CN108359666B - 一种nudC基因及其在制备烟酸方面的应用 - Google Patents
一种nudC基因及其在制备烟酸方面的应用 Download PDFInfo
- Publication number
- CN108359666B CN108359666B CN201810023635.1A CN201810023635A CN108359666B CN 108359666 B CN108359666 B CN 108359666B CN 201810023635 A CN201810023635 A CN 201810023635A CN 108359666 B CN108359666 B CN 108359666B
- Authority
- CN
- China
- Prior art keywords
- nicotinic acid
- nudc
- gene
- application
- ala
- 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
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 title claims abstract description 122
- 239000011664 nicotinic acid Substances 0.000 title claims abstract description 62
- 235000001968 nicotinic acid Nutrition 0.000 title claims abstract description 60
- 229960003512 nicotinic acid Drugs 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 23
- 241000894006 Bacteria Species 0.000 claims abstract description 15
- 238000004519 manufacturing process Methods 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 241000187480 Mycobacterium smegmatis Species 0.000 claims description 33
- 239000013598 vector Substances 0.000 claims description 12
- 101150057554 nucC gene Proteins 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 20
- 238000003786 synthesis reaction Methods 0.000 abstract description 6
- 230000007547 defect Effects 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 3
- 239000013612 plasmid Substances 0.000 description 30
- 108020004414 DNA Proteins 0.000 description 13
- 239000000706 filtrate Substances 0.000 description 11
- ITQTTZVARXURQS-UHFFFAOYSA-N 3-methylpyridine Chemical compound CC1=CC=CN=C1 ITQTTZVARXURQS-UHFFFAOYSA-N 0.000 description 10
- GZPHSAQLYPIAIN-UHFFFAOYSA-N 3-pyridinecarbonitrile Chemical compound N#CC1=CC=CN=C1 GZPHSAQLYPIAIN-UHFFFAOYSA-N 0.000 description 8
- 238000001502 gel electrophoresis Methods 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 230000003647 oxidation Effects 0.000 description 7
- 238000007254 oxidation reaction Methods 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 101150087140 nudC gene Proteins 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 238000010276 construction Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 239000003513 alkali Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 206010029400 Nicotinic acid deficiency Diseases 0.000 description 3
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 3
- 108010033272 Nitrilase Proteins 0.000 description 3
- 208000002141 Pellagra Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 229910017604 nitric acid Inorganic materials 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000012074 organic phase Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- NTSLROIKFLNUIJ-UHFFFAOYSA-N 5-Ethyl-2-methylpyridine Chemical compound CCC1=CC=C(C)N=C1 NTSLROIKFLNUIJ-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 230000002210 biocatalytic effect Effects 0.000 description 2
- 238000010170 biological method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.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 OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 2
- 229960002064 kanamycin sulfate Drugs 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N nicotinic acid amide Natural products NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 108010089198 phenylalanyl-prolyl-arginine Proteins 0.000 description 2
- FSSOYPTUNGNSQS-UHFFFAOYSA-N pyridine-3-carboxylic acid;sulfuric acid Chemical compound [H+].[O-]S([O-])(=O)=O.OC(=O)C1=CC=C[NH+]=C1 FSSOYPTUNGNSQS-UHFFFAOYSA-N 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 108010084932 tryptophyl-proline Proteins 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000019160 vitamin B3 Nutrition 0.000 description 2
- 239000011708 vitamin B3 Substances 0.000 description 2
- DOLJMUVMVVSXRX-UHFFFAOYSA-N 2-hydroxy-5-[(7-hydroxy-8-methyl-6-nitro-2-oxochromene-3-carbonyl)amino]benzoic acid Chemical compound O=C1OC=2C(C)=C(O)C([N+]([O-])=O)=CC=2C=C1C(=O)NC1=CC=C(O)C(C(O)=O)=C1 DOLJMUVMVVSXRX-UHFFFAOYSA-N 0.000 description 1
- HTMOATKEZDESFW-UHFFFAOYSA-N 2-methylpyridine;sulfuric acid Chemical compound OS(O)(=O)=O.CC1=CC=CC=N1 HTMOATKEZDESFW-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- KQFRUSHJPKXBMB-BHDSKKPTSA-N Ala-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)C)C(O)=O)=CNC2=C1 KQFRUSHJPKXBMB-BHDSKKPTSA-N 0.000 description 1
- 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 1
- BEMGNWZECGIJOI-WDSKDSINSA-N Ala-Gly-Glu Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O BEMGNWZECGIJOI-WDSKDSINSA-N 0.000 description 1
- OEVCHROQUIVQFZ-YTLHQDLWSA-N Ala-Thr-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](C)C(O)=O OEVCHROQUIVQFZ-YTLHQDLWSA-N 0.000 description 1
- MCYJBCKCAPERSE-FXQIFTODSA-N Arg-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N MCYJBCKCAPERSE-FXQIFTODSA-N 0.000 description 1
- ITVINTQUZMQWJR-QXEWZRGKSA-N Arg-Asn-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O ITVINTQUZMQWJR-QXEWZRGKSA-N 0.000 description 1
- OHYQKYUTLIPFOX-ZPFDUUQYSA-N Arg-Glu-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O OHYQKYUTLIPFOX-ZPFDUUQYSA-N 0.000 description 1
- DGFXIWKPTDKBLF-AVGNSLFASA-N Arg-His-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CCCN=C(N)N)N DGFXIWKPTDKBLF-AVGNSLFASA-N 0.000 description 1
- YKBHOXLMMPZPHQ-GMOBBJLQSA-N Arg-Ile-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O YKBHOXLMMPZPHQ-GMOBBJLQSA-N 0.000 description 1
- PFOYSEIHFVKHNF-FXQIFTODSA-N Asn-Ala-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PFOYSEIHFVKHNF-FXQIFTODSA-N 0.000 description 1
- SRUUBQBAVNQZGJ-LAEOZQHASA-N Asn-Gln-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)N)N SRUUBQBAVNQZGJ-LAEOZQHASA-N 0.000 description 1
- WONGRTVAMHFGBE-WDSKDSINSA-N Asn-Gly-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N WONGRTVAMHFGBE-WDSKDSINSA-N 0.000 description 1
- BYLSYQASFJJBCL-DCAQKATOSA-N Asn-Pro-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O BYLSYQASFJJBCL-DCAQKATOSA-N 0.000 description 1
- VWADICJNCPFKJS-ZLUOBGJFSA-N Asn-Ser-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O VWADICJNCPFKJS-ZLUOBGJFSA-N 0.000 description 1
- BFOYULZBKYOKAN-OLHMAJIHSA-N Asp-Asp-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BFOYULZBKYOKAN-OLHMAJIHSA-N 0.000 description 1
- VIRHEUMYXXLCBF-WDSKDSINSA-N Asp-Gly-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O VIRHEUMYXXLCBF-WDSKDSINSA-N 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- IOLWXFWVYYCVTJ-NRPADANISA-N Cys-Val-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CS)N IOLWXFWVYYCVTJ-NRPADANISA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 206010013954 Dysphoria Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 241000427940 Fusarium solani Species 0.000 description 1
- WQWMZOIPXWSZNE-WDSKDSINSA-N Gln-Asp-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O WQWMZOIPXWSZNE-WDSKDSINSA-N 0.000 description 1
- MWERYIXRDZDXOA-QEWYBTABSA-N Gln-Ile-Phe Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O MWERYIXRDZDXOA-QEWYBTABSA-N 0.000 description 1
- FGWRYRAVBVOHIB-XIRDDKMYSA-N Gln-Pro-Trp Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)N)N)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O FGWRYRAVBVOHIB-XIRDDKMYSA-N 0.000 description 1
- NHMRJKKAVMENKJ-WDCWCFNPSA-N Gln-Thr-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O NHMRJKKAVMENKJ-WDCWCFNPSA-N 0.000 description 1
- 208000005232 Glossitis Diseases 0.000 description 1
- UTKUTMJSWKKHEM-WDSKDSINSA-N Glu-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O UTKUTMJSWKKHEM-WDSKDSINSA-N 0.000 description 1
- KKCUFHUTMKQQCF-SRVKXCTJSA-N Glu-Arg-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O KKCUFHUTMKQQCF-SRVKXCTJSA-N 0.000 description 1
- WLIPTFCZLHCNFD-LPEHRKFASA-N Glu-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)O)N)C(=O)O WLIPTFCZLHCNFD-LPEHRKFASA-N 0.000 description 1
- QIQABBIDHGQXGA-ZPFDUUQYSA-N Glu-Ile-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QIQABBIDHGQXGA-ZPFDUUQYSA-N 0.000 description 1
- CBWKURKPYSLMJV-SOUVJXGZSA-N Glu-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CCC(=O)O)N)C(=O)O CBWKURKPYSLMJV-SOUVJXGZSA-N 0.000 description 1
- LXXLEUBUOMCAMR-NKWVEPMBSA-N Gly-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)CN)C(=O)O LXXLEUBUOMCAMR-NKWVEPMBSA-N 0.000 description 1
- JPWIMMUNWUKOAD-STQMWFEESA-N Gly-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)CN JPWIMMUNWUKOAD-STQMWFEESA-N 0.000 description 1
- UPADCCSMVOQAGF-LBPRGKRZSA-N Gly-Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CNC(=O)CN)C(O)=O)=CNC2=C1 UPADCCSMVOQAGF-LBPRGKRZSA-N 0.000 description 1
- ORXZVPZCPMKHNR-IUCAKERBSA-N Gly-His-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CNC=N1 ORXZVPZCPMKHNR-IUCAKERBSA-N 0.000 description 1
- 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 1
- XINDHUAGVGCNSF-QSFUFRPTSA-N His-Ala-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O XINDHUAGVGCNSF-QSFUFRPTSA-N 0.000 description 1
- SOFSRBYHDINIRG-QTKMDUPCSA-N His-Arg-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC1=CN=CN1)N)O SOFSRBYHDINIRG-QTKMDUPCSA-N 0.000 description 1
- LYSMQLXUCAKELQ-DCAQKATOSA-N His-Asp-Arg Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N LYSMQLXUCAKELQ-DCAQKATOSA-N 0.000 description 1
- LSQHWKPPOFDHHZ-YUMQZZPRSA-N His-Asp-Gly Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)O)N LSQHWKPPOFDHHZ-YUMQZZPRSA-N 0.000 description 1
- KAXZXLSXFWSNNZ-XVYDVKMFSA-N His-Ser-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O KAXZXLSXFWSNNZ-XVYDVKMFSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- JRHFQUPIZOYKQP-KBIXCLLPSA-N Ile-Ala-Glu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O JRHFQUPIZOYKQP-KBIXCLLPSA-N 0.000 description 1
- TZCGZYWNIDZZMR-UHFFFAOYSA-N Ile-Arg-Ala Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(C)C(O)=O)CCCN=C(N)N TZCGZYWNIDZZMR-UHFFFAOYSA-N 0.000 description 1
- NKRJALPCDNXULF-BYULHYEWSA-N Ile-Asp-Gly Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O NKRJALPCDNXULF-BYULHYEWSA-N 0.000 description 1
- PPSQSIDMOVPKPI-BJDJZHNGSA-N Ile-Cys-Leu Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(=O)O PPSQSIDMOVPKPI-BJDJZHNGSA-N 0.000 description 1
- NYEYYMLUABXDMC-NHCYSSNCSA-N Ile-Gly-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)O)N NYEYYMLUABXDMC-NHCYSSNCSA-N 0.000 description 1
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-UHFFFAOYSA-N 0.000 description 1
- LJHGALIOHLRRQN-DCAQKATOSA-N Leu-Ala-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LJHGALIOHLRRQN-DCAQKATOSA-N 0.000 description 1
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 1
- PJYSOYLLTJKZHC-GUBZILKMSA-N Leu-Asp-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(N)=O PJYSOYLLTJKZHC-GUBZILKMSA-N 0.000 description 1
- DZQMXBALGUHGJT-GUBZILKMSA-N Leu-Glu-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O DZQMXBALGUHGJT-GUBZILKMSA-N 0.000 description 1
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 1
- DBSLVQBXKVKDKJ-BJDJZHNGSA-N Leu-Ile-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O DBSLVQBXKVKDKJ-BJDJZHNGSA-N 0.000 description 1
- IAJFFZORSWOZPQ-SRVKXCTJSA-N Leu-Leu-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IAJFFZORSWOZPQ-SRVKXCTJSA-N 0.000 description 1
- RMLLCGYYVZKKRT-CIUDSAMLSA-N Met-Ser-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O RMLLCGYYVZKKRT-CIUDSAMLSA-N 0.000 description 1
- 108010066427 N-valyltryptophan Proteins 0.000 description 1
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 1
- 241000187654 Nocardia Species 0.000 description 1
- 241000277334 Oncorhynchus Species 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- BFYHIHGIHGROAT-HTUGSXCWSA-N Phe-Glu-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BFYHIHGIHGROAT-HTUGSXCWSA-N 0.000 description 1
- APJPXSFJBMMOLW-KBPBESRZSA-N Phe-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 APJPXSFJBMMOLW-KBPBESRZSA-N 0.000 description 1
- JXQVYPWVGUOIDV-MXAVVETBSA-N Phe-Ser-Ile Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JXQVYPWVGUOIDV-MXAVVETBSA-N 0.000 description 1
- LTAWNJXSRUCFAN-UNQGMJICSA-N Phe-Thr-Arg Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O LTAWNJXSRUCFAN-UNQGMJICSA-N 0.000 description 1
- YUPRIZTWANWWHK-DZKIICNBSA-N Phe-Val-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N YUPRIZTWANWWHK-DZKIICNBSA-N 0.000 description 1
- FZHBZMDRDASUHN-NAKRPEOUSA-N Pro-Ala-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1)C(O)=O FZHBZMDRDASUHN-NAKRPEOUSA-N 0.000 description 1
- FYQSMXKJYTZYRP-DCAQKATOSA-N Pro-Ala-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 FYQSMXKJYTZYRP-DCAQKATOSA-N 0.000 description 1
- 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 1
- ZVEQWRWMRFIVSD-HRCADAONSA-N Pro-Phe-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)N3CCC[C@@H]3C(=O)O ZVEQWRWMRFIVSD-HRCADAONSA-N 0.000 description 1
- PKHDJFHFMGQMPS-RCWTZXSCSA-N Pro-Thr-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PKHDJFHFMGQMPS-RCWTZXSCSA-N 0.000 description 1
- 241000589776 Pseudomonas putida Species 0.000 description 1
- 241000187693 Rhodococcus rhodochrous Species 0.000 description 1
- MMGJPDWSIOAGTH-ACZMJKKPSA-N Ser-Ala-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O MMGJPDWSIOAGTH-ACZMJKKPSA-N 0.000 description 1
- HBOABDXGTMMDSE-GUBZILKMSA-N Ser-Arg-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O HBOABDXGTMMDSE-GUBZILKMSA-N 0.000 description 1
- XXNYYSXNXCJYKX-DCAQKATOSA-N Ser-Leu-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(O)=O XXNYYSXNXCJYKX-DCAQKATOSA-N 0.000 description 1
- PQEQXWRVHQAAKS-SRVKXCTJSA-N Ser-Tyr-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)N)CC1=CC=C(O)C=C1 PQEQXWRVHQAAKS-SRVKXCTJSA-N 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- DNUJCLUFRGGSDJ-YLVFBTJISA-N Trp-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC1=CNC2=CC=CC=C21)N DNUJCLUFRGGSDJ-YLVFBTJISA-N 0.000 description 1
- OTWIOROMZLNAQC-XIRDDKMYSA-N Trp-His-Asp Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(O)=O OTWIOROMZLNAQC-XIRDDKMYSA-N 0.000 description 1
- SLLKXDSRVAOREO-KZVJFYERSA-N Val-Ala-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)N)O SLLKXDSRVAOREO-KZVJFYERSA-N 0.000 description 1
- AAOPYWQQBXHINJ-DZKIICNBSA-N Val-Gln-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N AAOPYWQQBXHINJ-DZKIICNBSA-N 0.000 description 1
- MDYSKHBSPXUOPV-JSGCOSHPSA-N Val-Gly-Phe Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N MDYSKHBSPXUOPV-JSGCOSHPSA-N 0.000 description 1
- UMPVMAYCLYMYGA-ONGXEEELSA-N Val-Leu-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O UMPVMAYCLYMYGA-ONGXEEELSA-N 0.000 description 1
- DLRZGNXCXUGIDG-KKHAAJSZSA-N Val-Thr-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N)O DLRZGNXCXUGIDG-KKHAAJSZSA-N 0.000 description 1
- 229930003537 Vitamin B3 Natural products 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- ZYTHLJLPPSSDIP-UHFFFAOYSA-N anileridine dihydrochloride Chemical compound Cl.Cl.C1CC(C(=O)OCC)(C=2C=CC=CC=2)CCN1CCC1=CC=C(N)C=C1 ZYTHLJLPPSSDIP-UHFFFAOYSA-N 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 108010059459 arginyl-threonyl-phenylalanine Proteins 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000002781 deodorant agent Substances 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 108010054813 diprotin B Proteins 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 229960005436 inositol nicotinate Drugs 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 229960003350 isoniazid Drugs 0.000 description 1
- QRXWMOHMRWLFEY-UHFFFAOYSA-N isoniazide Chemical compound NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- MFZCIDXOLLEMOO-GYSGTQPESA-N myo-inositol hexanicotinate Chemical compound O([C@H]1[C@@H]([C@H]([C@@H](OC(=O)C=2C=NC=CC=2)[C@@H](OC(=O)C=2C=NC=CC=2)[C@@H]1OC(=O)C=1C=NC=CC=1)OC(=O)C=1C=NC=CC=1)OC(=O)C=1C=NC=CC=1)C(=O)C1=CC=CN=C1 MFZCIDXOLLEMOO-GYSGTQPESA-N 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000003300 oropharynx Anatomy 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 208000035824 paresthesia Diseases 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- KFLRWGSAMLBHBV-UHFFFAOYSA-M sodium;pyridine-3-carboxylate Chemical compound [Na+].[O-]C(=O)C1=CC=CN=C1 KFLRWGSAMLBHBV-UHFFFAOYSA-M 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 229940124549 vasodilator Drugs 0.000 description 1
- 239000003071 vasodilator agent Substances 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种nudC基因及其在制备烟酸方面的应用。所述nudC基因的核苷酸序列如SEQ.ID.NO.1所示，其编码蛋白nudC蛋白的氨基酸序列如SEQ.ID.NO.2所示。利用本发明发现的nudC基因构建的工程菌可以一步直接获得烟酸，不仅避免了现有烟酸化学合成过程中的各种缺点，而且生产条件简单，无污染，简便易行，易放大，成本低，适合于大规模工业化生产应用，有很大的推广价值。
Description
技术领域
本发明涉及烟酸的合成技术领域，更具体地，涉及一种nudC基因及其在制备烟酸方面的应用。
背景技术
烟酸即3-吡啶甲酸，也称作为维生素B3，属于维生素B族，是一种水溶性维生素，是人体必需的13种维生素之一。烟酸是机体组织中重要的递氢体和抗糙皮病的因子，有维持皮肤和神经健康，促进消化的作用。若其缺乏时，可产生糙皮病，表现为皮炎、舌炎、口咽、腹泻及烦躁、失眠感觉异常等症状。与烟酰胺统称维生素PP，用于抗糙皮病，亦可用作血扩张药。作为医药中间体，用于异烟肼、烟酰胺、尼可刹及烟酸肌醇酯等的生产。烟酸也用于粮食制品，肉品添加剂和饲料添加剂以预防糙皮病。特别是饲料中添加烟酸后可提高畜禽的抗病能力，加快生长速度，提高饲料的利用率，可大量节省饲料，降低饲养成本。美国曾用添加烟酸的饲料喂养奶牛，与对照组相比，产奶量可提高15％-20％。另外烟酸还可作为形成活性污泥的生化激素，空气和废气的除臭剂。烟酸在染料工业、感光材料工业、染发助剂、洗涤剂等方面也有一定的应用。
目前工业上主要用化学合成法生产烟酸，合成方法主要有液相法（高锰酸钾氧化法和硝酸氧化法）和气相法（臭氧氧化法、氨氧化法和空气氧化法），所用原料主要为3-甲基吡啶、2-甲基-5-乙基吡啶、3-氰基吡啶以及哇琳等。目前最常用的方法是以3-甲基吡啶为原料用空气氧化法合成烟酸。将3-甲基吡啶、空气和氨气按比例通入流化床反应器中，在290～360℃、V2O5催化下反应生成烟腈；再于氢氧化钠水溶液中、160℃下高压水解生成烟酸钠，最后用盐酸酸化得烟酸。在这个合成过程中需要经过烟腈这一部中间产物，烟腈通过化学反应生成烟酸需要高温高压、强酸强碱的条件，对设备具有一定的要求而且也会腐蚀设备，且对环境也不友好。清华紫光公司的专利CN114288A报道了用3-甲基吡啶一步催化生成烟酸，其过程为将3-甲基吡啶与硫酸反应生成甲基吡啶硫酸盐，然后在氧化剂硝酸的作用下氧化生成烟酸硫酸盐，烟酸硫酸盐加入碱溶液中可制得烟酸，原料收率为90％左右。此过程中需要硫酸、硝酸、碱溶液等强酸强碱物质，污染严重。
在烟酸化学合成过程中，必须具备特定的高温高压环境或者采用强酸、强碱或化学催化剂处理，反应选择性不高，副产物多，产品的收率不高，环境污染大。相对而言，生物法制备烟酸具有底物选择性高、催化效率高、反应条件温和、环境污染小等特点。此外，生物法制备易放大，成本低，适合于大规模工业化生产应用。目前有报道用微生物枯草芽孢杆菌、玫瑰色红球菌、诺卡氏菌、茄病镰刀菌以及恶臭假单胞菌发酵来生物催化3-氰基吡啶制备烟酸。但是，生物催化法制备烟酸主要是依赖于微生物发酵产腈水解酶，通过此酶来催化3-氰基吡啶转化为烟酸，仍然需要添加原料3-氰基吡啶。
发明内容
本发明的目的是为了克服上述现有烟酸制备技术的不足，提供一种nudC基因的新应用，利用该基因构建的工程菌能够直接生产烟酸。
本发明的第一个目的是提供一种nudC基因。
本发明的第二个目的是提供一种nudC蛋白。
本发明的第三个目的是提供一种含有nudC基因的重组载体。
本发明的第四个目的是提供一种含有nudC基因的重组工程菌
本发明的第五个目的是提供所述的nudC基因在烟酸制备中的应用。
本发明的第六个目的是提供所述的nudC蛋白在烟酸制备中的应用。
本发明的第七个目的是提供所述的重组载体在烟酸的制备中的应用。
本发明的第八个目的是提供所述的重组工程菌在制备烟酸中的应用。
本发明的第八个目的是提供一种生产烟酸的方法。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
本发明发现过表达nudC基因的菌株的培养基滤液中可以分离检测到烟酸，说明nudC基因可以用于烟酸的制备。因此如下方案均应在本发明的保护范围之内：
一种nudC基因，其核苷酸序列如SEQ.ID.NO.1所示。
一种nudC蛋白，其氨基酸序列如SEQ.ID.NO.2所示。
一种重组载体，载体中连接有所述的nudC基因。
优选地，所述载体为pMV361或pMV261。
一种重组工程菌，是将SEQ.ID.NO.1所示序列重组入受体菌所得，也属于本发明的保护范围。
所述受体菌为耻垢分枝杆菌，将SEQ.ID.NO.1所示序列重组进去后能够正常表达即可。
所述的nudC基因在制备烟酸中的应用，也属于本发明的保护范围。
所述的nudC蛋白在制备烟酸中的应用，也属于本发明的保护范围。
所述的重组载体在制备烟酸中的应用，也属于本发明的保护范围。
所述重组工程菌在制备烟酸中的应用，也属于本发明的保护范围。
一种生产烟酸的方法，是将上述重组工程菌进行培养，从培养液中收集纯化烟酸。
与现有技术相比，本发明具有如下有益效果：
本发明发现了一种nudC基因的新应用，利用该基因构建的重组质粒及工程菌可以一步直接获得烟酸，而不依赖于添加3-氰基吡啶，不仅避免了现有烟酸化学合成过程中的各种缺点，而且生产条件简单，无污染，简便易行，易放大，成本低，适合于大规模工业化生产应用。
附图说明
图1为pMV361-NudCms质粒经EcoRⅠ和HindⅢ限制性内切酶酶切后核酸凝胶电泳，M：DNA marker；1～3：阳性质粒。
图 2为pMV361-NudCms质粒示意图
图3为PCR扩增产物核酸凝胶电泳；M：DNA marker；1～3：阳性克隆子。
图4为对照菌株和过表达菌株培养液滤液及烟酸标准品经HPLC分离色谱图；A：对照菌株耻垢分枝杆菌mc2155培养液滤液；B：过表达NudC基因的耻垢分枝杆菌mc2155-pMV261-NudCms培养液滤液；C：烟酸标准品。
图5为pMV261-NudCms质粒经BamHⅠ和HindⅢ限制性内切酶酶切后核酸凝胶电泳，M：DNA marker；1-3：阳性质粒。
图6为pMV261-NudCms质粒示意图。
图7为PCR扩增产物核酸凝胶电泳；M：DNA marker；1～3：阳性克隆子。
图8为对照菌株和过表达菌株培养液滤液及烟酸标准品经HPLC分离色谱图；A为对照菌株耻垢分枝杆菌mc2155培养液滤液；B：过表达NudC基因的耻垢分枝杆菌mc2155-pMV261-NudCms培养液滤液；C：烟酸标准品。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1 nudC基因的扩增及构建质粒pMV361-NudCms
1、nudC基因的扩增
以耻垢分枝杆菌mc2155基因组DNA为模板，使用引物对5'-ATCGGAATTCATGAGCGAACACCGCACGT-3'/5'-TGCAAAGCTTTCAGTCGAGTGCGGCCCAGG-3'，PCR扩增nudC基因ORF序列。PCR产物经核酸凝胶电泳分离目的DNA片段，使用Omega公司胶回试剂盒回收目的DNA片段，经过测序后得到nudC基因ORF序列如SEQ ID NO：1所示，其编码的蛋白质的氨基酸序列如SEQ ID NO：2。回收后的DNA均使用EcoRⅠ和HindⅢ限制性内切酶酶切并通过Omega公司Cycle-pure回收试剂盒回收酶切后的DNA备用。
2、酶切
LB培养含pMV361质粒的大肠杆菌DH5α菌株，使用Omega公司质粒抽提试剂盒抽提质粒，获得的pMV361质粒使用EcoRⅠ和HindⅢ限制性内切酶酶切后进行核酸凝胶电泳并使用Omega公司胶回收试剂盒回收线性化的质粒。
3、连接
酶切回收的DNA片段和经同样酶切回收的线性化质粒pMV361经T4 DNA连接酶16℃反应过夜，连接产物转化大肠杆菌DH5α感受态细胞并涂布LB固体平板（含100μg/ml硫酸卡那霉素），平板放置37℃恒温培养箱培养过夜。
4、阳性质粒的筛选
挑取平板上长出的单克隆菌落接种LB液体培养基，37℃摇床200rpm震荡过夜培养。然后使用Omega公司质粒抽提试剂盒抽提质粒，获得的质粒使用EcoRⅠ和HindⅢ限制性内切酶酶切后经核酸凝胶电泳检测验证，nudC基因ORF序列连接到pMV361载体上（如图1）。同时验证正确的质粒pMV361-NudCms经擎科公司测序验证序列无突变。构建成功的pMV361-NudCms质粒图谱示意图如图2。
实施例2 构建NudC过表达耻垢分枝杆菌菌株
1、制备耻垢分枝杆菌mc2155感受态细胞
制备耻垢分枝杆菌mc2155感受态细胞，挑取新鲜的耻垢分枝杆菌mc2155单菌落接种于5 ml 7H9液体培养基中，37℃ 200rpm振荡培养至对数生长期(OD0.5-1.0)；培养物以1:100的比例接种于新鲜的100 ml 7H9液体培养基中，37℃过夜培养至OD600到0.6左右，于4℃，5000 rpm离心10 min收集菌体，将菌体用预冷的10%无菌甘油至少洗涤两次，最后加入10 ml（适量）预冷的10%的甘油，吹匀菌体后，分装置-80℃冻存备用。
2、转化耻垢分枝杆菌mc2155
取构建正确的阳性质粒pMV361-NudCms的DNA加入200 µl的耻垢分枝杆菌mc2155电转感受态细胞中，冰上孵育10 min，然后转入2 mm BTX电转杯中，擦净电转杯外壁上的水，然后使用BTX ECM630电转化仪电击，电击参数是：电压2.5 kV, 电阻1000 Ω，电容25 µF。电击完后，立即加入1 ml 7H9液体培养基，37℃培养箱孵育过夜后取适量培养液涂布7H10固体平板（含50μg/ml硫酸卡那霉素），平板置37℃培养箱培养3～5天。
3、筛选阳性重组菌
挑取平板上长出来的单克隆子，接种到5mL 7H9液体培养基中培养2-3天至对数生长期，离心收集菌体并使用微生物基因组提取试剂盒（Omega）提取基因组DNA，使用引物对5'-GTGGCAGCGAGGACAACTTG-3'，5'-GATGCCTGGCAGTCGATCGTAC-3'，以提取的基因组DNA为模板，PCR验证pMV361-NudCms质粒转入耻垢分枝杆菌中（PCR结果如图3）。将验证正确的NudCms过表达的菌株命名为耻垢分枝杆菌mc2155-pMV361-NudCms。
实施例3耻垢分枝杆菌mc2155-pMV361-NudCms分泌烟酸
1、耻垢分枝杆菌mc2155-pMV361-NudCms接种7H9液体培养基，37℃培养至对数生长期后，使用Millipore 0.22μm无菌滤器过滤培养液并收集过滤的滤液，然后使用Millipore3-kDa超滤浓缩管去除滤液中的蛋白质等大分子并收集过滤后滤液。获取的滤液使用高效液相色谱仪（HPLC）分离并分析烟酸。
HPLC 具体的条件：
设备型号：Thermo Fisher Scientific UltiMate 3000 ultrahigh-performanceliquid chromatography
色谱柱：Thermo Fisher，Hypersil Gold aQ，150×4.6 mm，3 μm，柱温：30℃；
流动相：水相：去离子水（含0.1%甲酸）；
有机相：甲醇；
梯度洗脱：0 min 时，95%水相+5%有机相；
30 min 时，5%水相+95%有机相；
流速：0.3 mL/min；
进样量：10 μL；
波长：284 nm。
2、HPLC结果显示耻垢分枝杆菌mc2155-pMV361-NudCms培养液中含有烟酸（如图4）。
实施例4 构建质粒pMV261-NudCms
按照实施例1的方法进行质粒pMV261-NudCms的构建。其中，将载体更换为pMV261质粒。获得的质粒使用BamHⅠ和HindⅢ限制性内切酶酶切后经核酸凝胶电泳检测验证，nudC基因ORF序列连接到pMV261载体上（如图5）。同时验证正确的质粒pMV261-NudCms经擎科公司测序验证序列无突变，构建成功的pMV261-NudCms质粒图谱如图6。
实施例5 构建NudC过表达耻垢分枝杆菌菌株
按照实施例2的方法构建NudC过表达耻垢分枝杆菌菌株，PCR验证pMV261-NudCms质粒转入耻垢分枝杆菌中（PCR结果如图7）。将验证正确的NudCms过表达的菌株命名为耻垢分枝杆菌mc2155-pMV261-NudCms。
实施例6重组耻垢分枝杆菌mc2155-pMV261-NudCms分泌烟酸
耻垢分枝杆菌mc2155-pMV261-NudCms接种7H9液体培养基，按照实施例3的方法培养重组耻垢分枝杆菌并，利用HPLC分离并分析烟酸。HPLC结果显示耻垢分枝杆菌mc2155-pMV261-NudCms培养液中含有烟酸（如图8）。
序列表
<110> 上海晶诺生物科技有限公司
<120> 一种nudC基因及其在制备烟酸方面的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 936
<212> DNA
<213> 耻垢分枝杆菌(Mycobacterium smegmatis)
<400> 1
atgagcgaac accgcacgtt cgggctccgt aacgtcccgc tgctgtcccg ggtcggcgcc 60
gatcgcgccg ataccttgcg caccgacgtc gacgccgccc tggcgggctg gcccgacgcg 120
ctggtgctac gcgtggaccg ccgcaaccag gtgctcatcg ccaacggtca ggtggtgctc 180
ggtgaggccg gcgcactcgg agaccggccg cccgagcacg cggtgttcct gggacgtctg 240
caggacggca ggcacgtatg gggtatccgg gcggatctgg aggcgcccga ggatgccgac 300
ctggggaccg aggtgctcga cctgcgccgg gccgggcaga tcttcgacga caccagcgcc 360
cagttggtgg cgaccgccac ggcgctgctc aactggcatg acaacgcgcg gcacagcgcg 420
atcgacgggg cgccgacccg gcccgccaag ggcggctggt cgcgcgtcaa cccgctgacc 480
ggccacgagg agttcccgcg gatcgacccc gccatcatct gcctggtgca cgacgggcat 540
gaccgggcgg tgctggcccg tcagacgctg tggccggagc ggttgttctc gatcctggcc 600
ggcttcgtcg aggccggcga gtcgttcgag acatgcgtgc agcgcgagat cgccgaggag 660
atcgggctca cggtcaccga cgtgcagtac ctcggcagtc agccgtggcc gttcccgcgc 720
tcgctcatgg tgggattcca cgcgatcggc gacccggagc agccgttctc ctacaacgac 780
ggcgagatcg ccgaggccgc gtggttcacc cgcgatgaga tccgcgcggc actcgatcag 840
ggggactgga acagcgactc gccgtcacgg ctcctgctgc caggctccat ctcgatcgcc 900
cgcgagatca tcgaatcctg ggccgcactc gactga 936
<210> 2
<211> 311
<212> PRT
<213> 耻垢分枝杆菌(Mycobacterium smegmatis)
<400> 2
Met Ser Glu His Arg Thr Phe Gly Leu Arg Asn Val Pro Leu Leu Ser
1 5 10 15
Arg Val Gly Ala Asp Arg Ala Asp Thr Leu Arg Thr Asp Val Asp Ala
20 25 30
Ala Leu Ala Gly Trp Pro Asp Ala Leu Val Leu Arg Val Asp Arg Arg
35 40 45
Asn Gln Val Leu Ile Ala Asn Gly Gln Val Val Leu Gly Glu Ala Gly
50 55 60
Ala Leu Gly Asp Arg Pro Pro Glu His Ala Val Phe Leu Gly Arg Leu
65 70 75 80
Gln Asp Gly Arg His Val Trp Gly Ile Arg Ala Asp Leu Glu Ala Pro
85 90 95
Glu Asp Ala Asp Leu Gly Thr Glu Val Leu Asp Leu Arg Arg Ala Gly
100 105 110
Gln Ile Phe Asp Asp Thr Ser Ala Gln Leu Val Ala Thr Ala Thr Ala
115 120 125
Leu Leu Asn Trp His Asp Asn Ala Arg His Ser Ala Ile Asp Gly Ala
130 135 140
Pro Thr Arg Pro Ala Lys Gly Gly Trp Ser Arg Val Asn Pro Leu Thr
145 150 155 160
Gly His Glu Glu Phe Pro Arg Ile Asp Pro Ala Ile Ile Cys Leu Val
165 170 175
His Asp Gly His Asp Arg Ala Val Leu Ala Arg Gln Thr Leu Trp Pro
180 185 190
Glu Arg Leu Phe Ser Ile Leu Ala Gly Phe Val Glu Ala Gly Glu Ser
195 200 205
Phe Glu Thr Cys Val Gln Arg Glu Ile Ala Glu Glu Ile Gly Leu Thr
210 215 220
Val Thr Asp Val Gln Tyr Leu Gly Ser Gln Pro Trp Pro Phe Pro Arg
225 230 235 240
Ser Leu Met Val Gly Phe His Ala Ile Gly Asp Pro Glu Gln Pro Phe
245 250 255
Ser Tyr Asn Asp Gly Glu Ile Ala Glu Ala Ala Trp Phe Thr Arg Asp
260 265 270
Glu Ile Arg Ala Ala Leu Asp Gln Gly Asp Trp Asn Ser Asp Ser Pro
275 280 285
Ser Arg Leu Leu Leu Pro Gly Ser Ile Ser Ile Ala Arg Glu Ile Ile
290 295 300
Glu Ser Trp Ala Ala Leu Asp
305 310
Claims (6)
1.nudC基因在耻垢分支杆菌中制备烟酸的应用，其特征在于，其核苷酸序列如SEQ IDNO：1所示。
2.nudC蛋白在耻垢分支杆菌中制备烟酸的应用，其特征在于，其氨基酸序列如SEQ IDNO：2所示。
3.一种重组载体在耻垢分支杆菌中制备烟酸的应用，其特征在于，所述载体中连接有nudC基因，所述nudC基因的核苷酸序列如SEQ ID NO：1所示。
4.根据权利要求3所述的应用，其特征在于，所述载体为pMV361或pMV261。
5.一种重组工程菌在制备烟酸中的应用，其特征在于，所述重组工程菌为将SEQ IDNO：1所示的nudC基因序列重组入耻垢分支杆菌所得。
6.一种生产烟酸的方法，其特征在于，将权利要求5中所述重组工程菌进行培养，从培养液中收集纯化烟酸。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810023635.1A CN108359666B (zh) | 2018-01-10 | 2018-01-10 | 一种nudC基因及其在制备烟酸方面的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810023635.1A CN108359666B (zh) | 2018-01-10 | 2018-01-10 | 一种nudC基因及其在制备烟酸方面的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108359666A CN108359666A (zh) | 2018-08-03 |
| CN108359666B true CN108359666B (zh) | 2021-04-20 |
Family
ID=63011252
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810023635.1A Active CN108359666B (zh) | 2018-01-10 | 2018-01-10 | 一种nudC基因及其在制备烟酸方面的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108359666B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551748B (zh) * | 2019-08-27 | 2023-04-25 | 佛山科学技术学院 | 一种产烟酸的超级重组耻垢分枝杆菌及其构建方法 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009045116A1 (en) * | 2007-10-05 | 2009-04-09 | University Of Otago | Detection of volatile compounds as markers for mycobacteria tuberculosis |
-
2018
- 2018-01-10 CN CN201810023635.1A patent/CN108359666B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009045116A1 (en) * | 2007-10-05 | 2009-04-09 | University Of Otago | Detection of volatile compounds as markers for mycobacteria tuberculosis |
Non-Patent Citations (3)
| Title |
|---|
| Comparative analysis of mycobacterial NADH pyrophosphatase isoforms reveals a novel mechanism for isoniazid and ethionamide inactivation;Xu-De Wang等;《Molecular Microbiology》;20111103;第82卷(第6期);第1375-1391页 * |
| Mycobacterium smegmatis strain INHR1, complete genome，CP009495.1;Mohan,A.等;《NCBI GenBank》;20150226;第1-2页 * |
| 异烟肼与乙硫异烟胺在分枝杆菌中的代谢新途径;王绪德等;《2012年鄂粤微生物学学术年会——湖北省暨武汉微生物学会成立六十年庆祝大会论文集湖北省科学技术协会会议论文集》;20121008;第1页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108359666A (zh) | 2018-08-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114107160B (zh) | 一种烟酰胺核糖激酶基因工程菌及其应用 | |
| CN112143764B (zh) | 一种生物酶催化制备布瓦西坦中间体化合物的方法 | |
| CN107254429A (zh) | 一种高产重组腈水解酶的枯草芽孢杆菌及其应用方法 | |
| CN118726326A (zh) | 一种酚酸脱羧酶突变体及其制备和应用 | |
| CN114934062B (zh) | 一种高效表达d-阿洛酮糖3-差向异构酶的工程菌及应用 | |
| CN113355366A (zh) | 一种多酶级联制备2-苯乙醇的方法 | |
| CN108359666B (zh) | 一种nudC基因及其在制备烟酸方面的应用 | |
| CN108384739B (zh) | 一种产烟酸的整合型重组耻垢分枝杆菌及其构建方法 | |
| CN108330094B (zh) | 一种产烟酸的游离型重组耻垢分枝杆菌及其构建方法 | |
| CN109371070A (zh) | 一种高产α-酮异戊酸的方法 | |
| CN119932003B (zh) | 一种塔格糖-4-差向异构酶突变体及其在制备d-塔格糖中的应用 | |
| CN114891707A (zh) | 重组菌株及其全细胞催化生产胆红素的方法 | |
| CN118931901B (zh) | 基因的启动子及应用 | |
| CN108424871B (zh) | 一种分泌烟酸的突变耻垢分枝杆菌及其构建方法 | |
| CN118056902B (zh) | 合成HMOs的成熟多肽序列及应用 | |
| CN115975999B (zh) | 一种腈水解酶及其在2-羟基-4-甲硫基丁酸合成中的应用 | |
| CN114940964B (zh) | 工程菌及其高效催化cdca生产udca的方法 | |
| CN101892228B (zh) | 一种高丙烯酰胺和丙烯腈耐受性产腈水合酶工程菌及应用 | |
| CN116904380A (zh) | 一种生产麦角硫因基因工程菌的构建及应用 | |
| CN110551748B (zh) | 一种产烟酸的超级重组耻垢分枝杆菌及其构建方法 | |
| CN118056901B (zh) | 合成寡糖的成熟多肽序列及应用 | |
| CN118056903B (zh) | 一种合成寡糖的成熟多肽 | |
| CN119876103B (zh) | 一种腈水解酶及其在蛋氨酸合成中的用途 | |
| CN120118870A (zh) | 一种基因工程菌及其用于合成(r)-2-溴-2-氯苯乙醇的方法和相关酶 | |
| CN114854717A (zh) | 一种脂肪酶及其编码基因与应用 |
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 |