
FIG. 1 is a schematic diagram showing the construction of intracellular autonomous expression plasmids pTEF1-opt (X4) and pGAP-opt (X4).
FIG. 2 shows the gel electrophoresis patterns of PCR products of the transformants of engineering yeasts GS115/pTEF1-opt (X4) and GS115/pGAP-opt (X4), M being 2000DNA Marke.
FIG. 3 shows growth curves of GS115/pTEF1-opt (X4) and GS115/pGAP-opt (X4) in 6 different carbon sources, glycerin: glycerol, sorbitol: sorbitol, maltose: maltose, glucose: glucose, glucose: sucrose, methyl: methanol.
FIG. 4 is a 3D plot of response surface three-factor interactions of GS115/pGAP-opt (X4) (upper) and GS115/pTEF1-opt (X4) (lower).
FIG. 5 shows Western blot (top) and quantitative analysis (bottom) of GS115/pGAP-opt (X4) and GS115/pTEF1-opt (X4).
FIG. 6 is a graph showing the phenotype of GS115/pTEF1-opt (X4) lysate degradation of tetracycline in three different aqueous environments, tap water: tap water, lake water: lake water, livestock sewage: cultivation sewage, and antibacterial: tetracyclines, enzymes: purified tetracycline degrading enzyme Tet (X).
FIG. 7 is a graph showing the results of liquid phase detection of GS115/pTEF1-opt (X4) lysate degradation of tetracycline in three different aqueous environments, tap water: tap water, lake water: lake water, livestock sewage: cultivation sewage, and antibacterial: tetracyclines, enzymes: purified tetracycline degrading enzyme Tet (X).
FIG. 8 shows the variation of the copy number of drug resistant genes at different stages of GS115/pTEF1-opt (X4) lysate.
FIG. 9 shows the results of the natural transformation (left) and the conjugation transfer (right) experiments of GS115/pTEF1-opt (X4) and LHM 10-1.
FIG. 10 is a gel electrophoresis chart of PCR products of three transformants of LHM 10-1.
FIG. 11 is an external view of a Tet (X) enzyme preparation.
FIG. 12 shows the phenotypic results of Enzyme preparation on tetracycline degradation in aquaculture wastewater, enzyme being purified degrading Enzyme Tet (X) as positive control; the anti-biological is tetracycline Antibiotic as a control.
FIG. 13 shows the results of HPLC detection of tetracycline degradation in the cultivation wastewater by the enzyme preparation, and the anti-biological is tetracycline Antibiotic as a control.
FIG. 14 is the results of monitoring the storage time and degradation efficiency of the enzyme preparation at room temperature (25 ℃), 4 ℃, -20 ℃, -80 ℃.
FIG. 15 is an effect of ambient temperature and pH on degradation properties of an enzyme preparation.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The test methods used in the embodiment of the invention are all conventional methods unless specified otherwise; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
EXAMPLE 1 construction of intracellular expression engineering Yeast
(1) Modification of intracellular autonomous expression plasmids
As shown in FIG. 1, in order to obtain a plasmid capable of autonomous intracellular expression, the present invention completes plasmid transformation in two steps, first by replacing the inducible promoter AOX1 of plasmid pPIC9K-opt (X4) (refer to CN 202010010949.5) with TEF1 and GAP promoters, and then deleting the secretion signal peptide in the plasmid to achieve autonomous intracellular expression.
1. Construction of pTEF1 alpha-opt (X4) and pGAP alpha-opt (X4)
(1) The framework regions other than the promoter of the expression plasmid pPIC9K-opt (X4) were amplified using the primers PIC9K-infuF/R with the pPIC9K-opt (X4) as template.
PIC9K-infuF：tcgaataataactgttatttttcagtgt
PIC9K-infuR：ggatccaaacgatgagatttc
The PCR amplification system (50. Mu.L) was: 10. Mu.L of double pure water, 25. Mu.L of 2 XKOD Buffer, 10. Mu.L of dNTPs, 1.5. Mu.L of upstream primer, 1.5. Mu.L of downstream primer, 1. Mu.L of KOD enzyme and 1. Mu.L of template.
The reaction conditions for PCR amplification were: pre-denaturation at 94℃for 3min; denaturation at 98℃for 10s, annealing at 58℃for 30s, extension at 68℃for 5min for 35 cycles; final extension at 68℃for 7min; preserving at 4 ℃.
The PCR product is separated and identified by 1.0% agarose gel electrophoresis, and then the fragment is purified and recovered.
(2) The TEF1 and GAP promoters were amplified using the primers TEF1-infuF/R, GAP-infuF/R, respectively, using pCEV-G4-KM and pGAPZαA as templates.
TEF1-infuF：ctgaaaaataacagttattattcgacacacaccatagcttcaaaatg
TEF1-infuR：gaaggaaatctcatcgtttggatcccttagattagattgctatgctttc
GAP-infuF：ctgaaaaataacagttattattcgatttttgtagaaatgtcttggtgtc
GAP-infuR：gaaggaaatctcatcgtttggatccatagttgttcaattgattgaaatag
The PCR amplification system (50. Mu.L) was: 10. Mu.L of double pure water, 25. Mu.L of 2 XKOD Buffer, 10. Mu.L of dNTPs, 1.5. Mu.L of upstream primer, 1.5. Mu.L of downstream primer, 1. Mu.L of KOD enzyme and 1. Mu.L of template.
The reaction conditions for PCR amplification were: pre-denaturation at 94℃for 3min; denaturation at 98℃for 10s, annealing at 58℃for 30s, extension at 68℃for 30s for 35 cycles; final extension at 68℃for 7min; preserving at 4 ℃.
The PCR product is separated and identified by 2.0% agarose gel electrophoresis, and then the fragment is purified and recovered.
(3) The fragments recovered in (1) and (2) were subjected to homologous recombination, respectively, in a recombination system (10. Mu.L) of: amount of homologous recombinase 5 μl, fragment material: amount of carrier material = 1:1, supplementing water to 10 mu L, and incubating at 50 ℃ for 30min. 10 mu L of recombinant product is sucked and transformed into DH5 alpha H competence, positive transformants are screened by using an Amp100 medicine plate, and the sequencing verification of the positive transformants is correct.
2. Construction of pTEF1-opt (X4) and pGAP-opt (X4)
The skeleton regions other than the secretion signal peptides of pTEF 1. Alpha. -opt (X4) and pGAP. Alpha. -opt (X4) were amplified using primers TEF1-F/R, GAP-F/R using pTEF 1. Alpha. -opt (X4) and pGAP. Alpha. -opt (X4) as templates.
TEF1-F：caatctaatctaagggatccaaacgatgtccaacaaggagaagcaa
TEF1-R：cgtttggatcccttagattagattgctatgct
GAP-F：aattgaacaactatggatccaaacgatgtccaacaaggagaagcaaat
GAP-R：cgtttggatccatagttgttcaattgatt
The PCR amplification system (50. Mu.L) was: 10. Mu.L of double pure water, 25. Mu.L of 2 XKOD Buffer, 10. Mu.L of dNTPs, 1.5. Mu.L of upstream primer, 1.5. Mu.L of downstream primer, 1. Mu.L of KOD enzyme and 1. Mu.L of template.
The reaction conditions for PCR amplification were: pre-denaturation at 94℃for 3min; denaturation at 98℃for 10s, annealing at 56℃for 30s, extension at 68℃for 5min for 35 cycles; final extension at 68℃for 7min; preserving at 4 ℃.
The PCR product is separated and identified by 1.0% agarose gel electrophoresis, and then the fragment is purified and recovered. The recovered product was chemically transferred to DH 5. Alpha. H competent cells, and positive transformants were selected using Amp100 plates and verified using DeltaTEF 1-F/Deltaopt (X4) -R and DeltaGAP-F/Deltaopt (X4) -R, respectively.
△TEF1-F：cgtttggatcccttagattagattgctatgct
△GAP-F：tgagattattggaaaccaccag
△opt(X4)-R：gcttctctgtcgttgtctctttc
The PCR amplification system (50. Mu.L) was: 20. Mu.L of double pure water, 25. Mu.L of Taq MIX, 2. Mu.L of upstream primer, 2. Mu.L of downstream primer and 1. Mu.L of template.
The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 8min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 68℃for 30s, for a total of 35 cycles; final extension at 72℃for 5min; preserving at 4 ℃.
The PCR products are separated and identified by 2.0% agarose gel electrophoresis, and the sequencing of positive transformants is verified to be correct.
(2) Transformation of engineered yeasts
The transformation and selection of the intracellular expression plasmids pTEF1-opt (X4) and pGAP-opt (X4) were carried out in the resuscitating laboratory-stored Pichia pastoris GS115 and described with reference to the Invitrogen Pichia expression kit. Positive transformants were verified using DeltaTEF 1-F/Deltaopt (X4) -R and DeltaGAP-F/Deltaopt (X4) -R, respectively.
△TEF1-F：cgtttggatcccttagattagattgctatgct
△GAP-F：tgagattattggaaaccaccag
△opt(X4)-R：gcttctctgtcgttgtctctttc
The PCR amplification system (50. Mu.L) was: 20. Mu.L of double pure water, 25. Mu.L of Taq MIX, 2. Mu.L of upstream primer, 2. Mu.L of downstream primer and 1. Mu.L of template.
The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 8min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 68℃for 30s, for a total of 35 cycles; final extension at 72℃for 5min; preserving at 4 ℃.
The PCR products were identified by 2.0% agarose gel electrophoresis (FIG. 2), and positive transformants (single colonies were picked directly as templates for colony PCR) were sequenced and verified to be correct.
Example 2 optimization and screening of engineering Yeast
(1) Screening of optimal carbon sources
In order to find the optimal carbon source for growth of the engineering yeasts, GS115/pTEF1-opt (X4) and GS115/pGAP-opt (X4) were inoculated into YPD broth respectively, incubated at 30℃and 220rpm overnight, the bacterial cells were washed 2 times in PBS the following day, and OD was adjusted after PBS was resuspended 600 To 0.5. Subsequently, the two strains were each transferred to six different carbon sources (2% concentration) in modified YPD medium, and OD was continuously monitored 600 And finally judging that the optimal carbon sources of the two strains are glucose according to the trend of the growth curve (figure 3).
(2) Response surface optimization fermentation parameters
The invention optimizes the response surface of two strains of GS115/pTEF1-opt (X4) and GS115/pGAP-opt (X4), optimizes the fermentation temperature (15 ℃, 22.5 ℃,30 ℃), pH (4, 6, 8) and glucose concentration (0.5%, 2% and 3.5%), carries out 17 times of combined experiments on each strain to obtain a three-factor interaction 3D graph (figure 4), predicts the optimal condition according to the equation of a computer model, and shows that the GS115/pTEF1-opt (X4) and GS115/pGAP-opt (X4) almost grow at 30 ℃,2.0% glucose concentration and pH6.8, so that the subsequent experiments are carried out under the fermentation condition.
(3) Western blot verification of protein expression levels
According to the above test results, GS115/pTEF1-opt (X4) and GS115/pGAP-opt (X4) were cultured under fermentation at 30℃and 2.0% glucose concentration and pH6.8, respectively, for 4 days, 15000g was centrifuged for 5min, 100. Mu.L of yeast lysate was added to the pellet, and intracellular proteins were extracted for Western blotting by reference (https:// doi.org/10.1038/s 41467-020-19984-3), normalized by GAPDH as a reference protein, and the murine His mab was used to incubate the degrading enzyme Tet (X), and the results showed that the protein expression level of the TEF1 promoter was significantly higher than that of the GAP promoter (FIG. 5). Therefore, GS115/pTEF1-opt (X4) was selected as the application strain by screening.
EXAMPLE 3 degradation of tetracycline by engineered Yeast on samples of different Environment
(1) Phenotype verification of microbial degradation experiment
In order to evaluate whether engineering bacteria GS115/pTEF1-opt (X4) have degradability in actual environment samples, bacterial sediment fermented and cultured under optimal conditions is prepared according to 10 9 The CFU dose was added to 5mL of tap water, lake water and livestock breeding wastewater containing 8. Mu.g/mL of tetracycline, respectively, and the mixture was physically crushed. Each lysis suspension was then incubated in a dark environment at 37℃and 180rpm for 12h, after which time 15000g was centrifuged for 5min. The tetracycline antibiotics are used as blank control, the empty bacteria are used as negative control, and the purified tetracycline degrading enzyme is used as positive control. mu.L of the supernatant of each of the incubations was pipetted onto MH agar plates coated with Bacillus stearothermophilus ATCC7953 and the plates were incubated at 60℃for 5-6h. The degradation condition of the tetracycline is reflected by the diameter of the inhibition zone, and the degradation efficiency of the tetracycline is inversely proportional to the diameter of the inhibition zone. The results show that the GS115/pTEF1-opt (X4) lysate can degrade tetracycline in the 3 environmental matrices described above (FIG. 6), with a wide range of applicability.
(2) Degradation efficiency determination
(1) Extraction of tetracycline drugs
Taking 1mL of the incubation liquid, centrifuging at 15000g of 4 ℃ for 15min, sucking 500 mu L of supernatant, adding into a new 2mL centrifuge tube, then adding 500 mu L of extracting solution, uniformly mixing, adding 125 mu L of chloroform and 125 mu L of n-hexane, uniformly mixing for 10min by vortex, performing ultrasonic treatment for 10min, centrifuging at 15000g of the supernatant at 4 ℃ for 15min, sucking the supernatant, and filtering with a 0.22 mu m filter membrane for later use.
(2) HPLC detection of tetracycline concentration
Detection equipment: an Shimadzu LC-20AT liquid chromatograph,
detection conditions: a (acetonitrile): b (0.01M sodium dihydrogen phosphate) =19:81 as mobile phase, flow rate: 1mL/min, column temperature 30 ℃, sample injection volume 10 μL, detection wavelength 357nm.
According to the standard curve, the tetracycline content in each sample to be tested is quantified, the tetracycline residual rate is = (the tetracycline content of the experimental group/the tetracycline content of the blank group) X100%, and experimental results show that the GS115/pTEF1-opt (X4) lysate can degrade 98.8+/-0.4%, 77.6+/-13.2% and 87.3+/-0.6% of tetracycline in tap water, lake water and livestock breeding sewage respectively (figure 7).
Example 4 engineering Yeast biosafety assessment
(1) Gene copy number monitoring
Lysates of GS115/pTEF1-opt (X4) (high pressure fragmentation) were put into pure water containing tetracycline, 1mL of the mixture after 0h of cleavage and 12h and 24h of degradation was sucked up and centrifuged at 15000g for 5min, the supernatant was taken as a template for RT-qPCR and absolute quantification was performed using primers RTopt (X4) -F/R, and pPIC9K-opt (X4) was used as a standard plasmid for the construction of a standard plasmid.
RTopt(X4)-F：cattcaggctgacatccatca
RTopt(X4)-R：aagtgcaaagcaccgttgttg
The PCR amplification system (20. Mu.L) was: 2X ChamQ SYBR Color qPCR Master Mix. Mu.L, 0.4. Mu.L of the upstream primer, 0.4. Mu.L of the downstream primer, 1. Mu.L of the template, and 8.2. Mu.L of double pure water.
The reaction conditions for PCR amplification were: 95 ℃ for 30s; for a total of 40 cycles at 95℃for 10s and 60℃for 30 s.
The results showed that the copy number of drug resistance gene released from GS115/pTEF1-opt (X4) lysate decreased to undetectable levels at 24h with the extension of tetracycline degradation time (FIG. 8).
(2) Natural transformation experiment
Culturing the above strains in LB broth overnight with standard strains ATCC700603, ATCC14028 and ATCC25922 as recipient strain, sucking 100 μl of bacterial liquid the next day, centrifuging, removing supernatant, re-suspending and washing precipitate with 1mL PBS for 1 time, adding 1mL PBS, and adjusting OD 600 Until 0.1, and pricking on ice for standby. The genome of the engineering yeast GS115/pTEF1-opt (X4) was extracted by pipetting 7. Mu.L and mixed with an equal volume of the above-mentioned bacterial liquid, and the mixture was then dropped onto a modified LB agar plate, incubated at 37℃for 18 hours, and then colonies on the medium were eluted with 1mL of PBS and spread on a plate containing the sameScreening was performed on LB agar with 4 or 8. Mu.g/mL tigecycline. Bacterial LHM10-1 was used as a control. The results indicate that natural transformation is a difficult task, and neither the bacterial nor engineered yeast drug-resistant genes are taken up by the standard strain to produce the corresponding drug-resistant bacteria (FIG. 9).
(3) Bond transfer experiments
pGEN: : the plasmid of bleR (obtained by inserting the ble resistance gene into the sequence of the multiple cloning site of pGEN) was transferred to the standard strains ATCC700603, ATCC14028, ATCC25922 as recipient bacteria, and the following experiments were performed using the engineering yeast GS115/pTEF1-opt (X4) as donor bacteria, and the above bacteria were first cultivated separately, followed by the following recipient bacteria: donor bacteria = 3:1, and the mixture was dropped onto 0.22 μm of LB medium and cultured at 37℃for 18 hours. Then scraping thalli into PBS, uniformly mixing, diluting to 10-6 by a multiple ratio, sucking 100 mu L of each diluted solution, coating onto LB agar containing 4 or 8 mu g/mL tigecycline and 200 mu g/mL bleomycin, screening, and using a primer ZX-F/R for genotype verification of the screened transformant, wherein bacterial LHM10-1 is used as a control.
ZX-F：cgggattgttacaaacttattatg
ZX-R：ttgaaagtacctgtttcttcaac
The PCR amplification system (50. Mu.L) was: 20. Mu.L of double pure water, 25. Mu.L of Taq MIX, 2. Mu.L of upstream primer, 2. Mu.L of downstream primer and 1. Mu.L of template.
The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 8min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 68℃for 30s, for a total of 35 cycles; final extension at 72℃for 5min; preserving at 4 ℃.
The PCR products were identified by 2.0% agarose gel electrophoresis.
The experimental results show that only cultures incubated with LHM10-1 with the standard strain can be grown on double plates and that PCR verifies that both contain the tetracycline resistance gene (FIG. 10). Bacteria were demonstrated to be able to pass on drug resistance genes to standard strains (fig. 9), but live engineered yeasts were unable to pass on drug resistance genes to standard strains due to species segregation, a relatively safer degradation strategy.
EXAMPLE 5 preparation of Tet (X) preparation as a Tetracycline antibiotic-degrading enzyme
(1) Expression of degrading enzyme Tet (X)
The invention selects GS115/pTEF1-opt (X4) which can express tetracycline degrading enzyme Tet (X) in cells and is constructed as described above as an experimental strain. GS115/pTEF1-opt (X4) was recovered from the glycerol-retaining plates on YPD plates at 30℃for 4d cultivation. A single colony was dipped and inoculated into the modified YPD medium. The preparation method of the modified YPD medium comprises weighing 1g of yeast extract, adding 2g of tryptone into 80ml of pure water, autoclaving at 121 ℃ for 20min, cooling to room temperature, and adding 10mL of 2.0% glucose solution and 10mL of 1M potassium phosphate buffer with pH of 6.8 into the above solution. The medium after inoculation was incubated for 4 days at 30℃in a shaker at 220 rpm.
(2) Preparation of degrading enzyme Tet (X) preparation
(1) The fermentation broth was centrifuged at 15000g for 5min at 4℃and the supernatant was discarded.
(2) To the precipitate was added 30mL of pure water, the precipitate was washed by vortexing, centrifuged at 15000g at 4℃for 5min, and the supernatant was discarded.
(3) The operation was repeated (2)1 times.
(4) Resuspending the bacterial precipitate with sterilized water to adjust the bacterial load to 10 9 CFU/mL, split into 2mL centrifuge tubes, 1mL per tube, and then centrifuged at 15000g at 4℃for 5min, and the supernatant discarded.
(5) The pellet was resuspended in 1mL of sterile water and the resuspension was aspirated and added to a penicillin bottle containing 4mL of sterile water and pricked on ice for use.
(6) The ultra-high pressure low temperature breaker was started, the pressure was adjusted to 1222bar, the condensing temperature was set to 4 ℃, and the machine was washed 3 times with 30mL pure water.
(7) And (3) adding the bacterial liquid in the step (5) to a feeding port of a crusher, and collecting the crushed liquid at a collecting position until all the bacterial liquid flows out uniformly. The crushing was repeated 4 times.
(8) The penicillin bottle containing the crushed liquid is quickly transferred to an ultralow temperature (-80 ℃) refrigerator to be frozen for more than 6 hours.
(9) Setting parameters of a freeze dryer, condensing chamber temperature of-64 ℃, vacuum temperature of-75 ℃, vacuum pressure of 0.0012mbar and freeze drying time of 60h.
The rubber plug of the penicillin bottle in the step (8) is opened and is quickly transferred to a freeze dryer after a warm pump, and the instrument is operated according to the parameters in the step (9) in a light-proof state.
EXAMPLE 6 use of Tet (X) preparation as a Tetracycline antibiotic-degrading enzyme
(1) Evaluation of biological Activity of Tet (X) enzyme preparation
The enzyme preparation prepared in example 5 was added to 5mL of culture wastewater containing 8. Mu.g/mL of tetracycline, the sample was placed in a thermostatic shaker at 37℃and 220rpm and incubated for 12h in the absence of light, 1mL of the sample solution was sucked up and placed in a 2mL centrifuge tube after 12h, 15000g was centrifuged at 4℃for 5min, and the supernatant was dropped on MH agar plate coated with Bacillus stearothermophilus ATCC7953 and incubated at 60℃for 5h, which showed that the zone of inhibition from the GS115/pTEF1-opt (X4) expressed enzyme preparation group completely disappeared, demonstrating that the enzyme preparation had bioactivity that could degrade the tetracycline drug in livestock and poultry culture wastewater (FIG. 12).
To further confirm accurate degradation efficiency, the tetracycline content in the liquid was measured using a high performance liquid phase established standard curve.
Residual rate of tetracycline= (tetracycline concentration of sample 12 h/tetracycline concentration of control 12 h) X100%
The results show that the use of the enzyme preparation reduces the tetracycline residue in the livestock and poultry breeding wastewater to 12.7+/-11.9% (figure 13), which proves that the enzyme preparation prepared by the research has high-efficiency tetracycline degradation performance.
(2) Evaluation of storage Properties of Tet (X) enzyme preparation
The enzyme preparation prepared in example 5 was stored at room temperature (25 ℃), at-20℃and-80℃at four different temperatures, and the preparation was taken out on days 0, 2, 4, 7 and 14, respectively, and the degradation properties of the preparation were monitored with reference to the evaluation method of the biological activity of the Tet (X) enzyme preparation in example 6. The results show (FIG. 14), the enzyme preparation is stored at room temperature for 14 days, and the degradation efficiency of tetracycline in livestock and poultry breeding sewage is 86.5+/-2.2%. Likewise, at other temperature conditions, tetracycline degradation efficiencies exceeding 76.0.+ -. 1.7% were observed over all time periods.
(3) Evaluation of environmental resistance of Tet (X) enzyme preparation
(1) Influence of pH on enzyme preparation
Hydrochloric acid and sodium hydroxide are used for adjusting the pH value of the livestock and poultry raising sewage to 5, 8 and 11, and then tetracycline is artificially added into the sewage to a final concentration of 8 mug/mL. The enzyme preparation prepared in example 5 was put into 5mL of the above prepared solution, incubated in a constant temperature shaker at 37℃and 220rpm for 12 hours in the absence of light, 1mL of the sample solution was sucked after 12 hours and placed in a 2mL centrifuge tube, and centrifuged at 15000g and 4℃for 5 minutes, and the supernatant was taken. The tetracycline content in the supernatant was measured using a high performance liquid phase established standard curve.
Degradation rate of tetracycline = 1- (tetracycline concentration for sample 12 h/tetracycline concentration for control 12 h) X100%
(2) Influence of ambient temperature on enzyme preparation
The enzyme preparation prepared in example 5 is respectively put into 5mL of livestock and poultry raising sewage containing 8 mu g/mL of tetracycline, the solutions are respectively placed into three different temperature shaking tables of 17 ℃,37 ℃ and 47 ℃ for 220rp light-shielding incubation for 12 hours, 1mL of sample solution is sucked after 12 hours, the sample solution is placed into a 2mL centrifuge tube, 15000g is centrifuged for 5 minutes at 4 ℃, and the supernatant is taken. The tetracycline content in the supernatant was measured using a high performance liquid phase established standard curve.
Degradation rate of tetracycline = 1- (tetracycline concentration for sample 12 h/tetracycline concentration for control 12 h) X100%
The results of the high performance liquid phase showed that the enzyme preparation showed an efficiency of tetracycline degradation exceeding 88.6.+ -. 3.6% in low temperature (17 ℃), high temperature (47 ℃), acidic (pH 5) or basic (pH 11) environments, and excellent resistance to pH and temperature of the environment (FIG. 15).
Compared with the literature, the pure enzyme is directly added into sewage for degradation, and when aiming at the sewage environment with relatively complex physicochemical properties, the stability of the enzyme is greatly disturbed, so that the degradation effect cannot be fully exerted. The instability of enzymes makes this approach difficult to apply in environments where the physicochemical properties are relatively complex in practice. The enzyme preparation has better effect in sewage, especially livestock and poultry breeding sewage, and has better degradation performance due to the cracking of yeast in the preparation process, besides exogenously expressed enzyme, other biological matrixes from the yeast, such as polysaccharide such as glucan and the like, are doped in the preparation, and the composite material based on the biological matrixes has better degradation performance.
It should be understood that the foregoing description of the specific embodiments is merely illustrative of the invention, and is not intended to limit the invention, and that any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (8)
1. A construction method of a genetically engineered plasmid for intracellular expression of tetracycline degrading enzyme Tet (X) is characterized in that the promoter of pPIC9K-opt (X4) plasmid is replaced by GAP or TEF1 promoter, and the sequence for expressing secretion signal peptide in the plasmid is deleted.
2. A genetically engineered yeast for intracellular expression of tetracycline degrading enzyme Tet (X), characterized in that the plasmid of the genetically engineered yeast is constructed by the method of claim 1, and the expression host is Pichia pastoris GS115.
3. The use of genetically engineered yeasts and/or biological agents prepared from genetically engineered yeasts as defined in claim 2 for degrading tetracycline antibiotic residues.
4. The method according to claim 3, wherein the genetically engineered yeast is cultured in an expanded state, the cells are collected, and the cells are lysed and then the sample containing the tetracycline antibiotic is introduced.
5. The use according to claim 4, wherein the sample is livestock breeding wastewater.
6. A tetracycline antibiotic degrading enzyme Tet (X) preparation, which is characterized by being prepared from the genetically engineered saccharomycete according to claim 2.
7. The process for preparing tetracycline antibiotic degrading enzyme Tet (X) preparation of claim 6, wherein said genetically engineered saccharomycetes are expanded and cultured, and the thallus is collected, and then the thallus is cracked, broken and freeze-dried.
8. The method according to claim 7, wherein the YPD medium used in the expansion culture is prepared by adding 1g of yeast extract and 2g of tryptone to 80ml of pure water, autoclaving at 121℃for 20min, cooling to room temperature, adding 10ml of 2.0% glucose solution and 10ml of 1M potassium phosphate buffer at pH6.8, and expanding culture conditions: the culture was continued for 4d at 30℃in a 220rpm shaker.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310255638.9A CN116334115A (en) | 2023-03-16 | 2023-03-16 | Genetically engineered saccharomycete for intracellular expression of tetracycline degrading enzyme Tet (X) and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310255638.9A CN116334115A (en) | 2023-03-16 | 2023-03-16 | Genetically engineered saccharomycete for intracellular expression of tetracycline degrading enzyme Tet (X) and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116334115A true CN116334115A (en) | 2023-06-27 |
Family
ID=86892392
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310255638.9A Pending CN116334115A (en) | 2023-03-16 | 2023-03-16 | Genetically engineered saccharomycete for intracellular expression of tetracycline degrading enzyme Tet (X) and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116334115A (en) |
-
2023
- 2023-03-16 CN CN202310255638.9A patent/CN116334115A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10577615B2 (en) | Genetically engineered Candida utilis capable of degrading and utilizing kitchen waste and construction method therefor | |
| CN1225125A (en) | Stable recombinant yeasts for fermenting xylose to ethanol | |
| Yang et al. | Two-stage fermentation enhanced single-cell protein production by Yarrowia lipolytica from food waste | |
| US20170226539A1 (en) | Genetically recombinant saccharomyces cerevisiae for degrading kitchen waste | |
| JP7181542B2 (en) | A novel strain of Pseudozyma antactica | |
| CN106957805B (en) | Bacillus GBacilus-9 strain with bacteriostatic effect and separation method and application thereof | |
| CN101418276B (en) | Host cell and method for efficient expression and secretion thereof in recombinant protein | |
| CN107287229A (en) | A kind of method of utilization bacillus efficient secretory expression foreign protein | |
| CN116334115A (en) | Genetically engineered saccharomycete for intracellular expression of tetracycline degrading enzyme Tet (X) and application thereof | |
| CN114891806B (en) | Citrobacter welchii yqjH gene knockout mutant strain and application thereof | |
| CN108004154B (en) | Sporobolomyces yunnanensis 17wy1, microbial preparation thereof and application thereof in wheat powdery mildew prevention and treatment | |
| CN114480436B (en) | Method, strain and application for improving insecticidal toxicity of destruxins of Metarrhizium anisopliae | |
| AU2020100601A4 (en) | Recombinant broad-spectrum Metarhizium and production method and application thereof | |
| RU2658977C1 (en) | Method for producing protein fodder additive | |
| CN109554321B (en) | Genetically engineered bacterium for high-yield lipopeptide and application thereof | |
| Wang et al. | High-level secretory production of lysostaphin in Escherichia coli mutant by codon optimization and atmospheric and room temperature plasma mutagenesis | |
| Ladygin | Efficient transformation of mutant cells of Chlamydomonas reinhardtii by electroporation | |
| CN115011497A (en) | Pediococcus acidilactici and application thereof | |
| CN113122461A (en) | Single cell protein producing strain and its application | |
| CN109897863A (en) | A kind of method for transformation of Lactobacillus brevis | |
| CN101215525B (en) | Method for separating microorganism capable of producing ferulic acid esterase | |
| CN117887592B (en) | Aspergillus oryzae JAAS-32 and application thereof in preparation of hermetia illucens plasma protein peptide | |
| CN116240123B (en) | Paenibacillus polymyxa, microecological preparation and preparation method thereof | |
| CN115851630B (en) | Tetracycline antibiotic degrading enzyme, coding gene and application | |
| CN117904009B (en) | Bacillus subtilis applicable to non-grain bio-based carbon source and fermentation production method thereof |
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 |