CN114958877B - 去乙酰氧基头孢菌素c合成酶突变体及其编码基因与应用 - Google Patents
去乙酰氧基头孢菌素c合成酶突变体及其编码基因与应用 Download PDFInfo
- Publication number
- CN114958877B CN114958877B CN202210671678.7A CN202210671678A CN114958877B CN 114958877 B CN114958877 B CN 114958877B CN 202210671678 A CN202210671678 A CN 202210671678A CN 114958877 B CN114958877 B CN 114958877B
- Authority
- CN
- China
- Prior art keywords
- mutation
- mutant
- ala
- thr
- cephalosporin
- 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.)
- Active
Links
- HOKIDJSKDBPKTQ-GLXFQSAKSA-N Cephalosporin C Natural products S1CC(COC(=O)C)=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CCC[C@@H](N)C(O)=O)[C@@H]12 HOKIDJSKDBPKTQ-GLXFQSAKSA-N 0.000 title claims abstract description 67
- 108090000364 Ligases Proteins 0.000 title claims abstract description 38
- 102000003960 Ligases Human genes 0.000 title claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 31
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 43
- 230000035772 mutation Effects 0.000 claims abstract description 27
- 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 claims abstract description 25
- 239000004471 Glycine Substances 0.000 claims abstract description 24
- 108010005802 desacetoxycephalosporin C synthetase Proteins 0.000 claims abstract description 20
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000004473 Threonine Substances 0.000 claims abstract description 18
- 229940056360 penicillin g Drugs 0.000 claims abstract description 12
- 108020004705 Codon Proteins 0.000 claims description 48
- 241000228431 Acremonium chrysogenum Species 0.000 claims description 13
- 239000013604 expression vector Substances 0.000 claims description 11
- 229930182817 methionine Natural products 0.000 claims description 8
- 230000014509 gene expression Effects 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 7
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 6
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 6
- 235000009582 asparagine Nutrition 0.000 claims description 6
- 229960001230 asparagine Drugs 0.000 claims description 6
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 6
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 5
- 239000012620 biological material Substances 0.000 claims description 5
- 235000018417 cysteine Nutrition 0.000 claims description 5
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 5
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 4
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 3
- 229940124587 cephalosporin Drugs 0.000 claims description 3
- 108020004707 nucleic acids Proteins 0.000 claims 3
- 150000007523 nucleic acids Chemical class 0.000 claims 3
- 102000039446 nucleic acids Human genes 0.000 claims 3
- 150000001413 amino acids Chemical group 0.000 abstract description 33
- 241000187433 Streptomyces clavuligerus Species 0.000 abstract description 27
- 102000004190 Enzymes Human genes 0.000 abstract description 23
- 108090000790 Enzymes Proteins 0.000 abstract description 23
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 abstract description 6
- 230000003197 catalytic effect Effects 0.000 abstract description 4
- 238000002741 site-directed mutagenesis Methods 0.000 abstract description 3
- 230000009466 transformation Effects 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 abstract description 2
- LZAJKCZTKKKZNT-PMNGPLLRSA-N trichothecene Chemical compound C12([C@@]3(CC[C@H]2OC2C=C(CCC23C)C)C)CO1 LZAJKCZTKKKZNT-PMNGPLLRSA-N 0.000 abstract 2
- 229930013292 trichothecene Natural products 0.000 abstract 2
- 239000013612 plasmid Substances 0.000 description 34
- 229940024606 amino acid Drugs 0.000 description 21
- 235000001014 amino acid Nutrition 0.000 description 21
- 238000000034 method Methods 0.000 description 21
- 229940088598 enzyme Drugs 0.000 description 20
- 230000000694 effects Effects 0.000 description 19
- 229960002449 glycine Drugs 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 18
- 230000008569 process Effects 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 235000008521 threonine Nutrition 0.000 description 15
- 108091008146 restriction endonucleases Proteins 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 229920001817 Agar Polymers 0.000 description 11
- 241001619326 Cephalosporium Species 0.000 description 11
- 239000008272 agar Substances 0.000 description 11
- 239000000499 gel Substances 0.000 description 11
- 239000000047 product Substances 0.000 description 10
- 210000001938 protoplast Anatomy 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 238000001962 electrophoresis Methods 0.000 description 9
- 238000000855 fermentation Methods 0.000 description 9
- 230000004151 fermentation Effects 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 239000002773 nucleotide Substances 0.000 description 9
- 125000003729 nucleotide group Chemical group 0.000 description 9
- 101710104123 Deacetoxycephalosporin C synthase Proteins 0.000 description 8
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 8
- VHIZXDZMTDVFGX-DCAQKATOSA-N Val-Ser-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N VHIZXDZMTDVFGX-DCAQKATOSA-N 0.000 description 8
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 8
- 108010000761 leucylarginine Proteins 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 235000006109 methionine Nutrition 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- 239000013600 plasmid vector Substances 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 5
- MIFYHUACUWQUKT-UHFFFAOYSA-N Isopenicillin N Natural products OC(=O)C1C(C)(C)SC2C(NC(=O)CCCC(N)C(O)=O)C(=O)N21 MIFYHUACUWQUKT-UHFFFAOYSA-N 0.000 description 5
- 229950008644 adicillin Drugs 0.000 description 5
- 235000004554 glutamine Nutrition 0.000 description 5
- 229960002743 glutamine Drugs 0.000 description 5
- 229930027917 kanamycin Natural products 0.000 description 5
- 229960000318 kanamycin Drugs 0.000 description 5
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 5
- 229930182823 kanamycin A Natural products 0.000 description 5
- 229960004452 methionine Drugs 0.000 description 5
- 238000005457 optimization Methods 0.000 description 5
- -1 oxo cephalosporin C Chemical compound 0.000 description 5
- MIFYHUACUWQUKT-GPUHXXMPSA-N penicillin N Chemical compound OC(=O)[C@H]1C(C)(C)S[C@@H]2[C@H](NC(=O)CCC[C@@H](N)C(O)=O)C(=O)N21 MIFYHUACUWQUKT-GPUHXXMPSA-N 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 4
- GSCLWXDNIMNIJE-ZLUOBGJFSA-N Ala-Asp-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O GSCLWXDNIMNIJE-ZLUOBGJFSA-N 0.000 description 4
- MKZCBYZBCINNJN-DLOVCJGASA-N Ala-Asp-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MKZCBYZBCINNJN-DLOVCJGASA-N 0.000 description 4
- WKOBSJOZRJJVRZ-FXQIFTODSA-N Ala-Glu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WKOBSJOZRJJVRZ-FXQIFTODSA-N 0.000 description 4
- PNALXAODQKTNLV-JBDRJPRFSA-N Ala-Ile-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O PNALXAODQKTNLV-JBDRJPRFSA-N 0.000 description 4
- ZBLQIYPCUWZSRZ-QEJZJMRPSA-N Ala-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CC=CC=C1 ZBLQIYPCUWZSRZ-QEJZJMRPSA-N 0.000 description 4
- VJVQKGYHIZPSNS-FXQIFTODSA-N Ala-Ser-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N VJVQKGYHIZPSNS-FXQIFTODSA-N 0.000 description 4
- JJHBEVZAZXZREW-LFSVMHDDSA-N Ala-Thr-Phe Chemical compound C[C@@H](O)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](Cc1ccccc1)C(O)=O JJHBEVZAZXZREW-LFSVMHDDSA-N 0.000 description 4
- OMSKGWFGWCQFBD-KZVJFYERSA-N Ala-Val-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OMSKGWFGWCQFBD-KZVJFYERSA-N 0.000 description 4
- OTOXOKCIIQLMFH-KZVJFYERSA-N Arg-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N OTOXOKCIIQLMFH-KZVJFYERSA-N 0.000 description 4
- IASNWHAGGYTEKX-IUCAKERBSA-N Arg-Arg-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(O)=O IASNWHAGGYTEKX-IUCAKERBSA-N 0.000 description 4
- MTANSHNQTWPZKP-KKUMJFAQSA-N Arg-Gln-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCN=C(N)N)N)O MTANSHNQTWPZKP-KKUMJFAQSA-N 0.000 description 4
- NYZGVTGOMPHSJW-CIUDSAMLSA-N Arg-Glu-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N NYZGVTGOMPHSJW-CIUDSAMLSA-N 0.000 description 4
- HNJNAMGZQZPSRE-GUBZILKMSA-N Arg-Pro-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O HNJNAMGZQZPSRE-GUBZILKMSA-N 0.000 description 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- JQSWHKKUZMTOIH-QWRGUYRKSA-N Asn-Gly-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N JQSWHKKUZMTOIH-QWRGUYRKSA-N 0.000 description 4
- DPSUVAPLRQDWAO-YDHLFZDLSA-N Asn-Tyr-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC(=O)N)N DPSUVAPLRQDWAO-YDHLFZDLSA-N 0.000 description 4
- UWOPETAWXDZUJR-ACZMJKKPSA-N Asp-Cys-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(O)=O UWOPETAWXDZUJR-ACZMJKKPSA-N 0.000 description 4
- QQXOYLWJQUPXJU-WHFBIAKZSA-N Asp-Cys-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)NCC(O)=O QQXOYLWJQUPXJU-WHFBIAKZSA-N 0.000 description 4
- UCHSVZYJKJLPHF-BZSNNMDCSA-N Asp-Phe-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O UCHSVZYJKJLPHF-BZSNNMDCSA-N 0.000 description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- VRJZMZGGAKVSIQ-SRVKXCTJSA-N Cys-Tyr-Ser Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O VRJZMZGGAKVSIQ-SRVKXCTJSA-N 0.000 description 4
- RZSLYUUFFVHFRQ-FXQIFTODSA-N Gln-Ala-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O RZSLYUUFFVHFRQ-FXQIFTODSA-N 0.000 description 4
- DHNWZLGBTPUTQQ-QEJZJMRPSA-N Gln-Asp-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)N)N DHNWZLGBTPUTQQ-QEJZJMRPSA-N 0.000 description 4
- NPTGGVQJYRSMCM-GLLZPBPUSA-N Gln-Gln-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NPTGGVQJYRSMCM-GLLZPBPUSA-N 0.000 description 4
- ZNTDJIMJKNNSLR-RWRJDSDZSA-N Gln-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N ZNTDJIMJKNNSLR-RWRJDSDZSA-N 0.000 description 4
- MKRDNSWGJWTBKZ-GVXVVHGQSA-N Gln-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N MKRDNSWGJWTBKZ-GVXVVHGQSA-N 0.000 description 4
- VTTSANCGJWLPNC-ZPFDUUQYSA-N Glu-Arg-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VTTSANCGJWLPNC-ZPFDUUQYSA-N 0.000 description 4
- XIKYNVKEUINBGL-IUCAKERBSA-N Glu-His-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)NCC(O)=O XIKYNVKEUINBGL-IUCAKERBSA-N 0.000 description 4
- SJJHXJDSNQJMMW-SRVKXCTJSA-N Glu-Lys-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O SJJHXJDSNQJMMW-SRVKXCTJSA-N 0.000 description 4
- GMVCSRBOSIUTFC-FXQIFTODSA-N Glu-Ser-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O GMVCSRBOSIUTFC-FXQIFTODSA-N 0.000 description 4
- VIPDPMHGICREIS-GVXVVHGQSA-N Glu-Val-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O VIPDPMHGICREIS-GVXVVHGQSA-N 0.000 description 4
- RPLLQZBOVIVGMX-QWRGUYRKSA-N Gly-Asp-Phe Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RPLLQZBOVIVGMX-QWRGUYRKSA-N 0.000 description 4
- MBOAPAXLTUSMQI-JHEQGTHGSA-N Gly-Glu-Thr Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MBOAPAXLTUSMQI-JHEQGTHGSA-N 0.000 description 4
- GAFKBWKVXNERFA-QWRGUYRKSA-N Gly-Phe-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 GAFKBWKVXNERFA-QWRGUYRKSA-N 0.000 description 4
- DBUNZBWUWCIELX-JHEQGTHGSA-N Gly-Thr-Glu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DBUNZBWUWCIELX-JHEQGTHGSA-N 0.000 description 4
- SYOJVRNQCXYEOV-XVKPBYJWSA-N Gly-Val-Glu Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SYOJVRNQCXYEOV-XVKPBYJWSA-N 0.000 description 4
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 4
- ZPVJJPAIUZLSNE-DCAQKATOSA-N His-Arg-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O ZPVJJPAIUZLSNE-DCAQKATOSA-N 0.000 description 4
- CDGLBYSAZFIIJO-RCOVLWMOSA-N Ile-Gly-Gly Chemical compound CC[C@H](C)[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O CDGLBYSAZFIIJO-RCOVLWMOSA-N 0.000 description 4
- IBMVEYRWAWIOTN-UHFFFAOYSA-N L-Leucyl-L-Arginyl-L-Proline Natural products CC(C)CC(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O IBMVEYRWAWIOTN-UHFFFAOYSA-N 0.000 description 4
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 4
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 4
- KAFOIVJDVSZUMD-DCAQKATOSA-N Leu-Gln-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-DCAQKATOSA-N 0.000 description 4
- UHNQRAFSEBGZFZ-YESZJQIVSA-N Leu-Phe-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N2CCC[C@@H]2C(=O)O)N UHNQRAFSEBGZFZ-YESZJQIVSA-N 0.000 description 4
- ICYRCNICGBJLGM-HJGDQZAQSA-N Leu-Thr-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(O)=O ICYRCNICGBJLGM-HJGDQZAQSA-N 0.000 description 4
- FMFNIDICDKEMOE-XUXIUFHCSA-N Leu-Val-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FMFNIDICDKEMOE-XUXIUFHCSA-N 0.000 description 4
- HKXSZKJMDBHOTG-CIUDSAMLSA-N Lys-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CCCCN HKXSZKJMDBHOTG-CIUDSAMLSA-N 0.000 description 4
- MCNGIXXCMJAURZ-VEVYYDQMSA-N Met-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCSC)N)O MCNGIXXCMJAURZ-VEVYYDQMSA-N 0.000 description 4
- SXWQMBGNFXAGAT-FJXKBIBVSA-N Met-Gly-Thr Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O SXWQMBGNFXAGAT-FJXKBIBVSA-N 0.000 description 4
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 4
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 4
- IWRZUGHCHFZYQZ-UFYCRDLUSA-N Phe-Arg-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 IWRZUGHCHFZYQZ-UFYCRDLUSA-N 0.000 description 4
- ALHULIGNEXGFRM-QWRGUYRKSA-N Phe-Cys-Gly Chemical compound OC(=O)CNC(=O)[C@H](CS)NC(=O)[C@@H](N)CC1=CC=CC=C1 ALHULIGNEXGFRM-QWRGUYRKSA-N 0.000 description 4
- AXIOGMQCDYVTNY-ACRUOGEOSA-N Phe-Phe-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 AXIOGMQCDYVTNY-ACRUOGEOSA-N 0.000 description 4
- CZQZSMJXFGGBHM-KKUMJFAQSA-N Phe-Pro-Gln Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O CZQZSMJXFGGBHM-KKUMJFAQSA-N 0.000 description 4
- MVIJMIZJPHQGEN-IHRRRGAJSA-N Phe-Ser-Val Chemical compound CC(C)[C@@H](C([O-])=O)NC(=O)[C@H](CO)NC(=O)[C@@H]([NH3+])CC1=CC=CC=C1 MVIJMIZJPHQGEN-IHRRRGAJSA-N 0.000 description 4
- BSKMOCNNLNDIMU-CDMKHQONSA-N Phe-Thr-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O BSKMOCNNLNDIMU-CDMKHQONSA-N 0.000 description 4
- SGCZFWSQERRKBD-BQBZGAKWSA-N Pro-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]1CCCN1 SGCZFWSQERRKBD-BQBZGAKWSA-N 0.000 description 4
- FKKHDBFNOLCYQM-FXQIFTODSA-N Pro-Cys-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O FKKHDBFNOLCYQM-FXQIFTODSA-N 0.000 description 4
- FMLRRBDLBJLJIK-DCAQKATOSA-N Pro-Leu-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 FMLRRBDLBJLJIK-DCAQKATOSA-N 0.000 description 4
- XYSXOCIWCPFOCG-IHRRRGAJSA-N Pro-Leu-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XYSXOCIWCPFOCG-IHRRRGAJSA-N 0.000 description 4
- OYEDZGNMSBZCIM-XGEHTFHBSA-N Ser-Arg-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OYEDZGNMSBZCIM-XGEHTFHBSA-N 0.000 description 4
- SMIDBHKWSYUBRZ-ACZMJKKPSA-N Ser-Glu-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O SMIDBHKWSYUBRZ-ACZMJKKPSA-N 0.000 description 4
- CKDXFSPMIDSMGV-GUBZILKMSA-N Ser-Pro-Val Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O CKDXFSPMIDSMGV-GUBZILKMSA-N 0.000 description 4
- VGQVAVQWKJLIRM-FXQIFTODSA-N Ser-Ser-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O VGQVAVQWKJLIRM-FXQIFTODSA-N 0.000 description 4
- XJDMUQCLVSCRSJ-VZFHVOOUSA-N Ser-Thr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O XJDMUQCLVSCRSJ-VZFHVOOUSA-N 0.000 description 4
- VVKVHAOOUGNDPJ-SRVKXCTJSA-N Ser-Tyr-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O VVKVHAOOUGNDPJ-SRVKXCTJSA-N 0.000 description 4
- LHEZGZQRLDBSRR-WDCWCFNPSA-N Thr-Glu-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O LHEZGZQRLDBSRR-WDCWCFNPSA-N 0.000 description 4
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 4
- KZSYAEWQMJEGRZ-RHYQMDGZSA-N Thr-Leu-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O KZSYAEWQMJEGRZ-RHYQMDGZSA-N 0.000 description 4
- ABWNZPOIUJMNKT-IXOXFDKPSA-N Thr-Phe-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O ABWNZPOIUJMNKT-IXOXFDKPSA-N 0.000 description 4
- QNXZCKMXHPULME-ZNSHCXBVSA-N Thr-Val-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N)O QNXZCKMXHPULME-ZNSHCXBVSA-N 0.000 description 4
- JTMZSIRTZKLBOA-NWLDYVSISA-N Trp-Thr-Gln Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O JTMZSIRTZKLBOA-NWLDYVSISA-N 0.000 description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 4
- DMWNPLOERDAHSY-MEYUZBJRSA-N Tyr-Leu-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DMWNPLOERDAHSY-MEYUZBJRSA-N 0.000 description 4
- LMKKMCGTDANZTR-BZSNNMDCSA-N Tyr-Phe-Asp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(O)=O)C(O)=O)C1=CC=C(O)C=C1 LMKKMCGTDANZTR-BZSNNMDCSA-N 0.000 description 4
- XYBNMHRFAUKPAW-IHRRRGAJSA-N Tyr-Ser-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC1=CC=C(C=C1)O)N XYBNMHRFAUKPAW-IHRRRGAJSA-N 0.000 description 4
- DDRBQONWVBDQOY-GUBZILKMSA-N Val-Ala-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O DDRBQONWVBDQOY-GUBZILKMSA-N 0.000 description 4
- RYQUMYBMOJYYDK-NHCYSSNCSA-N Val-Pro-Glu Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)O)N RYQUMYBMOJYYDK-NHCYSSNCSA-N 0.000 description 4
- 108010044940 alanylglutamine Proteins 0.000 description 4
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 4
- 108010013835 arginine glutamate Proteins 0.000 description 4
- 239000002131 composite material Substances 0.000 description 4
- 229960002433 cysteine Drugs 0.000 description 4
- 108010016616 cysteinylglycine Proteins 0.000 description 4
- 108010093176 deacetoxycephalosporin C synthetase Proteins 0.000 description 4
- 108010054813 diprotin B Proteins 0.000 description 4
- 238000003208 gene overexpression Methods 0.000 description 4
- 108010078144 glutaminyl-glycine Proteins 0.000 description 4
- 108010049041 glutamylalanine Proteins 0.000 description 4
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 4
- 108010010147 glycylglutamine Proteins 0.000 description 4
- 108010081551 glycylphenylalanine Proteins 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 235000014304 histidine Nutrition 0.000 description 4
- 229960002885 histidine Drugs 0.000 description 4
- 108010034529 leucyl-lysine Proteins 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 108010012581 phenylalanylglutamate Proteins 0.000 description 4
- 108010031719 prolyl-serine Proteins 0.000 description 4
- 102200087311 rs74315345 Human genes 0.000 description 4
- 108010071207 serylmethionine Proteins 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 108010061238 threonyl-glycine Proteins 0.000 description 4
- 229960004799 tryptophan Drugs 0.000 description 4
- 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 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108700010070 Codon Usage Proteins 0.000 description 3
- 108030001962 Deacetoxycephalosporin-C synthases Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 3
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- 229960005261 aspartic acid Drugs 0.000 description 3
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 210000004671 cell-free system Anatomy 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 3
- 235000014705 isoleucine Nutrition 0.000 description 3
- 229960000310 isoleucine Drugs 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 102220156356 rs886046630 Human genes 0.000 description 3
- 235000004400 serine Nutrition 0.000 description 3
- 229960001153 serine Drugs 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 239000002132 β-lactam antibiotic Substances 0.000 description 3
- 229940124586 β-lactam antibiotics Drugs 0.000 description 3
- FMNBYVSGRCXWEK-FOHZUACHSA-N Asn-Thr-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O FMNBYVSGRCXWEK-FOHZUACHSA-N 0.000 description 2
- 239000004470 DL Methionine Substances 0.000 description 2
- 229920002430 Fibre-reinforced plastic Polymers 0.000 description 2
- 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 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- 239000002211 L-ascorbic acid Substances 0.000 description 2
- 235000000069 L-ascorbic acid Nutrition 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- GXWRTSIVLSQACD-RCWTZXSCSA-N Pro-Thr-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@@H]1CCCN1)O GXWRTSIVLSQACD-RCWTZXSCSA-N 0.000 description 2
- 241000933812 Streptomyces flaveus Species 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 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 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 2
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- IYNDLOXRXUOGIU-LQDWTQKMSA-M benzylpenicillin potassium Chemical compound [K+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 IYNDLOXRXUOGIU-LQDWTQKMSA-M 0.000 description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- NNQIJOYQWYKBOW-UHFFFAOYSA-N desacetoxycephalosphorin G Natural products S1CC(C)=C(C(O)=O)N2C(=O)C(NC(=O)CCCC(N)C(O)=O)C12 NNQIJOYQWYKBOW-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000011151 fibre-reinforced plastic Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 235000005772 leucine Nutrition 0.000 description 2
- 229960003136 leucine Drugs 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 235000018977 lysine Nutrition 0.000 description 2
- 229960003646 lysine Drugs 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- FFEARJCKVFRZRR-UHFFFAOYSA-N methionine Chemical compound CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000006225 natural substrate Substances 0.000 description 2
- 235000008729 phenylalanine Nutrition 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000006049 ring expansion reaction Methods 0.000 description 2
- 102200031779 rs3204853 Human genes 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 235000012424 soybean oil Nutrition 0.000 description 2
- 239000003549 soybean oil Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- KPGXRSRHYNQIFN-UHFFFAOYSA-L 2-oxoglutarate(2-) Chemical compound [O-]C(=O)CCC(=O)C([O-])=O KPGXRSRHYNQIFN-UHFFFAOYSA-L 0.000 description 1
- AGIJRRREJXSQJR-UHFFFAOYSA-N 2h-thiazine Chemical group N1SC=CC=C1 AGIJRRREJXSQJR-UHFFFAOYSA-N 0.000 description 1
- 241001328642 Acremonium chrysogenum ATCC 11550 Species 0.000 description 1
- 101001063937 Acremonium chrysogenum Deacetoxycephalosporin C hydroxylase Proteins 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- 241000123346 Chrysosporium Species 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 102220645180 Protocadherin gamma-A4_G92Q_mutation Human genes 0.000 description 1
- 241000931888 Pyxis Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 101100370749 Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) trpC1 gene Proteins 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical group C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- OXSWKJLAKXNIFG-UHFFFAOYSA-N azane sulfuric acid Chemical compound N.N.N.OS(O)(=O)=O OXSWKJLAKXNIFG-UHFFFAOYSA-N 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 108010083912 bleomycin N-acetyltransferase Proteins 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229960004841 cefadroxil Drugs 0.000 description 1
- NBFNMSULHIODTC-CYJZLJNKSA-N cefadroxil monohydrate Chemical compound O.C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CC=C(O)C=C1 NBFNMSULHIODTC-CYJZLJNKSA-N 0.000 description 1
- 229960002588 cefradine Drugs 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- ZAIPMKNFIOOWCQ-UEKVPHQBSA-N cephalexin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CC=CC=C1 ZAIPMKNFIOOWCQ-UEKVPHQBSA-N 0.000 description 1
- 229940106164 cephalexin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- RDLPVSKMFDYCOR-UEKVPHQBSA-N cephradine Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CCC=CC1 RDLPVSKMFDYCOR-UEKVPHQBSA-N 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 108010031946 deacetoxycephalosporin C hydroxylase Proteins 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- XQGPKZUNMMFTAL-UHFFFAOYSA-L dipotassium;hydrogen phosphate;trihydrate Chemical compound O.O.O.[K+].[K+].OP([O-])([O-])=O XQGPKZUNMMFTAL-UHFFFAOYSA-L 0.000 description 1
- 238000004090 dissolution Methods 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
- 229940079593 drug Drugs 0.000 description 1
- 230000009144 enzymatic modification Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 101150073906 gpdA gene Proteins 0.000 description 1
- 101150095733 gpsA gene Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000010030 laminating Methods 0.000 description 1
- 239000012939 laminating adhesive Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229940124588 oral cephalosporin Drugs 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000013215 result calculation Methods 0.000 description 1
- 102220049425 rs587784285 Human genes 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 description 1
- 235000010378 sodium ascorbate Nutrition 0.000 description 1
- 229960005055 sodium ascorbate Drugs 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 101150016309 trpC gene Proteins 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 150000003952 β-lactams Chemical class 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
- C12P35/06—Cephalosporin C; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
- C12Y114/11026—Deacetoxycephalosporin-C hydroxylase (1.14.11.26)
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了去乙酰氧基头孢菌素C合成酶突变体及其编码基因与应用，属于酶工程技术领域。本发明去乙酰氧基头孢菌素C合成酶突变体氨基酸序列以SEQ ID NO.1为参考，具有位于第72位苏氨酸T72、第92位甘氨酸G92中的至少一个突变；第72位苏氨酸突变为除其自身以外任意一种天然氨基酸，第92位甘氨酸突变为除其自身以外任意一种天然氨基酸。本发明通过对棒状链霉菌的去乙酰氧基头孢菌素C合成酶进行定点突变和基因工程改造，提高了该酶对青霉素G的催化活性，并且可提高产黄支顶孢霉头孢菌素C的产量，适于商业和工业应用。
Description
技术领域
本发明属于酶工程技术领域，具体涉及去乙酰氧基头孢菌素C合成酶突变体及其编码基因与应用。
背景技术
去乙酰氧基头孢菌素C合成酶(Deacetoxycephalosporin C synthase，DAOCS)是β-内酰胺类抗生素生物合成过程中的关键酶，其催化五元噻唑环扩环生成六元噻嗪环，从而得到相应的β-内酰胺类代谢产物。
头孢菌素C(CPC)是丝状真菌产黄支顶孢霉产生的一种重要的天然β-内酰胺类抗生素。在产黄支顶孢霉基因组上存在编码双功能酶——去乙酰氧基头孢菌素C合成酶(扩环酶)/去乙酰基头孢菌素C合成酶(羟化酶)(Deacetoxycephalosporin/deacetylcephalosporin C synthase，DAOC/DACS)的cefEF基因，是产黄支顶孢霉合成CPC的限速酶，催化青霉素N(penicillin N，PeN)发生扩环反应生成去乙氧基头孢菌素C(DAOC)，然后催化DAOC发生羟基化反应生成去乙酰基头孢菌素C(DACS)。CPC和Pen N是产黄支顶孢霉所分泌的主要天然产物，在CPC发酵生产过程中，产黄支顶孢霉还会同时分泌大量的Pen N；这两种化合物的分泌取决于他们在细胞水平上的生物合成；因此，提升产黄支顶孢霉扩环酶酶活可能有利于副产物Pen N向产物CPC的转化。
另外，7-氨基-3-去乙酰氧基头孢烷酸(7-ADCA)是生产口服头孢类药物的重要中间体原料，主要用于合成头孢氨苄、头孢拉定、头孢羟氨苄等抗生素。以青霉素G钾盐为原料化学合成7-ADCA是目前在工业生产中普遍采用的方法，然而其合成过程中需要多步化学扩环，反应条件要求高、各步反应产物收率低、生产结束化学污染较重，因此，人们试图用无污染且更经济的酶转化方法来实现头孢菌素母核的合成。青霉素G产量大、价格低，相对有效的方式是利用去乙酰氧基头孢菌素C合成酶，即青霉素扩环酶使青霉素G扩环得到G-7-ADCA(7-苯乙酰基脱乙酰氧基头孢菌素C)，再去除其7位侧链得到7-ADCA。然而，野生型DAOCS的天然底物是目前尚无法大规模工业合成的青霉素N(penicillin N，PeN)；该酶对于大量存在的非天然底物青霉素G的反应活性很低，因此实际转化率也很低，限制了去乙酰氧基头孢菌素C合成酶在工业生产上的应用。所以，对DAOCS进行酶学改造，增强其对大量廉价青霉素G的催化活性，增强其反应稳定性，成为将其引入工业化生产必须解决的首要问题。
发明内容
本发明公开了去乙酰氧基头孢菌素C合成酶突变体及其编码基因与应用，通过对棒状链霉菌中的去乙酰氧基头孢菌素C合成酶进行定点突变可提高酶的催化活性，进而有利于β-内酰胺类抗生素的合成和转化。
为实现上述目的，本发明采用如下技术方案：
去乙酰氧基头孢菌素C合成酶突变体，该突变体的氨基酸序列以SEQ ID NO.1(野生型去乙酰氧基头孢菌素C合成酶)为参考：MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTMRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDCEPLLRFRYFPQVPEHRSAEEQPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGAFTDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDVSLDGETATFQDWIGGNYVNIRRTSKA；具有位于第72位苏氨酸T72、第92位甘氨酸G92中的至少一个突变；第72位苏氨酸突变为除其自身以外任意一种天然氨基酸，第92位甘氨酸突变为除其自身以外任意一种天然氨基酸。
进一步地，编码野生型去乙酰氧基头孢菌素C合成酶的核苷酸序列如SEQ ID NO.2所示：atggacacgacggtgcccaccttcagcctggccgaactccagcagggcctgcaccaggacgagttccgcaggtgtctgagggacaagggcctcttctatctgacggactgcggtctgaccgacaccgagctgaagtcggccaaggacctcgtcatcgacttcttcgagcacggcagcgaggcggagaagcgcgccgtcacctcgcccgtccccaccatgcgccgcggcttcaccgggctggagtcggagagcaccgcccagatcaccaataccggcagctactccgactactcgatgtgctactcgatgggcaccgcggacaacctcttcccgtccggtgacttcgagcggatctggacccagtacttcgaccgccagtacaccgcctcccgcgcggtcgcccgggaggtcctgcgggcgaccgggaccgagcccgacggcggggtcgaggccttcctcgactgcgagccgctgctgcggttccgctacttcccgcaggtccccgagcaccgcagcgccgaggagcagcccctgcggatggcgccgcactacgacctgtcgatggtcaccctcatccagcagacaccctgcgccaacggcttcgtcagcctccaggccgaggtcggcggcgcgttcacggacctgccctaccgtccggacgccgtcctcgtcttctgcggcgccatcgcgaccctggtgaccggcggccaggtcaaggccccccggcaccatgtcgcggccccccgcagggaccagatagcgggcagcagccgcacctccagtgtgttcttcctccgtcccaacgcggacttcaccttctccgtcccgctggcgcgcgagtgcggcttcgatgtcagcctggacggcgagaccgccacgttccaggattggatcgggggcaactacgtgaacatccgccgcacatccaaggcatag。
进一步地，去乙酰氧基头孢菌素C合成酶单位点突变体氨基酸序列如SEQ ID NO.3所示：MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPXMRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDCEPLLRFRYFPQVPEHRSAEEQPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGAFTDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDVSLDGETATFQDWIGGNYVNIRRTSKA，其中第72位X代表除苏氨酸以外的天然氨基酸。
进一步地，编码去乙酰氧基头孢菌素C合成酶单位点突变体的核苷酸序列如SEQID NO.4所示：atggacacgacggtgcccaccttcagcctggccgaactccagcagggcctgcaccaggacgagttccgcaggtgtctgagggacaagggcctcttctatctgacggactgcggtctgaccgacaccgagctgaagtcggccaaggacctcgtcatcgacttcttcgagcacggcagcgaggcggagaagcgcgccgtcacctcgcccgtccccnnnatgcgccgcggcttcaccgggctggagtcggagagcaccgcccagatcaccaataccggcagctactccgactactcgatgtgctactcgatgggcaccgcggacaacctcttcccgtccggtgacttcgagcggatctggacccagtacttcgaccgccagtacaccgcctcccgcgcggtcgcccgggaggtcctgcgggcgaccgggaccgagcccgacggcggggtcgaggccttcctcgactgcgagccgctgctgcggttccgctacttcccgcaggtccccgagcaccgcagcgccgaggagcagcccctgcggatggcgccgcactacgacctgtcgatggtcaccctcatccagcagacaccctgcgccaacggcttcgtcagcctccaggccgaggtcggcggcgcgttcacggacctgccctaccgtccggacgccgtcctcgtcttctgcggcgccatcgcgaccctggtgaccggcggccaggtcaaggccccccggcaccatgtcgcggccccccgcagggaccagatagcgggcagcagccgcacctccagtgtgttcttcctccgtcccaacgcggacttcaccttctccgtcccgctggcgcgcgagtgcggcttcgatgtcagcctggacggcgagaccgccacgttccaggattggatcgggggcaactacgtgaacatccgccgcacatccaaggcatag，第72位氨基酸的密码子序列nnn代表除野生型中苏氨酸密码子acc以外的其他天然氨基酸密码子序列。
进一步地，去乙酰氧基头孢菌素C合成酶单位点突变体氨基酸序列如SEQ ID NO.5所示：MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTMRRGFTGLESESTAQITNTXSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDCEPLLRFRYFPQVPEHRSAEEQPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGAFTDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDVSLDGETATFQDWIGGNYVNIRRTSKA，其中第92位X代表除甘氨酸以外的天然氨基酸。
进一步地，编码去乙酰氧基头孢菌素C合成酶单位点突变体的核苷酸序列如SEQID NO.6所示：atggacacgacggtgcccaccttcagcctggccgaactccagcagggcctgcaccaggacgagttccgcaggtgtctgagggacaagggcctcttctatctgacggactgcggtctgaccgacaccgagctgaagtcggccaaggacctcgtcatcgacttcttcgagcacggcagcgaggcggagaagcgcgccgtcacctcgcccgtccccaccatgcgccgcggcttcaccgggctggagtcggagagcaccgcccagatcaccaataccnnnagctactccgactactcgatgtgctactcgatgggcaccgcggacaacctcttcccgtccggtgacttcgagcggatctggacccagtacttcgaccgccagtacaccgcctcccgcgcggtcgcccgggaggtcctgcgggcgaccgggaccgagcccgacggcggggtcgaggccttcctcgactgcgagccgctgctgcggttccgctacttcccgcaggtccccgagcaccgcagcgccgaggagcagcccctgcggatggcgccgcactacgacctgtcgatggtcaccctcatccagcagacaccctgcgccaacggcttcgtcagcctccaggccgaggtcggcggcgcgttcacggacctgccctaccgtccggacgccgtcctcgtcttctgcggcgccatcgcgaccctggtgaccggcggccaggtcaaggccccccggcaccatgtcgcggccccccgcagggaccagatagcgggcagcagccgcacctccagtgtgttcttcctccgtcccaacgcggacttcaccttctccgtcccgctggcgcgcgagtgcggcttcgatgtcagcctggacggcgagaccgccacgttccaggattggatcgggggcaactacgtgaacatccgccgcacatccaaggcatag，第92位氨基酸的密码子序列nnn代表除野生型中甘氨酸密码子ggc以外的其他天然氨基酸密码子序列。
进一步地，去乙酰氧基头孢菌素C合成酶双位点突变体氨基酸序列如SEQ ID NO.7所示：MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPXMRRGFTGLESESTAQITNTXSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDCEPLLRFRYFPQVPEHRSAEEQPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGAFTDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDVSLDGETATFQDWIGGNYVNIRRTSKA，其中第72位X代表除苏氨酸以外的天然氨基酸，第92位X代表除甘氨酸以外的天然氨基酸。
进一步地，编码去乙酰氧基头孢菌素C合成酶双位点突变体的核苷酸序列如SEQID NO.8所示：atggacacgacggtgcccaccttcagcctggccgaactccagcagggcctgcaccaggacgagttccgcaggtgtctgagggacaagggcctcttctatctgacggactgcggtctgaccgacaccgagctgaagtcggccaaggacctcgtcatcgacttcttcgagcacggcagcgaggcggagaagcgcgccgtcacctcgcccgtccccnnnatgcgccgcggcttcaccgggctggagtcggagagcaccgcccagatcaccaataccnnnagctactccgactactcgatgtgctactcgatgggcaccgcggacaacctcttcccgtccggtgacttcgagcggatctggacccagtacttcgaccgccagtacaccgcctcccgcgcggtcgcccgggaggtcctgcgggcgaccgggaccgagcccgacggcggggtcgaggccttcctcgactgcgagccgctgctgcggttccgctacttcccgcaggtccccgagcaccgcagcgccgaggagcagcccctgcggatggcgccgcactacgacctgtcgatggtcaccctcatccagcagacaccctgcgccaacggcttcgtcagcctccaggccgaggtcggcggcgcgttcacggacctgccctaccgtccggacgccgtcctcgtcttctgcggcgccatcgcgaccctggtgaccggcggccaggtcaaggccccccggcaccatgtcgcggccccccgcagggaccagatagcgggcagcagccgcacctccagtgtgttcttcctccgtcccaacgcggacttcaccttctccgtcccgctggcgcgcgagtgcggcttcgatgtcagcctggacggcgagaccgccacgttccaggattggatcgggggcaactacgtgaacatccgccgcacatccaaggcatag，第72位氨基酸的密码子序列nnn代表除野生型中苏氨酸密码子acc以外的其他天然氨基酸密码子序列，第92位氨基酸的密码子序列nnn代表除野生型中甘氨酸密码子ggc以外的其他天然氨基酸密码子序列。
优选地，突变体的氨基酸序列以SEQ ID NO.1为参考，第72位苏氨酸突变为色氨酸、甘氨酸、谷氨酰胺、天冬酰胺、天冬氨酸、亮氨酸、组氨酸、异亮氨酸、赖氨酸或蛋氨酸。
更优选地，第72位苏氨酸突变为色氨酸或甘氨酸。
最优选地，第72位苏氨酸突变为色氨酸。
优选地，突变体的氨基酸序列以SEQ ID NO.1为参考，第92位甘氨酸突变为蛋氨酸、半胱氨酸、丝氨酸、天冬酰胺、谷氨酰胺或组氨酸。
更优选地，第92位甘氨酸突变为蛋氨酸或半胱氨酸。
最优选地，第92位甘氨酸突变为蛋氨酸。
优选地，突变体的氨基酸序列以SEQ ID NO.1为参考，第72位苏氨酸突变为色氨酸、甘氨酸、谷氨酰胺、天冬酰胺、天冬氨酸、异亮氨酸或组氨酸；第92位甘氨酸突变为蛋氨酸、半胱氨酸、丝氨酸、天冬酰胺、谷氨酰胺、苯丙氨酸、苏氨酸或丙氨酸。
更优选地，上述突变体为双位点突变体T72D/G92C、T72D/G92H、T72G/G92C、T72G/G92M、T72G/G92S、T72I/G92M、T72N/G92M、T72Q/G92C、T72Q/G92H、T72Q/G92M、T72Q/G92S、T72W/G92C、T72W/G92F、T72W/G92H、T72W/G92M、T72W/G92N、T72W/G92Q、T72W/G92S或T72W/G92T。
更优选地，上述突变体为双位点突变体T72G/G92M、T72N/G92M、T72Q/G92M、T72W/G92C、T72W/G92F、T72W/G92H、T72W/G92M、T72W/G92N、T72W/G92S或T72W/G92T。
最优选地，上述突变体为双位点突变体T72W/G92M。
进一步地，上述突变体的制备可利用本领域已知的技术，先构建含有野生型去乙酰氧基头孢菌素C合成酶基因的载体质粒，然后选择定点突变的位点以及突变后的氨基酸种类，合成相应的引物，以含野生型去乙酰氧基头孢菌素C合成酶基因的载体质粒为模板，PCR扩增突变DNA片段，分离纯化，然后通过PCR将所得片段扩增为全长突变基因，再将该全长突变基因克隆到适当的载体上并转化适当的宿主细胞，经培养筛选出具有高酶活的阳性克隆，最后从阳性克隆中提取质粒DNA，进行DNA序列测定分析，以确定引入的突变。或者可通过基因合成的方式，合成整个含有突变酶分子DNA序列的质粒载体，然后转化适当的宿主细胞，经培养筛选出具有高酶活的阳性克隆。
制备去乙酰氧基头孢菌素C合成酶突变体的过程中，可采用任何合适的载体，例如，原核表达载体pET28、pRSET、pET-30a(+)、pGEMT-Easy等；真核表达载体pYD1、pYES2/GS等；还可选用pUC18/19、pBluscript-SK。
一种生物材料，为含有上述基因的表达载体，或含有上述表达载体的酶蛋白表达宿主细胞。
进一步地，上述基因可在原核细胞、真核细胞内进行表达，也可采用本领域已知的任何适当方法开展在原核细胞、真核细胞或无细胞体系进行胞外表达。上述表达载体的底盘***可以为原核微生物细胞、真核微生物细胞或无细胞体系；其中原核微生物可选用大肠杆菌、芽孢杆菌、谷氨酸棒杆菌、链霉菌等；真核微生物可选用酿酒酵母、毕赤巴斯德酵母、丝状真菌等；无细胞体系可来源于大肠杆菌裂解物、酵母细胞裂解物、麦胚抽提物、哺乳动物细胞裂解物等。
上述去乙酰氧基头孢菌素C合成酶突变体、基因、或生物材料在制备7-苯乙酰基脱乙酰氧基头孢菌素中的应用。
优选地，利用去乙酰氧基头孢菌素C合成酶突变体，以青霉素G为底物制备7-苯乙酰基脱乙酰氧基头孢菌素。
编码上述去乙酰氧基头孢菌素C合成酶突变体的基因经密码子优化后，在产黄支顶孢霉中异源表达提高头孢菌素C产量中的应用。
优选地，去乙酰氧基头孢菌素C合成酶突变体以青霉素N为底物。
综上所述，本发明通过对棒状链霉菌的去乙酰氧基头孢菌素C合成酶进行定点突变和基因工程改造，提高了该酶对青霉素G的催化活性，并且可提高产黄支顶孢霉头孢菌素C的产量，适于商业和工业应用。
附图说明
图1所示为棒状链霉菌去乙酰氧基头孢菌素C合成酶野生型表达质粒。
图2所示为棒状链霉菌去乙酰氧基头孢菌素C合成酶野生型蛋白电泳胶图；
M：marker；1：全细胞样品；2：细胞破壁后离心上清样品；3：细胞破壁后沉淀样品。
图3所示为棒状链霉菌去乙酰氧基头孢菌素C合成酶野生型及突变体G13571-56以青霉素G为底物转化生成G-7-ADCA的反应能力测试结果。
图4所示为经密码子优化后的高活性棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体G13571-56*大肠杆菌重组表达载体。
图5所示为重组质粒pBARGPE1-ScDAOCS-G13571-56*。
图6所示为阴性对照产黄支顶孢霉菌株ATCC11550-pBARGPE1、高活性棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体过表达菌株ATCC11550-pBARGPE1-ScDAOCS-G13571-56*的发酵CPC产量的HPLC叠加比较峰图。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：野生型去乙酰氧基头孢菌素C合成酶基因的获得及突变体文库的构建
根据NCBI网站公布的野生型棒状链霉菌(Streptomyces clavuligerus)去乙酰氧基头孢菌素C合成酶氨基酸序列SEQ ID NO.1(protein ID：AAA26715.1)及其编码基因核苷酸序列SEQ ID NO.2(GenBank ID：m32324，CDS序列1559至2494bp)设计突变体；突变体氨基酸序列如SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7所示，编码基因核苷酸序列如SEQ IDNO.4、SEQ ID NO.6、SEQ ID NO.8所示。
去乙酰氧基头孢菌素C合成酶野生型及突变体文库由常州基宇生物科技有限公司构建，包括去乙酰氧基头孢菌素C合成酶位点72苏氨酸(T72)的单点饱和突变、92位甘氨酸(G92)的单点饱和突变、72位苏氨酸和92位甘氨酸的部分双点组合突变(T72/G92)；克隆载体选用pET-30a(+)融合蛋白表达载体(卡那霉素抗性，空载体大小为5422bp)，克隆位点为NdeI-HindIII(限制性内切酶NdeI识别5'-CA↑TATG-3'的酶切位点，限制性内切酶HindIII识别5'-A↑AGCTT-3'的酶切位点)，宿主细胞为大肠杆菌TOP10感受态菌株(购自北京索莱宝科技有限公司，货号C1200)。
棒状链霉菌去乙酰氧基头孢菌素C合成酶文库野生型***片段核苷酸序列为SEQID NO.9:catatggacacgacggtgcccaccttcagcctggccgaactccagcagggcctgcaccaggacgagttccgcaggtgtctgagggacaagggcctcttctatctgacggactgcggtctgaccgacaccgagctgaagtcggccaaggacctcgtcatcgacttcttcgagcacggcagcgaggcggagaagcgcgccgtcacctcgcccgtccccaccatgcgccgcggcttcaccgggctggagtcggagagcaccgcccagatcaccaataccggcagctactccgactactcgatgtgctactcgatgggcaccgcggacaacctcttcccgtccggtgacttcgagcggatctggacccagtacttcgaccgccagtacaccgcctcccgcgcggtcgcccgggaggtcctgcgggcgaccgggaccgagcccgacggcggggtcgaggccttcctcgactgcgagccgctgctgcggttccgctacttcccgcaggtccccgagcaccgcagcgccgaggagcagcccctgcggatggcgccgcactacgacctgtcgatggtcaccctcatccagcagacaccctgcgccaacggcttcgtcagcctccaggccgaggtcggcggcgcgttcacggacctgccctaccgtccggacgccgtcctcgtcttctgcggcgccatcgcgaccctggtgaccggcggccaggtcaaggccccccggcaccatgtcgcggccccccgcagggaccagatagcgggcagcagccgcacctccagtgtgttcttcctccgtcccaacgcggacttcaccttctccgtcccgctggcgcgcgagtgcggcttcgatgtcagcctggacggcgagaccgccacgttccaggattggatcgggggcaactacgtgaacatccgccgcacatccaaggcatagtaaaagctt；棒状链霉菌去乙酰氧基头孢菌素C合成酶文库突变体***片段核苷酸序列为SEQ ID NO.10:catatggacacgacggtgcccaccttcagcctggccgaactccagcagggcctgcaccaggacgagttccgcaggtgtctgagggacaagggcctcttctatctgacggactgcggtctgaccgacaccgagctgaagtcggccaaggacctcgtcatcgacttcttcgagcacggcagcgaggcggagaagcgcgccgtcacctcgcccgtccccnnnatgcgccgcggcttcaccgggctggagtcggagagcaccgcccagatcaccaataccnnnagctactccgactactcgatgtgctactcgatgggcaccgcggacaacctcttcccgtccggtgacttcgagcggatctggacccagtacttcgaccgccagtacaccgcctcccgcgcggtcgcccgggaggtcctgcgggcgaccgggaccgagcccgacggcggggtcgaggccttcctcgactgcgagccgctgctgcggttccgctacttcccgcaggtccccgagcaccgcagcgccgaggagcagcccctgcggatggcgccgcactacgacctgtcgatggtcaccctcatccagcagacaccctgcgccaacggcttcgtcagcctccaggccgaggtcggcggcgcgttcacggacctgccctaccgtccggacgccgtcctcgtcttctgcggcgccatcgcgaccctggtgaccggcggccaggtcaaggccccccggcaccatgtcgcggccccccgcagggaccagatagcgggcagcagccgcacctccagtgtgttcttcctccgtcccaacgcggacttcaccttctccgtcccgctggcgcgcgagtgcggcttcgatgtcagcctggacggcgagaccgccacgttccaggattggatcgggggcaactacgtgaacatccgccgcacatccaaggcatagtaaaagctt。第72位氨基酸的密码子序列nnn代表除野生型中苏氨酸密码子acc以外的其他天然氨基酸密码子序列，第92位氨基酸的密码子序列nnn代表除野生型中甘氨酸密码子ggc以外的其他天然氨基酸密码子序列。其中，野生型酶表达质粒图谱如附图1所示，质粒大小为6192bp。
实施例2：去乙酰氧基头孢菌素C合成酶突变体宿主菌株培养及诱导表达
从-80℃低温冰箱中取出实施例1构建的去乙酰氧基头孢菌素C合成酶野生型或突变体表达菌株，在冰浴上缓慢溶解完全，然后在无菌操作台上从菌种冻存管中吸取0.5mL菌种冻存液于含有3mL无菌冷却LB液体培养基(含50μg/mL卡那霉素)中，37℃震荡培养12h。
培养结束后取出0.5mL转接于含50mL无菌LB培养基的250mL三角瓶中，37℃下220rpm震荡培养1.5h，取出分别加入终浓度为100μmol/L的异丙基-β-D-硫代半乳糖苷(IPTG，购自上海阿拉丁生化科技股份有限公司，货号：I104812-25g)诱导剂，25℃下继续220rpm振荡诱导培养10h。
培养结束进行SDS-PAGE蛋白电泳。
1)实验试剂按如下操作配制：
①30％储备胶溶液：丙烯酰胺(Acr)29.0g，亚甲双丙烯酰胺(Bis)1.0g，混后加ddH2O溶解，定容至100mL，棕色瓶存于室温。
②10×电泳缓冲液(pH＝8.3)：3.02g Tris，18.8g甘氨酸，10mL 10％SDS，加入ddH20溶解，定容至100mL。
③10％过硫酸铵(AP)：100mgAP加ddH20至1mL充分溶解。
④2×SDS电泳上样缓冲液：1mol/L的Tris-HCl(pH＝6.8)2.5mL，β-巯基乙醇1.0mL，SDS0.6g，甘油2.0mL，0.1％溴酚兰1.0mL，ddH2O 3.5mL。
2)聚丙烯酰胺凝胶的配制：
①12mL分离胶(12％)的配制：ddH2O，3.96mL；30％储备胶，4.8mL；1.5mol/L的Tris-HCl，3mL；10％SDS，0.12mL；10％AP，0.12mL。上述溶液混匀后加入TEMED(N,N,N',N'-四甲基乙二胺)10μL，混匀后灌入玻璃板间，以水饱和正丁醇封顶，注意使液面平，凝胶完全聚合需30-60min。
②6ml积层胶(4％)的配制：ddH2O，4.2mL；30％储备胶，0.99mL；1mol/L的Tris-HCl，0.75mL；10％SDS，0.06mL；10％AP，0.06mL；TEMED，6μL。将分离胶上的液体倒去，超纯水冲洗干净，用滤纸吸干。加入上述混合液，立即将梳子***玻璃板间，完全聚合需15-30min。
3)样品处理：将样品加入等量的2×SDS上样缓冲液，100℃加热5min，离心12000g×1min，同时将蛋白Marker作平行处理。
4)上样：取10uL处理后的样品加入胶槽中，并加入20μL蛋白Marker作对照。
5)电泳：采用垂直式电泳槽装置，在电泳槽中加入1×电泳缓冲液，连接电源，负极在上，正极在下，电泳时，积层胶电压150V，分离胶电压200V，电泳至溴酚兰行至电泳槽下端停止。约1h后将蛋白胶从玻璃板中取出，使用pyxis蛋白快速处理***进行染脱色。脱色后的蛋白胶在图像处理***下将脱色好的凝胶摄像，凝胶可保存于7％乙酸溶液中。
棒状链霉菌去乙酰氧基头孢菌素C合成酶野生型蛋白电泳胶图如附图2所示。
实施例3：去乙酰氧基头孢菌素C合成酶突变体以青霉素G为底物转化生成G-7-ADCA的反应能力测试
1)底盘宿主细胞的破壁：按照实施例2方法对去乙酰氧基头孢菌素C合成酶野生型及突变体宿主菌株进行诱导培养，培养结束后取下摇瓶，吸取4mL于15mL离心管中，6000rpm条件下离心5min。离心结束后弃去上清并收集菌体，用4ml Tris-硫酸铵缓冲液(各50mmol/L，pH＝7.4，加入终浓度1mmol/L的DTT)重悬。菌体重悬后将离心管置于冰浴上进行超声波破碎(超声参数：45％功率，超声3s，停止2s，超声工作总计15min)。
2)酶与底物反应能力测试：菌体重悬液超声破碎后吸取2.4mL转移至50mL离心管中，加入终浓度为2mmol/L FeSO4，2mmol/L维生素C钠，5mmol/Lα-酮戊二酸，5mmol/L青霉素G钾；30℃下静置反应30min。反应结束后，加入1.2mL乙腈(分析纯)溶液终止反应，获得的反应液进行HPLC检测。
HPLC检测仪器：分析天平sartorius BSA224S(0.0001g)、赛默飞U3000高效液相色谱仪、超声波清洗仪、pH计为sartorius PB-10、安捷伦C18色谱柱(4.6×250mm，粒径5μm)。
HPLC检测试剂：
①20mmoL/L磷酸盐溶液(pH＝3.50)：取4.5644g三水合磷酸氢二钾加水溶解成1000mL，用磷酸调节pH至3.50。
②流动相：20mmoL/L磷酸盐溶液(pH＝3.5)：甲醇(50％：50％)。
③***适应性溶液：取G-7-ADCA标准品10mg与青霉素G标准品、α-酮戊二酸及L-抗坏血酸各2mg置于50ml容量瓶中，用流动相超声溶解并定容，摇匀。超声过程中应控制超声波内水温在2～8℃。
④对照溶液：取G-7-ADCA标准品10mg置于50ml容量瓶中，用pH＝3.5的磷酸盐溶液超声溶解并定容，摇匀。α-酮戊二酸、硫酸亚铁和L-抗坏血酸分别配制成溶液，用流动相稀释至500μM、200μM以及400μM。超声过程中应控制超声波内水温在2～8℃。
⑤供试试液：反应液用甲醇稀释4倍，混匀。
HPLC色谱条件：柱温30℃，溶解温度2～8℃，检测波长225nm，流速0.65mL/min，进样量5μL。
HPLC检测结果计算：
C样——供试样品中G-7-ADCA的浓度，mg/mL；
C标——标准品溶液中G-7-ADCA的浓度，mg/mL；
A样——供试样品中G-7-ADCA峰面积；
A标——G-7-ADCA标准品峰面积；
相对酶活计算：
相对酶活＝(C突变体/C野生型)×100％。
C突变体——去乙酰氧基头孢菌素C合成酶突变体酶反应后的供试样品中G-7-ADCA的浓度，mg/mL；
C野生型——去乙酰氧基头孢菌素C合成酶野生型酶反应后的供试样品中G-7-ADCA的浓度，mg/mL；
结果如表1及图3所示。
表1去乙酰氧基头孢菌素C合成酶突变体相对酶活
实施例4：高活性棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体密码子优化
遗传信息是由三联体密码子记载的。由于密码子的简并性，大多数氨基酸是由2～6种同义密码子编码。不同的物种编码同种氨基酸所利用的密码子种类与使用频率存在差别，这种现象称为密码子偏好性(Codon Usage Bias)。原核微生物和真核微生物密码子偏好性存在明显的差异。因此，本实施例中将原核微生物棒状链霉菌去乙酰氧基头孢菌素C合成酶高活性突变体G13571-56基因的密码子优化为适合丝状真菌产黄支顶孢霉表达的密码子序列。
本实施例中，丙氨酸密码子序列优化为gcc半胱氨酸密码子序列优化为tgc、天冬氨酸密码子序列优化为gac、谷氨酸密码子序列优化为gag、苯丙氨酸密码子序列优化为ttc、甘氨酸密码子序列优化为ggc、组氨酸密码子序列优化为cac、异亮氨酸密码子序列优化为atc、赖氨酸密码子序列优化为aag、亮氨酸密码子序列优化为ctc、天冬酰胺密码子序列优化为aac、脯氨酸密码子序列优化为ccc、谷氨酰胺密码子序列优化为cag、精氨酸密码子序列优化为cgc、丝氨酸密码子序列优化为agc、苏氨酸密码子序列优化为acc、缬氨酸密码子序列优化为gtc、酪氨酸密码子序列优化为taa。
经密码子优化后的棒状链霉菌去乙酰氧基头孢菌素C合成酶高活性突变体基因G13571-56*的核苷酸序列如SEQ ID NO.11所示(因密码子序列优化而改变)：
实施例5：经密码子偏好性优化后的高活性棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体大肠杆菌重组表达载体的构建、转化和验证
pET-28a(+)质粒载体全长5369bp，含有卡那霉素抗性。利用限制性内切酶(均购自上海生工生物工程股份有限公司)NdeI(酶切识别位点：5'-CA↑TATG-3'，上海生工生物工程有限公司，货号：B600120-0500)和HindIII(5'-A↑AGCTT-3'，上海生工生物工程股份有限公司，货号：B600184)对纯化得到的pET-28a(+)质粒进行双酶切，切掉63bp的多克隆位点序列。酶切反应体系为：质粒载体，3μg；Buffer，5μL；NdeI限制性内切酶，HindIII 0.5μL；限制性内切酶，0.5μL；ddwater，至50μL；37℃孵育3～4小时，每隔一段时间振荡一下并离心以防液滴蒸发至管盖上。酶切后的线性质粒载体经验证后切胶纯化备用。
G13571-56*基因序列合成由常州基宇生物科技有限公司完成，去乙酰氧基头孢菌素C合成酶突变体基因上游和下游分别添加限制性内切酶NdeI识别位点(5'-CA↑TATG-3')和HindIII识别位点(5'-A↑AGCTT-3')，合成***片段核苷酸序列如SEQ ID NO.12所示：
合成的***片段经限制性内切酶NdeI和HindIII酶切后，与经限制性内切酶NdeI和HindIII双酶切并纯化后得到的pET-28a(+)线性质粒进行融合蛋白表达载体进行酶连接反应。连接反应体系：退火产物，2μL；线性化质粒25ng；T4 DNA连接酶(上海生工生物工程股份有限公司，货号：B110041)0.5μL；连接buffer，5μL；ddwater补至10μL，16℃连接过夜。
连接产物经纯化验证后转化大肠杆菌感受态细胞：1μL表达载体质粒加入到100μL大肠杆菌BL21商业感受态细胞中，冰浴孵育30min，取出放置42℃水浴热激2min，再立即置于冰浴上3min，加入到650μL的LB液体培养基(37℃)中，37℃下200rpm振荡培养1h，室温下5000rpm离心3min，留大约150μL涂抗性平板(50μg/mL卡那霉素)，37℃培养箱静置培养过夜。挑取抗性平板上长好的单克隆菌落，接种到5mL含50μg/mL卡那霉素抗性的LB液体培养基中，37℃下200rpm振摇过夜。培养好后利用质粒提取试剂盒(天根质粒提取试剂盒，货号：DP103)提取质粒载体(如图4所示)，纯化后送测序公司测序。测序结果正确者-80℃低温冰箱保存备用。
实施例6：高活性棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体在产黄支顶孢霉中提高头孢菌素C产量的应用
1)基因过表达质粒载体pBARGPE1-ScDAOCS-G13571-56*的构建
产黄枝顶头孢霉异源过表达棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体G13571-56*，构建重组质粒pBARGPE1-ScDAOCS-G13571-56*的示意图见图5。
具体步骤如下：
①利用细菌质粒提取试剂盒提取质粒pET28-G13571-56*作为模板DNA。
②以步骤①获得的cDNA为模板，以PrimeSTAR高保真酶和引物G13571-56*-F/G13571-56*-R进行PCR扩增，并对PCR扩增产物进行测序验证，其中引物G13571-56*-F序列：5′-GGTTCCATGGACACGACGGTGCCCACCTTC-3′(SEQ ID NO.13，下划线为限制性内切酶BamHI的识别位点)和引物G13571-56*-R序列：5′-GGGCCCCTAGGCCTTGGAGGTGCGGCGGAT-3′(SEQ IDNO.14，下划线为限制性内切酶ApaI的识别位点)，获得约948bp的PCR扩增cDNA产物。
③将步骤②获得的PCR扩增产物和载体pEASY-Blunt进行连接，得到重组质粒pEASY-ScDAOCS-G13571-56*。
④用限制性内切酶BamHI和ApaI双酶切重组质粒pEASY-ScDAOCS-G13571-56*，回收约948bp的DNA片段ScDAOCS-G13571-56*。
⑤用限制性内切酶BamHI和ApaI双酶切质粒pBARGPE1-Hygro，回收约5936bp的载体骨架。
⑥将DNA片段ScDAOCS-G13571-56*和载体骨架进行连接，得到重组质粒pBARGPE1-ScDAOCS-G13571-56*。将重组质粒pBARGPE1-ScDAOCS-G13571-56*进行测序。根据测序结果，对重组质粒pBARGPE1-ScDAOCS-G13571-56*结构描述如下：将质粒pBARGPE1-Hygro的限制性内切酶BamHI和ApaI之间的小片段取代为序列表的序列SEQ ID No.11示的DNA分子，构建成用于在产黄支顶孢霉中进行棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体过表达的重组质粒pBARGPE1-ScDAOCS-G13571-56*。重组质粒pBARGPE1-ScDAOCS-G13571-56*具有gpdA启动子、经密码子优化后的棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体G13571-56*基因、trpC终止子和潮霉素抗性基因。
2)产黄枝顶头孢霉ATCC11550菌丝体的培养和原生质体制备
①从培养斜面上刮下适量产黄枝顶头孢霉菌ATCC11550孢子，分别接种于100mLYPS液体培养基(葡萄糖2％，酵母提取物0.5％，聚胨1％，MgSO4·7H2O 0.1％，K2HPO4·3H2O0.13％，pH＝7.0)中，28℃、230rpm振荡培养4～5d；
②8000rpm离心15min收集菌丝体，用无菌水洗涤一次；
③0.22μm无菌滤膜过滤50mL二硫苏糖醇(DTT，5mmol/L)溶液，重悬菌体，30℃、150rpm振荡孵育40～60min；
④8000rpm离心5min，用P Buffer(KCl 44.7g/L，MgCl2·6H2O 2.03g/L，CaCl22.78g/L)常温洗涤2次；
⑤0.22μm无菌滤膜过滤60mL Lysing酶解液(P Buffer配制，10mg/mL)，重悬菌体，30℃、150rpm振荡孵育3～4h；
⑥镜检，当大部分菌丝释放原生质体后，加入4倍体积的PBuffer，用灭菌的装填脱脂棉的针筒过滤除去残留的菌丝体；
⑦3000rpm离心5min，PBuffer洗涤2次，将原生质体悬浮于适量PBuffer中，使原生质体浓度>108CFU；
⑧将产黄枝顶头孢霉菌ATCC11550的原生质体分装于1.5mL离心管中，每管100μL。
3)产黄枝顶头孢霉原生质体转化及鉴定
采用PEG-CaCl2介导的原生质体转化法将构建的质粒转化产黄枝顶头孢霉，实验步骤如下：
①原生质体加入10μg的重组质粒pBARGPE1-ScDAOCS-G13571-56*DNA，轻轻混匀，冰浴30min；
②加入900μL 30％的PEG4000/CaCl2溶液(称取CaCl2·2H2O14.7g，90mL纯水溶解于100mL容量瓶中，再加入30g的PEG4000后用纯水定容至100mL，过滤除菌后分装储存至4℃)，25℃孵育15min；
③6000rpm×5min，尽量吸出PEG4000/CaCl2溶液，用PBuffer洗涤1次；
④使用100μL的PBuffer重悬原生质体，加入到45℃保温的上层软琼脂培养基(蛋白胨，1％；NaCl，0.5％；酵母提取物，0.3％；蔗糖，2％；琼脂，0.75％；自然pH)中，于旋涡振荡器上轻轻振荡混匀，然后倾倒在再生琼脂平板(可溶性淀粉2.4％，甘氨酸0.12％，聚胨0.4％，硫酸铵0.6％，磷酸二氢钾0.012％，硫酸钙0.8％，硫酸镁0.06％，酵母提取物0.03％，琼脂2％，pH＝8.5，按照250mL体积分装至500mL锥形瓶，121℃灭菌15min，冷却备用)上，迅速转动平板使软琼脂均匀覆盖在下层培养基表面；于28℃培养36h，覆盖含有潮霉素的NaCl软琼脂(NaCl，4％；琼脂，0.75％)，使平板中潮霉素终浓度为5.0μg/mL，NaCl软琼脂凝固后，于28℃培养；⑤培养7d后分别挑取菌株进行博来霉素抗性转化子斜面培养，7d后提取基因组DNA进行PCR验证，重组质粒转化成功。
4)高活性棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体基因过表达对产黄支顶孢霉CPC增产效果的验证
将转化后的产黄枝顶头孢霉ATCC11550原生质体细胞与软琼脂混合后涂布于再生琼脂培养基上生长。根据菌落的生长状况(菌落生长3～5d后，可以看见较稀疏单菌落)，在平板铺上含有潮霉素(终浓度为5.0μg/ml)的NaCl软琼脂。铺完抗生素后，28℃培养，期间应每天都注意观察转化子是否出现以及生长状况。若有转化子一般10-15d后其基本生长良好，可挑取至含有5.0μg/ml潮霉素的分单培养基上，28℃恒温培养5-7d。将得到的所有转化子进行摇瓶发酵培养，发酵5天后经HPLC检测得到发酵结果，发现CPC发酵单位显著提高，证明过表达高活性棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体基因对CPC产量起到上调作用。
5)产黄枝顶头孢霉的发酵和发酵产物CPC的检测
本实施例中的三角瓶的规格均为500mL，三角瓶底部带有直挡板，每个实验菌株有三个摇瓶重复。从培养10d的斜面上刮下适量产黄枝顶头孢霉孢子，以野生型出发菌株ATCC11550为对照，含转化子的菌株为实验组，其中转化子1为转有空质粒pBARGPE1-Hygro的产黄枝顶头孢霉菌菌株ATCC11550-pBRGPE1-Hygro，转化子2为转有高活性棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体基因过表达质粒pBARGPE1-ScDAOCS-G13571-56*的产黄枝顶头孢霉菌菌株ATCC11550-pBARGPE1-ScDAOCS-G13571-56*，分别接种于装有30mL种子培养基(葡萄糖，5g/L；蔗糖，35g/L；玉米浆10mL/L；硫酸铵，8g/L；DL-甲硫氨酸，0.5g/L；碳酸钙，5g/L；豆油，5mL/L；pH＝7.20±0.05；121℃×15min灭菌后冷却备用)的500mL摇瓶中，于旋转式摇床培养3d，转速为230rpmn，温度28℃。再以10％(v/v)的接种量转接至装量为30mL发酵培养基(玉米淀粉，30g/L，麦芽糊精，60g/L；α-淀粉酶，0.2g/L；玉米浆，10mL/L；DL-甲硫氨酸，6g/L；尿素，2g/L；硫酸铵，11g/L；MgSO4·7H2O，3g/L；K2HPO4，9g/L；碳酸钙，5g/L；豆油，10mL/L；pH＝7.20±0.05；121℃×15min灭菌后冷却备用)的250mL摇瓶中，25℃，230rpm，培养7d。发酵液经普通滤纸、0.22μm过滤，收集滤液，稀释20倍后进行HPLC检测。
HPLC检测使用美国Agilent 1260HPLC检测器，C18色谱柱，柱温40℃，流动相为甲醇:0.2％(w/v)磷酸二氢钠＝5:95，流速为1.0mL/min，检测波长为254nm，进样量10μL，分析时间9min。根据峰面积和标准品的有效含量计算出样品中CPC含量。
检测结果如图6所示，野生型出发对照菌株ATCC11550的CPC产量为2679.45±138.9mg/L，转有空质粒pBARGPE1-Hygro转化子的突变型对照菌株ATCC11550-pBRGPE1-Hygro的CPC产量为2591±214.6mg/L，转有高活性棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体酶过表达质粒pBARGPE1-ScDAOCS-G13571-56*转化子的突变型菌株ATCC11550-pBARGPE1-ScDAOCS-G13571-56*的CPC产量为4357.4±231.9mg/L。对数据进行统计分析后可知，野生型对照菌株ATCC11550与转有空质粒pBRGPE1-Hygro的对照菌株ATCC11550-pBRGPE1-Hygro的CPC产量无显著性差异，而与转有高活性棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体基因过表达质粒pBARGPE1-ScDAOCS-G13571-56*的实验突变菌株ATCC11550-pBARGPE1-ScDAOCS-G13571-56*有显著性差异(p＝0.011)；转有空质粒pBRGPE1-Hygro的对照菌株ATCC11550-pBRGPE1-Hygro与转有高活性棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体基因过表达质粒pBARGPE1-ScDAOCS-G13571-56*的实验突变菌株ATCC11550-pBARGPE1-ScDAOCS-G13571-56之间同样有显著性差异(p＝0.024)，后者CPC产量提高了68.2％。上述实验结果证明，对外源高活性棒状链霉菌去乙酰氧基头孢菌素C合成酶突变体基因在产黄支顶孢霉ATCC11550中进行过表达，能够有效促进CPC产量的提高。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对上述实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
序列表
<110> 河南省健康元生物医药研究院有限公司
<120> 去乙酰氧基头孢菌素C合成酶突变体及其编码基因与应用
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 311
<212> PRT
<213> Artificial
<400> 1
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe
20 25 30
Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys
35 40 45
Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Thr Met Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
100 105 110
Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr
115 120 125
Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln
165 170 175
Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Gly Gly Ala Phe Thr Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala
225 230 235 240
Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Val Pro Leu Ala Arg Glu Cys Gly Phe Asp Val Ser Leu Asp
275 280 285
Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn
290 295 300
Ile Arg Arg Thr Ser Lys Ala
305 310
<210> 2
<211> 936
<212> DNA
<213> Artificial
<400> 2
atggacacga cggtgcccac cttcagcctg gccgaactcc agcagggcct gcaccaggac 60
gagttccgca ggtgtctgag ggacaagggc ctcttctatc tgacggactg cggtctgacc 120
gacaccgagc tgaagtcggc caaggacctc gtcatcgact tcttcgagca cggcagcgag 180
gcggagaagc gcgccgtcac ctcgcccgtc cccaccatgc gccgcggctt caccgggctg 240
gagtcggaga gcaccgccca gatcaccaat accggcagct actccgacta ctcgatgtgc 300
tactcgatgg gcaccgcgga caacctcttc ccgtccggtg acttcgagcg gatctggacc 360
cagtacttcg accgccagta caccgcctcc cgcgcggtcg cccgggaggt cctgcgggcg 420
accgggaccg agcccgacgg cggggtcgag gccttcctcg actgcgagcc gctgctgcgg 480
ttccgctact tcccgcaggt ccccgagcac cgcagcgccg aggagcagcc cctgcggatg 540
gcgccgcact acgacctgtc gatggtcacc ctcatccagc agacaccctg cgccaacggc 600
ttcgtcagcc tccaggccga ggtcggcggc gcgttcacgg acctgcccta ccgtccggac 660
gccgtcctcg tcttctgcgg cgccatcgcg accctggtga ccggcggcca ggtcaaggcc 720
ccccggcacc atgtcgcggc cccccgcagg gaccagatag cgggcagcag ccgcacctcc 780
agtgtgttct tcctccgtcc caacgcggac ttcaccttct ccgtcccgct ggcgcgcgag 840
tgcggcttcg atgtcagcct ggacggcgag accgccacgt tccaggattg gatcgggggc 900
aactacgtga acatccgccg cacatccaag gcatag 936
<210> 3
<211> 311
<212> PRT
<213> Artificial
<400> 3
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe
20 25 30
Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys
35 40 45
Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Xaa Met Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
100 105 110
Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr
115 120 125
Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln
165 170 175
Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Gly Gly Ala Phe Thr Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala
225 230 235 240
Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Val Pro Leu Ala Arg Glu Cys Gly Phe Asp Val Ser Leu Asp
275 280 285
Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn
290 295 300
Ile Arg Arg Thr Ser Lys Ala
305 310
<210> 4
<211> 936
<212> DNA
<213> Artificial
<400> 4
atggacacga cggtgcccac cttcagcctg gccgaactcc agcagggcct gcaccaggac 60
gagttccgca ggtgtctgag ggacaagggc ctcttctatc tgacggactg cggtctgacc 120
gacaccgagc tgaagtcggc caaggacctc gtcatcgact tcttcgagca cggcagcgag 180
gcggagaagc gcgccgtcac ctcgcccgtc cccnnnatgc gccgcggctt caccgggctg 240
gagtcggaga gcaccgccca gatcaccaat accggcagct actccgacta ctcgatgtgc 300
tactcgatgg gcaccgcgga caacctcttc ccgtccggtg acttcgagcg gatctggacc 360
cagtacttcg accgccagta caccgcctcc cgcgcggtcg cccgggaggt cctgcgggcg 420
accgggaccg agcccgacgg cggggtcgag gccttcctcg actgcgagcc gctgctgcgg 480
ttccgctact tcccgcaggt ccccgagcac cgcagcgccg aggagcagcc cctgcggatg 540
gcgccgcact acgacctgtc gatggtcacc ctcatccagc agacaccctg cgccaacggc 600
ttcgtcagcc tccaggccga ggtcggcggc gcgttcacgg acctgcccta ccgtccggac 660
gccgtcctcg tcttctgcgg cgccatcgcg accctggtga ccggcggcca ggtcaaggcc 720
ccccggcacc atgtcgcggc cccccgcagg gaccagatag cgggcagcag ccgcacctcc 780
agtgtgttct tcctccgtcc caacgcggac ttcaccttct ccgtcccgct ggcgcgcgag 840
tgcggcttcg atgtcagcct ggacggcgag accgccacgt tccaggattg gatcgggggc 900
aactacgtga acatccgccg cacatccaag gcatag 936
<210> 5
<211> 311
<212> PRT
<213> Artificial
<400> 5
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe
20 25 30
Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys
35 40 45
Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Thr Met Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Xaa Ser Tyr Ser Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
100 105 110
Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr
115 120 125
Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln
165 170 175
Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Gly Gly Ala Phe Thr Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala
225 230 235 240
Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Val Pro Leu Ala Arg Glu Cys Gly Phe Asp Val Ser Leu Asp
275 280 285
Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn
290 295 300
Ile Arg Arg Thr Ser Lys Ala
305 310
<210> 6
<211> 936
<212> DNA
<213> Artificial
<400> 6
atggacacga cggtgcccac cttcagcctg gccgaactcc agcagggcct gcaccaggac 60
gagttccgca ggtgtctgag ggacaagggc ctcttctatc tgacggactg cggtctgacc 120
gacaccgagc tgaagtcggc caaggacctc gtcatcgact tcttcgagca cggcagcgag 180
gcggagaagc gcgccgtcac ctcgcccgtc cccaccatgc gccgcggctt caccgggctg 240
gagtcggaga gcaccgccca gatcaccaat accnnnagct actccgacta ctcgatgtgc 300
tactcgatgg gcaccgcgga caacctcttc ccgtccggtg acttcgagcg gatctggacc 360
cagtacttcg accgccagta caccgcctcc cgcgcggtcg cccgggaggt cctgcgggcg 420
accgggaccg agcccgacgg cggggtcgag gccttcctcg actgcgagcc gctgctgcgg 480
ttccgctact tcccgcaggt ccccgagcac cgcagcgccg aggagcagcc cctgcggatg 540
gcgccgcact acgacctgtc gatggtcacc ctcatccagc agacaccctg cgccaacggc 600
ttcgtcagcc tccaggccga ggtcggcggc gcgttcacgg acctgcccta ccgtccggac 660
gccgtcctcg tcttctgcgg cgccatcgcg accctggtga ccggcggcca ggtcaaggcc 720
ccccggcacc atgtcgcggc cccccgcagg gaccagatag cgggcagcag ccgcacctcc 780
agtgtgttct tcctccgtcc caacgcggac ttcaccttct ccgtcccgct ggcgcgcgag 840
tgcggcttcg atgtcagcct ggacggcgag accgccacgt tccaggattg gatcgggggc 900
aactacgtga acatccgccg cacatccaag gcatag 936
<210> 7
<211> 311
<212> PRT
<213> Artificial
<400> 7
Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly
1 5 10 15
Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe
20 25 30
Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys
35 40 45
Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg
50 55 60
Ala Val Thr Ser Pro Val Pro Xaa Met Arg Arg Gly Phe Thr Gly Leu
65 70 75 80
Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Xaa Ser Tyr Ser Asp
85 90 95
Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser
100 105 110
Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr
115 120 125
Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu
130 135 140
Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg
145 150 155 160
Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln
165 170 175
Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile
180 185 190
Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val
195 200 205
Gly Gly Ala Phe Thr Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val
210 215 220
Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala
225 230 235 240
Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser
245 250 255
Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr
260 265 270
Phe Ser Val Pro Leu Ala Arg Glu Cys Gly Phe Asp Val Ser Leu Asp
275 280 285
Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn
290 295 300
Ile Arg Arg Thr Ser Lys Ala
305 310
<210> 8
<211> 936
<212> DNA
<213> Artificial
<400> 8
atggacacga cggtgcccac cttcagcctg gccgaactcc agcagggcct gcaccaggac 60
gagttccgca ggtgtctgag ggacaagggc ctcttctatc tgacggactg cggtctgacc 120
gacaccgagc tgaagtcggc caaggacctc gtcatcgact tcttcgagca cggcagcgag 180
gcggagaagc gcgccgtcac ctcgcccgtc cccnnnatgc gccgcggctt caccgggctg 240
gagtcggaga gcaccgccca gatcaccaat accnnnagct actccgacta ctcgatgtgc 300
tactcgatgg gcaccgcgga caacctcttc ccgtccggtg acttcgagcg gatctggacc 360
cagtacttcg accgccagta caccgcctcc cgcgcggtcg cccgggaggt cctgcgggcg 420
accgggaccg agcccgacgg cggggtcgag gccttcctcg actgcgagcc gctgctgcgg 480
ttccgctact tcccgcaggt ccccgagcac cgcagcgccg aggagcagcc cctgcggatg 540
gcgccgcact acgacctgtc gatggtcacc ctcatccagc agacaccctg cgccaacggc 600
ttcgtcagcc tccaggccga ggtcggcggc gcgttcacgg acctgcccta ccgtccggac 660
gccgtcctcg tcttctgcgg cgccatcgcg accctggtga ccggcggcca ggtcaaggcc 720
ccccggcacc atgtcgcggc cccccgcagg gaccagatag cgggcagcag ccgcacctcc 780
agtgtgttct tcctccgtcc caacgcggac ttcaccttct ccgtcccgct ggcgcgcgag 840
tgcggcttcg atgtcagcct ggacggcgag accgccacgt tccaggattg gatcgggggc 900
aactacgtga acatccgccg cacatccaag gcatag 936
<210> 9
<211> 948
<212> DNA
<213> Artificial
<400> 9
catatggaca cgacggtgcc caccttcagc ctggccgaac tccagcaggg cctgcaccag 60
gacgagttcc gcaggtgtct gagggacaag ggcctcttct atctgacgga ctgcggtctg 120
accgacaccg agctgaagtc ggccaaggac ctcgtcatcg acttcttcga gcacggcagc 180
gaggcggaga agcgcgccgt cacctcgccc gtccccacca tgcgccgcgg cttcaccggg 240
ctggagtcgg agagcaccgc ccagatcacc aataccggca gctactccga ctactcgatg 300
tgctactcga tgggcaccgc ggacaacctc ttcccgtccg gtgacttcga gcggatctgg 360
acccagtact tcgaccgcca gtacaccgcc tcccgcgcgg tcgcccggga ggtcctgcgg 420
gcgaccggga ccgagcccga cggcggggtc gaggccttcc tcgactgcga gccgctgctg 480
cggttccgct acttcccgca ggtccccgag caccgcagcg ccgaggagca gcccctgcgg 540
atggcgccgc actacgacct gtcgatggtc accctcatcc agcagacacc ctgcgccaac 600
ggcttcgtca gcctccaggc cgaggtcggc ggcgcgttca cggacctgcc ctaccgtccg 660
gacgccgtcc tcgtcttctg cggcgccatc gcgaccctgg tgaccggcgg ccaggtcaag 720
gccccccggc accatgtcgc ggccccccgc agggaccaga tagcgggcag cagccgcacc 780
tccagtgtgt tcttcctccg tcccaacgcg gacttcacct tctccgtccc gctggcgcgc 840
gagtgcggct tcgatgtcag cctggacggc gagaccgcca cgttccagga ttggatcggg 900
ggcaactacg tgaacatccg ccgcacatcc aaggcatagt aaaagctt 948
<210> 10
<211> 948
<212> DNA
<213> Artificial
<400> 10
catatggaca cgacggtgcc caccttcagc ctggccgaac tccagcaggg cctgcaccag 60
gacgagttcc gcaggtgtct gagggacaag ggcctcttct atctgacgga ctgcggtctg 120
accgacaccg agctgaagtc ggccaaggac ctcgtcatcg acttcttcga gcacggcagc 180
gaggcggaga agcgcgccgt cacctcgccc gtccccnnna tgcgccgcgg cttcaccggg 240
ctggagtcgg agagcaccgc ccagatcacc aataccnnna gctactccga ctactcgatg 300
tgctactcga tgggcaccgc ggacaacctc ttcccgtccg gtgacttcga gcggatctgg 360
acccagtact tcgaccgcca gtacaccgcc tcccgcgcgg tcgcccggga ggtcctgcgg 420
gcgaccggga ccgagcccga cggcggggtc gaggccttcc tcgactgcga gccgctgctg 480
cggttccgct acttcccgca ggtccccgag caccgcagcg ccgaggagca gcccctgcgg 540
atggcgccgc actacgacct gtcgatggtc accctcatcc agcagacacc ctgcgccaac 600
ggcttcgtca gcctccaggc cgaggtcggc ggcgcgttca cggacctgcc ctaccgtccg 660
gacgccgtcc tcgtcttctg cggcgccatc gcgaccctgg tgaccggcgg ccaggtcaag 720
gccccccggc accatgtcgc ggccccccgc agggaccaga tagcgggcag cagccgcacc 780
tccagtgtgt tcttcctccg tcccaacgcg gacttcacct tctccgtccc gctggcgcgc 840
gagtgcggct tcgatgtcag cctggacggc gagaccgcca cgttccagga ttggatcggg 900
ggcaactacg tgaacatccg ccgcacatcc aaggcatagt aaaagctt 948
<210> 11
<211> 936
<212> DNA
<213> Artificial
<400> 11
atggacacga cggtgcccac cttcagcctg gccgagctcc agcagggcct gcaccaggac 60
gagttccgca ggtgcctgag ggacaagggc ctcttctacc tgacggactg cggcctgacc 120
gacaccgagc tgaagtcggc caaggacctc gtcatcgact tcttcgagca cggcagcgag 180
gcggagaagc gcgccgtcac ctcgcccgtc ccctggatgc gccgcggctt caccggcctg 240
gagtcggaga gcaccgccca gatcaccaac accatgagct actccgacta ctcgatgtgc 300
tactcgatgg gcaccgcgga caacctcttc ccgtccggcg acttcgagcg catctggacc 360
cagtacttcg accgccagta caccgcctcc cgcgcggtcg cccgcgaggt cctgcgcgcg 420
accggcaccg agcccgacgg cggcgtcgag gccttcctcg actgcgagcc gctgctgcgg 480
ttccgctact tcccgcaggt ccccgagcac cgcagcgccg aggagcagcc cctgcggatg 540
gcgccgcact acgacctgtc gatggtcacc ctcatccagc agacgccctg cgccaacggc 600
ttcgtcagcc tccaggccga ggtcggcggc gcgttcacgg acctgcccta ccgcccggac 660
gccgtcctcg tcttctgcgg cgccatcgcg accctggtga ccggcggcca ggtcaaggcc 720
ccccgccacc acgtcgcggc cccccgcagg gaccagatcg cgggcagcag ccgcacctcc 780
agcgtgttct tcctccgccc caacgcggac ttcaccttct ccgtcccgct ggcgcgcgag 840
tgcggcttcg acgtcagcct ggacggcgag accgccacgt tccaggattg gatcggcggc 900
aactacgtga acatccgccg cacctccaag gcctag 936
<210> 12
<211> 948
<212> DNA
<213> Artificial
<400> 12
catatggaca cgacggtgcc caccttcagc ctggccgagc tccagcaggg cctgcaccag 60
gacgagttcc gcaggtgcct gagggacaag ggcctcttct acctgacgga ctgcggcctg 120
accgacaccg agctgaagtc ggccaaggac ctcgtcatcg acttcttcga gcacggcagc 180
gaggcggaga agcgcgccgt cacctcgccc gtcccctgga tgcgccgcgg cttcaccggc 240
ctggagtcgg agagcaccgc ccagatcacc aacaccatga gctactccga ctactcgatg 300
tgctactcga tgggcaccgc ggacaacctc ttcccgtccg gcgacttcga gcgcatctgg 360
acccagtact tcgaccgcca gtacaccgcc tcccgcgcgg tcgcccgcga ggtcctgcgc 420
gcgaccggca ccgagcccga cggcggcgtc gaggccttcc tcgactgcga gccgctgctg 480
cggttccgct acttcccgca ggtccccgag caccgcagcg ccgaggagca gcccctgcgg 540
atggcgccgc actacgacct gtcgatggtc accctcatcc agcagacgcc ctgcgccaac 600
ggcttcgtca gcctccaggc cgaggtcggc ggcgcgttca cggacctgcc ctaccgcccg 660
gacgccgtcc tcgtcttctg cggcgccatc gcgaccctgg tgaccggcgg ccaggtcaag 720
gccccccgcc accacgtcgc ggccccccgc agggaccaga tcgcgggcag cagccgcacc 780
tccagcgtgt tcttcctccg ccccaacgcg gacttcacct tctccgtccc gctggcgcgc 840
gagtgcggct tcgacgtcag cctggacggc gagaccgcca cgttccagga ttggatcggc 900
ggcaactacg tgaacatccg ccgcacctcc aaggcctagt aaaagctt 948
<210> 13
<211> 30
<212> DNA
<213> Artificial
<400> 13
ggttccatgg acacgacggt gcccaccttc 30
<210> 14
<211> 30
<212> DNA
<213> Artificial
<400> 14
gggcccctag gccttggagg tgcggcggat 30
Claims (8)
1.去乙酰氧基头孢菌素C合成酶突变体，其特征在于，所述突变体是在SEQ ID NO .1的基础上，对第92位的甘氨酸G92进行单突变，将第92位甘氨酸突变为蛋氨酸、半胱氨酸、丝氨酸、天冬酰胺、谷氨酰胺或组氨酸；
或，对第72位的苏氨酸T72和第92位的甘氨酸G92进行双突变，所述双突变选自：
（1）第72位T突变为D，且第92位G突变为A；
（2）第72位T突变为D，且第92位G突变为C；
（3）第72位T突变为D，且第92位G突变为H；
（4）第72位T突变为G，且第92位G突变为C；
（5）第72位T突变为G，且第92位G突变为M；
（6）第72位T突变为G，且第92位G突变为S；
（7）第72位T突变为I，且第92位G突变为M；
（8）第72位T突变为N，且第92位G突变为M；
（9）第72位T突变为Q，且第92位G突变为C；
（10）第72位T突变为Q，且第92位G突变为H；
（11）第72位T突变为Q，且第92位G突变为M；
（12）第72位T突变为Q，且第92位G突变为S；
（13）第72位T突变为W，且第92位G突变为C；
（14）第72位T突变为W，且第92位G突变为F；
（15）第72位T突变为W，且第92位G突变为H；
（16）第72位T突变为W，且第92位G突变为M；
（17）第72位T突变为W，且第92位G突变为N；
（18）第72位T突变为W，且第92位G突变为Q；
（19）第72位T突变为W，且第92位G突变为S；
或，
（20）第72位T突变为W，且第92位G突变为T。
2.编码权利要求1所述的去乙酰氧基头孢菌素C合成酶突变体的核酸。
3.一种生物材料，其特征在于，所述生物材料为含有权利要求2所述核酸的表达载体。
4.一种生物材料，其特征在于，所述生物材料为含有权利要求3中所述的表达载体的宿主细胞。
5.权利要求1所述的去乙酰氧基头孢菌素C合成酶突变体、权利要求2所述的核酸、或权利要求3或4所述生物材料在制备7-苯乙酰基脱乙酰氧基头孢菌素中的应用。
6.根据权利要求5所述的应用，其特征在于，利用去乙酰氧基头孢菌素C合成酶突变体，以青霉素G为底物制备7-苯乙酰基脱乙酰氧基头孢菌素。
7.经密码子优化后的编码权利要求1所述去乙酰氧基头孢菌素C合成酶突变体的基因的应用，所述应用为在产黄支顶孢霉中异源表达提高头孢菌素C产量。
8.根据权利要求7所述的应用，其特征在于，去乙酰氧基头孢菌素C合成酶突变体以青霉素G为底物。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210671678.7A CN114958877B (zh) | 2022-06-14 | 2022-06-14 | 去乙酰氧基头孢菌素c合成酶突变体及其编码基因与应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210671678.7A CN114958877B (zh) | 2022-06-14 | 2022-06-14 | 去乙酰氧基头孢菌素c合成酶突变体及其编码基因与应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114958877A CN114958877A (zh) | 2022-08-30 |
| CN114958877B true CN114958877B (zh) | 2024-02-20 |
Family
ID=82963617
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210671678.7A Active CN114958877B (zh) | 2022-06-14 | 2022-06-14 | 去乙酰氧基头孢菌素c合成酶突变体及其编码基因与应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114958877B (zh) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1284997A (zh) * | 1997-12-24 | 2001-02-21 | 埃西斯创新有限公司 | 修饰的脱乙酰氧基头孢菌素c合酶daocs及x射线结构 |
| WO2001085951A1 (en) * | 2000-05-09 | 2001-11-15 | Acs Dobfar Uk Limited | A modified expandase and uses thereof |
| CN1446908A (zh) * | 2002-03-26 | 2003-10-08 | 骏瀚生化股份有限公司 | 突变的青霉素扩环酶及用其制备7－氨基去乙酰氧基头胞菌素酸的方法 |
| CN101300360A (zh) * | 2005-07-12 | 2008-11-05 | 帝斯曼知识产权资产管理有限公司 | 突变体扩环酶及其在β-内酰胺化合物生产中的用途 |
| CN107304418A (zh) * | 2016-04-18 | 2017-10-31 | 百瑞全球有限公司 | 青霉素扩环酶突变体、编码该突变体的dna、含有该突变体的试剂盒及其应用 |
| CN113584008A (zh) * | 2021-07-30 | 2021-11-02 | 湖南福来格生物技术有限公司 | 一种青霉素g酰化酶突变体及其应用 |
-
2022
- 2022-06-14 CN CN202210671678.7A patent/CN114958877B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1284997A (zh) * | 1997-12-24 | 2001-02-21 | 埃西斯创新有限公司 | 修饰的脱乙酰氧基头孢菌素c合酶daocs及x射线结构 |
| WO2001085951A1 (en) * | 2000-05-09 | 2001-11-15 | Acs Dobfar Uk Limited | A modified expandase and uses thereof |
| CN1446908A (zh) * | 2002-03-26 | 2003-10-08 | 骏瀚生化股份有限公司 | 突变的青霉素扩环酶及用其制备7－氨基去乙酰氧基头胞菌素酸的方法 |
| CN101300360A (zh) * | 2005-07-12 | 2008-11-05 | 帝斯曼知识产权资产管理有限公司 | 突变体扩环酶及其在β-内酰胺化合物生产中的用途 |
| CN107304418A (zh) * | 2016-04-18 | 2017-10-31 | 百瑞全球有限公司 | 青霉素扩环酶突变体、编码该突变体的dna、含有该突变体的试剂盒及其应用 |
| CN113584008A (zh) * | 2021-07-30 | 2021-11-02 | 湖南福来格生物技术有限公司 | 一种青霉素g酰化酶突变体及其应用 |
Non-Patent Citations (1)
| Title |
|---|
| Deacetoxycephalosporin C synthase (expandase): Research progress and application potential;Xiaofan Niu等;《Synthetic andSystemsBiotechnology》;第6卷;396–401 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114958877A (zh) | 2022-08-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20130252310A1 (en) | Novel aldolase and production process of substituted alpha-keto acids | |
| CN109055327B (zh) | 醛酮还原酶突变体及其应用 | |
| CN111500566B (zh) | 一种海藻糖合成酶突变体及其制备方法和应用 | |
| CN110117601B (zh) | 灰树花葡聚糖合成酶、其编码基因及应用 | |
| CN112695021A (zh) | 一种α-糖苷酶基因突变体及在制备2-O-α-D-葡萄糖基-L-抗坏血酸中的应用 | |
| CN112980906B (zh) | 一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用 | |
| CN114807206A (zh) | 合成聚(3-羟基丁酸酯-co-4-羟基丁酸酯)的菌株及其构建方法和应用 | |
| CN103695405B (zh) | 一种新型β-内酰胺类抗生素合成酶的生产方法 | |
| CN113106028A (zh) | 高产头孢菌素c的基因工程菌构建方法及其应用 | |
| CN114958877B (zh) | 去乙酰氧基头孢菌素c合成酶突变体及其编码基因与应用 | |
| CN110129305B (zh) | 一种用于制备7-aca的头孢菌素c酰化酶突变体 | |
| CN114277068B (zh) | 一种r-3-羟基丁酸乙酯微生物发酵制备方法 | |
| CN107099523B (zh) | 头孢拉定合成酶突变体及其编码基因 | |
| CN113832087B (zh) | 一种利用大肠杆菌全生物合成丙二酸的方法 | |
| CN114736884A (zh) | 一种胞苷单磷酸激酶突变体及其基因和应用 | |
| CN114672525A (zh) | N-乙酰基-5-甲氧基色胺的生物合成方法及其应用 | |
| CN113403355A (zh) | 一种生物法生产l-5-甲基四氢叶酸的方法 | |
| CN114990138B (zh) | 基因、表达载体、菌株及在提高头孢菌素c产量中的应用 | |
| CN112410353A (zh) | 一种fkbS基因、含其的基因工程菌及其制备方法和用途 | |
| CN113493745B (zh) | 生产头孢菌素c的基因工程菌及其构建方法 | |
| CN113881737B (zh) | 利用基因工程菌和酵母耦合发酵大规模产cmp-唾液酸的方法 | |
| CN117603930B (zh) | 一种表达突变型西罗血红素合酶的重组菌 | |
| CN110467655B (zh) | 一种蛋白质及其应用 | |
| CN103374563A (zh) | 一种改良7-aca产生菌的方法 | |
| CN117965325A (zh) | 一种产黄青霉菌株及其应用 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |