CN114774336A - 一种产l-赖氨酸的枯草芽孢杆菌重组菌的构建及其应用 - Google Patents
一种产l-赖氨酸的枯草芽孢杆菌重组菌的构建及其应用 Download PDFInfo
- Publication number
- CN114774336A CN114774336A CN202210183889.6A CN202210183889A CN114774336A CN 114774336 A CN114774336 A CN 114774336A CN 202210183889 A CN202210183889 A CN 202210183889A CN 114774336 A CN114774336 A CN 114774336A
- Authority
- CN
- China
- Prior art keywords
- subtilis
- gene
- recombinant
- lysine
- hom
- 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.)
- Granted
Links
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 title claims abstract description 135
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 119
- 239000004472 Lysine Substances 0.000 title claims abstract description 68
- 235000019766 L-Lysine Nutrition 0.000 title claims abstract description 67
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 23
- 241000894006 Bacteria Species 0.000 title claims abstract description 18
- 238000010276 construction Methods 0.000 title claims abstract description 10
- 241000186226 Corynebacterium glutamicum Species 0.000 claims abstract description 31
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 23
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 23
- 101150035025 lysC gene Proteins 0.000 claims abstract description 19
- 230000037361 pathway Effects 0.000 claims abstract description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 8
- 239000006041 probiotic Substances 0.000 claims abstract description 8
- 235000018291 probiotics Nutrition 0.000 claims abstract description 8
- 230000000529 probiotic effect Effects 0.000 claims abstract description 7
- 101100115938 Lysinibacillus sphaericus dapdh gene Proteins 0.000 claims abstract description 5
- GMKMEZVLHJARHF-UHFFFAOYSA-N (2R,6R)-form-2.6-Diaminoheptanedioic acid Natural products OC(=O)C(N)CCCC(N)C(O)=O GMKMEZVLHJARHF-UHFFFAOYSA-N 0.000 claims abstract description 3
- 230000008859 change Effects 0.000 claims abstract description 3
- 238000009826 distribution Methods 0.000 claims abstract description 3
- GMKMEZVLHJARHF-SYDPRGILSA-N meso-2,6-diaminopimelic acid Chemical compound [O-]C(=O)[C@@H]([NH3+])CCC[C@@H]([NH3+])C([O-])=O GMKMEZVLHJARHF-SYDPRGILSA-N 0.000 claims abstract description 3
- 239000013612 plasmid Substances 0.000 claims description 33
- 238000000855 fermentation Methods 0.000 claims description 21
- 230000004151 fermentation Effects 0.000 claims description 21
- 102000004190 Enzymes Human genes 0.000 claims description 19
- 108090000790 Enzymes Proteins 0.000 claims description 19
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 238000004519 manufacturing process Methods 0.000 claims description 16
- 101150057904 ddh gene Proteins 0.000 claims description 13
- 235000001014 amino acid Nutrition 0.000 claims description 11
- 229940024606 amino acid Drugs 0.000 claims description 11
- 150000001413 amino acids Chemical class 0.000 claims description 11
- 101150109073 ldhD gene Proteins 0.000 claims description 11
- 238000001976 enzyme digestion Methods 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 108010064711 Homoserine dehydrogenase Proteins 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 8
- 238000011218 seed culture Methods 0.000 claims description 8
- 101100029904 Bacillus subtilis (strain 168) pksD gene Proteins 0.000 claims description 7
- 239000012634 fragment Substances 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- 108010029731 6-phosphogluconolactonase Proteins 0.000 claims description 6
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 claims description 6
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 230000004927 fusion Effects 0.000 claims description 6
- 108020001657 6-phosphogluconate dehydrogenase Proteins 0.000 claims description 5
- 108010055400 Aspartate kinase Proteins 0.000 claims description 5
- 108091033409 CRISPR Proteins 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 claims description 4
- 101150054365 dapdh gene Proteins 0.000 claims description 4
- 108010056578 diaminopimelate dehydrogenase Proteins 0.000 claims description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims description 4
- 235000016068 Berberis vulgaris Nutrition 0.000 claims description 3
- 241000335053 Beta vulgaris Species 0.000 claims description 3
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 241000193996 Streptococcus pyogenes Species 0.000 claims description 3
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 3
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 3
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- 229960003237 betaine Drugs 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 239000011616 biotin Substances 0.000 claims description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 229910052564 epsomite Inorganic materials 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 3
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 229960003966 nicotinamide Drugs 0.000 claims description 3
- 235000005152 nicotinamide Nutrition 0.000 claims description 3
- 239000011570 nicotinamide Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 235000019157 thiamine Nutrition 0.000 claims description 3
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 claims description 3
- 229960003495 thiamine Drugs 0.000 claims description 3
- 239000011721 thiamine Substances 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 2
- 229960000723 ampicillin Drugs 0.000 claims description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 2
- 235000013379 molasses Nutrition 0.000 claims description 2
- 102100031126 6-phosphogluconolactonase Human genes 0.000 claims 1
- 108010020020 phosphogluconate dehydrogenase (decarboxylating) Proteins 0.000 claims 1
- 241000831652 Salinivibrio sharmensis Species 0.000 abstract description 17
- 239000006227 byproduct Substances 0.000 abstract description 7
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 abstract description 6
- 238000012269 metabolic engineering Methods 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 241001052560 Thallis Species 0.000 abstract description 2
- 230000009466 transformation Effects 0.000 abstract description 2
- 241000385736 bacterium B Species 0.000 abstract 1
- 230000002349 favourable effect Effects 0.000 abstract 1
- 230000009897 systematic effect Effects 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 18
- 102100035172 Glucose-6-phosphate 1-dehydrogenase Human genes 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- 230000003834 intracellular effect Effects 0.000 description 14
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 13
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 230000009123 feedback regulation Effects 0.000 description 7
- 238000012795 verification Methods 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 6
- 238000010200 validation analysis Methods 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 102000004567 6-phosphogluconate dehydrogenase Human genes 0.000 description 3
- 101710088194 Dehydrogenase Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 230000037353 metabolic pathway Effects 0.000 description 3
- 230000004108 pentose phosphate pathway Effects 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 101100174653 Dictyostelium discoideum g6pd-2 gene Proteins 0.000 description 2
- 101100387232 Escherichia coli (strain K12) asd gene Proteins 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- 101150085516 ZWF1 gene Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 102000038379 digestive enzymes Human genes 0.000 description 2
- 108091007734 digestive enzymes Proteins 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000012262 fermentative production Methods 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- 101150014950 gnd gene Proteins 0.000 description 2
- 101150063051 hom gene Proteins 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 101150078419 zwf gene Proteins 0.000 description 2
- 101150026856 zwf2 gene Proteins 0.000 description 2
- 101150028074 2 gene Proteins 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 101150090724 3 gene Proteins 0.000 description 1
- 101150033839 4 gene Proteins 0.000 description 1
- 101150096316 5 gene Proteins 0.000 description 1
- 101150101112 7 gene Proteins 0.000 description 1
- -1 AK III Proteins 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 101100447432 Danio rerio gapdh-2 gene Proteins 0.000 description 1
- 101000779367 Escherichia coli (strain K12) Lysine-sensitive aspartokinase 3 Proteins 0.000 description 1
- 101150112014 Gapdh gene Proteins 0.000 description 1
- 108010026389 Gramicidin Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- 229930182844 L-isoleucine Natural products 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 229930195722 L-methionine Natural products 0.000 description 1
- GHSJKUNUIHUPDF-BYPYZUCNSA-N L-thialysine Chemical compound NCCSC[C@H](N)C(O)=O GHSJKUNUIHUPDF-BYPYZUCNSA-N 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 108010040201 Polymyxins Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- VFRROHXSMXFLSN-KCDKBNATSA-N aldehydo-D-galactose 6-phosphate Chemical compound OP(=O)(O)OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O VFRROHXSMXFLSN-KCDKBNATSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 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 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- RKLXDNHNLPUQRB-TVJUEJKUSA-N chembl564271 Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]2C(C)SC[C@H](N[C@@H](CC(N)=O)C(=O)NC(=O)[C@@H](NC2=O)CSC1C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NC(=C)C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@H]1NC(=O)C(=C\C)/NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]2NC(=O)CNC(=O)[C@@H]3CCCN3C(=O)[C@@H](NC(=O)[C@H]3N[C@@H](CC(C)C)C(=O)NC(=O)C(=C)NC(=O)CC[C@H](NC(=O)[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC=4C5=CC=CC=C5NC=4)CSC3)C(O)=O)C(C)SC2)C(C)C)C(C)SC1)C1=CC=CC=C1 RKLXDNHNLPUQRB-TVJUEJKUSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 229960004905 gramicidin Drugs 0.000 description 1
- ZWCXYZRRTRDGQE-SORVKSEFSA-N gramicidina Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@H](NC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](NC=O)C(C)C)CC(C)C)C(=O)NCCO)=CNC2=C1 ZWCXYZRRTRDGQE-SORVKSEFSA-N 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000007852 inverse PCR Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 235000018977 lysine Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229960004452 methionine Drugs 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229960000988 nystatin Drugs 0.000 description 1
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 235000019629 palatability Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 108010082567 subtilin Proteins 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229960002898 threonine Drugs 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- 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.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
-
- 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
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01003—Homoserine dehydrogenase (1.1.1.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01044—Phosphogluconate dehydrogenase (decarboxylating) (1.1.1.44)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01049—Glucose-6-phosphate dehydrogenase (1.1.1.49)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
- C12Y104/01016—Diaminopimelate dehydrogenase (1.4.1.16)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
- C12Y207/02004—Aspartate kinase (2.7.2.4)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Husbandry (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种产L‑赖氨酸的枯草芽孢杆菌重组菌的构建及其应用,属于基因工程技术领域。本发明应用基因工程方法,对饲料工业上常用的益生菌B.subtilis ACCC11025进行了系统的代谢工程改造。本发明发现,以来源C.glutamicum中lysC311、zwf243和gnd361替换B.subtilis中thrD、zwf和gnd(即:重组菌B.subtilis XH4),有利于L‑赖氨酸的合成,L‑赖氨酸产量达到20.3±1.9g/L。此外,将B.subtilis中hom替换成来源C.glutamicum中hom59(即:重组菌B.subtilis XH5),可实现显著降低副产物产量,L‑赖氨酸产量达到23.2±1.7g/L,且不影响菌体生长。在重组菌B.subtilis XH5中引入C.glutamicum中DapDH会改变二氨基庚二酸途径碳分布而促进L‑赖氨酸的合成,目标重组菌XH6中L‑赖氨酸产量达到25.6±2.3g/L。
Description
技术领域
本发明涉及一种产L-赖氨酸的枯草芽孢杆菌重组菌的构建及其应用,属于基因工程技术领域。
背景技术
L-赖氨酸是动物和人类无法自身合成的氨基酸,属于八大必需氨基酸之一。由于在谷类在加工过程中赖氨酸很容易被破坏,L-赖氨酸得率极低,故L-赖氨酸称为第一限制性氨基酸。因此,L-赖氨酸广泛应用在动物饲料中,具有平衡饲料中氨基酸组成,提高动物贵氨基酸的摄取和代谢,促进包括家畜、家禽和鱼类等在内的生长发育,进而提高饲料蛋白质的利用率,节约生产成本,减少环境污染。全球L-赖氨酸市场目前估计为每年220万吨,并以每年10%左右的速度增长,在世界范围内年产量是继L-谷氨酸后的第二位氨基酸。目前,工业上用于发酵生产L-赖氨酸的菌种主要有大肠杆菌(Escherichia coli)和谷氨酸棒杆菌(Corynebacterium glutamicum)。然而,E.coli和C.glutamicum直接应用于动物饲料中存在一些缺陷,如:存在内毒素、适口性差等。自2013年,国家规定的饲料添加剂目录里已明文规定饲料级L-赖氨酸中不得含有E.coli,而作为市场上饲用级L-赖氨酸产品规格主要是纯度为65%和70%。因此,我们急需开发食品安全性的微生物底盘细胞用于发酵生产饲用L-赖氨酸。
益生菌因其绿色、无残留、无污染的特性成为替代抗生素的最佳选择之一,受到国内外学者和饲料生产企业的广泛关注。枯草芽孢杆菌(Bacillus subtilis)是芽孢杆菌属的一种,已被美国食品和药品管理局(FDA)、美国饲料监察协会(AAFCO)和我国农业部认定为饲料安全性菌株,广泛用于饲料中。B.subtilis产生的枯草菌素、多粘菌素、制霉菌素、短杆菌肽等活性物质对致病菌或内源性感染的条件致病菌有明显抑制作用。同时,B.subtilis能合成消化性酶类,如蛋白酶、淀粉酶、脂肪酶、纤维素酶、果胶酶等,可降解饲料中复杂碳水化合物,提高饲料消化率,提高动物生产性能。此外,B.subtilis能刺激动物免疫器官的生长发育,激活T和B淋巴细胞,提高免疫球蛋白和抗体水平,增强细胞免疫和体液免疫功能,提高群体免疫力。有研究表明,B.subtilis可使生猪日增重提高6%~7%,饲料转化率提高3%~4%,也能提高肉鸡的抗氧化能力,从而提高肉鸡生长性能。此外,为了有效提高饲料中营养物质的利用率,通过紫外诱变或其他代谢改造B.subtilis选育高产消化性酶类的突变菌株已成为B.subtilis发酵饲料发展趋势。然而,虽然B.subtilis作为益生菌广泛用于饲料中,但是该菌株不能有效积累L-赖氨酸。因此,饲料生产工业上为了同时满足家畜或家禽对L-赖氨酸和B.subtilis的需求,需要同时添加上述两种物质,从而将饲料生产工艺复杂化。此外,L-赖氨酸和B.subtilis的添加在一定程度上增加了企业生产成本。因此,实现B.subtilis高效合成L-赖氨酸有利于简化饲料生产工艺,降低生产成本,提高企业行业竞争力。
随着B.subtilis的基因组注释已经解析,其发酵生产L-赖氨酸的生物合成途径和调节机制也已比较清晰。由图1可知,以葡萄糖为原料时,B.subtilis中有5个途径参与L-赖氨酸合成:糖酵解途径(天蓝色线条)、磷酸戊糖途径(蓝色线条)、三羧酸(TCA)循环(黑色线条)、CO2固定反应(紫色线条)和L-赖氨酸终端合成途径(绿色线条)。现阶段,针对E.coli和C.glutamicum的代谢工程改造合成L-赖氨酸已有非常多的报道,主要集中在以下几个方面:①解除合成途径中反馈调节,提高L-赖氨酸合成途径的效率;②阻断副产物的支路代谢途径,促进多代谢流进入L-赖氨酸合成途径;③强化合成途径关键酶表达水平,提高L-赖氨酸前体物供应;④拓宽菌株代谢底物谱,降低L-赖氨酸生产成本;⑤提高胞内NADPH有效供应水平,促进L-赖氨酸高效率合成。然而,针对B.subtilis的代谢工程改造合成L-赖氨酸却鲜有报道。
发明内容
为解决上述问题,本发明以饲料工业上常用的益生菌B.subtilis ACCC11025为出发菌株,采用CRISPR-Cas9基因编辑技术对磷酸戊糖途径、L-赖氨酸终端合成途径和L-赖氨酸竞争支路代谢途径进行重构,获得一株具有益生功能和L-赖氨酸高效合成能力的“双功能”枯草芽孢杆菌重组菌株。本发明结果为利用B.subtilis生产L-赖氨酸等饲用氨基酸提供参考,为实现简化饲料生产工艺提供了一定的理论基础。
本发明的第一个目的是提供一种代谢工程改造促进L-赖氨酸的合成的枯草芽孢杆菌重组菌,所述的重组菌以枯草芽孢杆菌ACCC11025为宿主,将B.subtilis中thrD、zwf和gnd用来源C.glutamicum中lysC311、zwf243和gnd361替换,且B.subtilis中hom替换成来源C.glutamicum中hom59,最后再引入C.glutamicum中DapDH改变二氨基庚二酸途径碳分布而促进L-赖氨酸的合成。
进一步地,所述的枯草芽孢杆菌包括B.subtilis XH0、B.subtilis XH1、B.subtilis XH2、B.subtilis XH3、B.subtilis XH4、B.subtilis XH5或B.subtilis XH6。
其中,B.subtilis XH0、B.subtilis XH1、B.subtilis XH2、B.subtilis XH3、B.subtilis XH4、B.subtilis XH5和B.subtilis XH6保藏于江苏星海生物科技有限公司。
进一步地,所述的编码天冬氨酸激酶的基因lysC311来源于C.glutamicum,核苷酸序列如SEQ ID NO.1所示。
进一步地,所述的编码葡萄糖-6-磷酸脱氢酶的基因zwf243来源于C.glutamicum,核苷酸序列如SEQ ID NO.2所示。
进一步地,所述的编码6-磷酸葡萄糖酸脱氢酶的基因gnd361来源于C.glutamicum。核苷酸序列如SEQ ID NO.3所示。
进一步地,所述的编码高丝氨酸脱氢酶的基因hom59来源于C.glutamicum,核苷酸序列如SEQ ID NO.4所示。
进一步地,所述的编码二氨基庚二酸脱氢酶的基因ddh来源于C.glutamicum,核苷酸序列如SEQ ID NO.5所示。
本发明的第二个目的是提供所述的重组菌的构建方法,包括如下步骤:
(1)天冬氨酸激酶基因lysC、葡萄糖-6-磷酸脱氢酶基因zwf、6-磷酸葡萄糖酸脱氢酶基因gnd和高丝氨酸脱氢酶基因hom的替换,以及异源表达DapDH基因ddh:以枯草芽孢杆菌ACCC11025为宿主,采用CRISPR-Cas9系统将基因thrD、zwf、gnd和hom替换为来源于C.glutamicum的lysC311、zwf243、gnd361和hom59,再异源引入C.glutamicum的DapDH基因ddh。
(2)重组质粒构建:以酿脓链球菌的基因组作为模板,将基因Spcas9进行PCR扩增,随后利用限制性内切酶对质粒pHT01和片段Spcas9进行双酶切并酶连,获得目标重组质粒pHT01-Cas9。再将B.subtilis的thrD、zwf、gnd、hom和pksD基因的sgRNA无缝连接到质粒pBE980b,获得五个带有靶向位点的质粒。再将C.glutamicum的lysC311基因、zwf243基因、gnd361基因、hom59基因和ddh基因与B.subtilis的thrD、zwf、gnd、hom和pksD基因的上下同源臂通过融合PCR进行基因融合,再依次酶切连接至质粒pBE980b后得到重组质粒;
(3)重组菌构建:将重组质粒pHT01-Cas9电转至B.subtilis ACCC11025感受态细胞中,通过氨苄抗性筛选得到带有Cas9蛋白的转化子。再将其余重组质粒电转至带有Cas9蛋白的B.subtilis ACCC11025感受态细胞中,筛选得到所述的重组菌。
本发明的第三个目的是提供所述的重组菌发酵生产L-赖氨酸的方法,所述方法是将所述重组菌单菌落接种至液体种子培养基,30~38℃,50~200r·min-1培养8-10h;以8~12%接种量将种子培养液转接至发酵培养基,发酵40h。
进一步地,所述的种子培养基为:葡萄糖5g/L,NaCl 5g/L,牛肉蛋白胨10g/L,酵母膏10g/L。
进一步地,所述的发酵培养基为:葡萄糖80g/L、玉米浆35g/L、甜菜糖蜜12g/L、NH4)2SO4 36g/L、MgSO4·7H2O 1.5g/L、K2HPO4 1g/L、KH2PO4 1g/L、FeSO4 0.02g/L、MnSO40.02g/L、甜菜碱0.05d/L、烟酰胺0.008g/L、硫胺素0.00045g/L、生物素0.00085g/L、CaCO340g/L。所有培养基用20%(m/v)NaOH调节pH 7.0-7.2。
本发明的第四个目的是利用B.subtilis生产L-赖氨酸等饲用氨基酸提供参考,为实现简化饲料生产工艺提供了一定的理论基础。
本发明的有益效果:
本发明首次以饲料工业上常用的益生菌B.subtilis ACCC11025为出发菌株,围绕胞内前体物和辅因子NADPH供应、副产物积累和优化终端合成途径对L-赖氨酸合成的影响,采用CRISPR-Cas9基因编辑技术对磷酸戊糖途径、L-赖氨酸终端合成途径和L-赖氨酸竞争支路代谢途径进行重构,获得一株具有益生功能和L-赖氨酸高效合成能力的“双功能”枯草芽孢杆菌重组菌株。实验结果表明,将B.subtilis中内源的参与L-赖氨酸合成的关键性酶替换成来源C.glutamicum中解除反馈调节作用的关键性酶(即:AK III、G6PD和6GPD),可有效的为L-赖氨酸合成提供前体物和辅因子NADPH,保证在B.subtilis中有效积累L-赖氨酸。此外,将B.subtilis中L-赖氨酸合成支路途径中的限速酶HSD替换成来源C.glutamicum中渗漏型HSD,可实现在不影响菌体生长的情况下降到副产物积累,从而保证在B.subtilis中高效清洁发酵生产L-赖氨酸。进一步实验结果表明,在B.subtilis中引入来自C.glutamicum中的脱氢酶途径,可以引导碳流进入脱氢酶途径,从而途径L-赖氨酸的高效合成。经上述对B.subtilis基因组进行一系列的遗传改造,最终获得的重组菌株B.subtilis XH6中L-赖氨酸产量达到25.6±2.3g/L。
附图说明
图1:以葡萄糖为碳源时B.subtilis中L-赖氨酸生物合成途径
缩写说明:椭圆圈里指示的是编码基因,其中红色圈表示替代基因;表示基因替换;基因zwf编码葡萄糖-6-磷酸脱氢酶(即:G6PD),基因gnd编码6-磷酸葡萄糖酸脱氢酶(即:6GPD),基因lysC编码天冬氨酸激酶(即:AKIII),基因hom编码高丝氨酸脱氢酶(即:HSD),基因ddh编码二氨基庚二酸脱氢酶(即:DapDH)。
图2:目的重组质粒和目的重组菌株的验证
图形说明:图2A中,泳道1 pBE980b-gnd361双酶切验证;泳道2 pBE980b-lysC311双酶切验证;泳道3pBE980b-zwf234双酶切验证;泳道4 pBE980b-hom59双酶切验证;泳道5pBE980b-ddh双酶切验证。图2B中,泳道1 B.subtilis ACCC11025对照;泳道2菌株XH3中基因gnd PCR验证;泳道3菌株XH4中基因gnd PCR验证;泳道4菌株XH2中基因zwfPCR验证;泳道5菌株XH6中基因ddh PCR验证;泳道6菌株XH1中基因lysC PCR验证;泳道7菌株XH5中基因hom PCR验证。
图3:基因lysC替换和基因替换后重组菌株生长情况
图形说明:(A)基因序列比对;(B)B.subtilis XH0和B.subtilis XH1在LB(B1)和LB+AEC(B2)中的生长情况。
图4:不同菌株L-赖氨酸和菌体生长发酵情况
图5:不同菌株胞内NADPH水平以及L-赖氨酸和菌体生长发酵情况
图6:菌株XL-3和XL-5菌体生长和L-赖氨酸合成情况
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
以下实施例中以枯草芽孢杆菌B.subtilis XH0为例。
底物与产物的定性与定量分析以及菌体生长情况的监测:发酵液于4℃和12000r/min离心5min后取上清,稀释100倍后,通过生物传感分析仪SBA-40C分析发酵液中葡萄糖和L-赖氨酸含量。酶活测定:采用超声波细胞破碎法处理细胞,离心取上清液获得粗酶液,该粗酶液保存于-20℃备用或立即用于酶活性的测定。HSD的测定采用吸光光度法进行。菌液浓度测定:定时取200μL发酵液,并用0.25mol/L的稀盐酸溶液稀释到5mL后,用紫外分光光度计于562nm下测定吸光度(即:OD562)。
表1:实施例中所用到的引物(下划线为酶切位点)
实施例1:质粒pHT01-Cas9、pBE980b-hom59、pBE980b-lysC311、pBE980b-zwf234、pBE980b-gnd361和pBE980b-ddh的构建
以酿脓链球菌的基因组作为模板,使用引物Spcas9-F和Spcas9-R将基因Spcas9进行PCR扩增,随后利用限制性内切酶XbaI和XamI对质粒pHT01和片段Spcas9进行双酶切并酶连,获得目标重组质粒pHT01-Cas9。
pBE980b-hom59、pBE980b-lysC311、pBE980b-zwf234、pBE980b-gnd361和pBE980b-ddh的构建以质粒pBE980b作为模板,利用引物hom-F、hom-R、lysC-F、lysC-R、zwf-F、zwf-R、gnd-F、gnd-R、pksD-F和pksD-R通过反向PCR扩增技术将20bp的sgRNA无缝连接到质粒pBE980b,获得五个带有靶向位点的质粒。
以B.subtilis ACCC11025基因组为模板,利用引物hom-L-F、hom-L-R、hom-R-F、hom-R-R、lysC-L-F、lysC-L-R、lysC-R-F、lysC-R-R、zwf-L-F、zwf-L-R、zwf-R-F、zwf-R-R、gnd-L-F、gnd-L-R、gnd-R-F、gnd-R-R、pksD-L-F、pksD-L-R、pksD-R-F和pksD-R-R分别对基因hom、lysC、zwf、gnd以及pksD的上下同源臂进行扩增,同时以C.glutamicum为基因组,利用引物Cghom-F、Cghom-R、CglysC-F、CglysC-R、Cgzwf-F、Cgzwf-R、Cggnd-F、Cggnd-R、Cgddh-F和Cgddh-R分别扩增基因hom、lysC、zwf、gnd以及ddh。将回收后的同源臂片段以及基因片段同时作为模板,通过融合PCR进行基因融合,利用限制性核酸内切酶对融合片段以及带有靶向位点的质粒pBE980b进行酶切酶连,获得重组质粒pBE980b-hom59、pBE980b-lysC311、pBE980b-zwf234、pBE980b-gnd361和pBE980b-ddh。
实施例2:目的重组菌株的筛选与鉴定
将重组质粒pHT01-Cas9、pBE980b-hom59、pBE980b-lysC311、pBE980b-zwf234、pBE980b-gnd361和pBE980b-ddh进行双酶切或PCR验证,从而确定目的重组质粒。从图2A可知,所选择的质粒都带有目的片段,为目的重组质粒。随后,将质粒pHT01-Cas9电转到B.subtilis ACCC11025,其次依次将目的重组质粒pBE980b-hom59、pBE980b-lysC311、pBE980b-zwf234、pBE980b-gnd361和pBE980b-ddh电转至带有Cas9蛋白的B.subtilisACCC11025感受态细胞中,经过PCR验证,确定目标重菌株菌株(图2B)。
实施例3:重组菌和出发菌株中的生长情况、G6PD和6GPD酶活力、胞内NADH浓度测定
生长情况测定:定时取200μL发酵液,并用0.25mol/L的稀盐酸溶液稀释到5mL后,用紫外分光光度计于562nm下测定吸光度(即:OD562)。
重组菌株B.subtilis XH1(即:XH0 thrD::lysC311)在添加有L-赖氨酸结构类似物S-(2-氨基乙基)-L-半胱氨酸(AEC)的LB固体培养基中正常生长,而出发菌株B.subtilisXH0则不能生长(图3B)。这些结果表明,将B.subtilis中thrD基因替换成lysC311成功实现解除L-赖氨酸对AK III的反馈调节作用。
需要指出的是,与出发菌株B.subtilis XH0相比,重组菌株B.subtilis XH1菌体生长并未受到明显抑制,最终菌体量为出发菌株的97.6%(OD562=38.4±3.5vs.OD562=37.5±4.1;图4B)。这些结果表明,将B.subtilis中AK III替换成来源于C.glutamicum中解除反馈调节的AK III可以实现改造B.subtilis合成L-赖氨酸。
G6PD和6GPD酶活力测定:采用超声波细胞破碎法处理细胞,离心取上清液获得粗酶液,该粗酶液保存于-20℃备用或立即用于酶活性的测定,具体方法参照Xu等人建立的方法进行(Xu JZ,et al.,Amino Acids,2014,46(9),2165-2175)。经测定,与出发菌株相比,重组菌株XH2和XH4中G6PD以及重组菌株XH3和XH4中6GPD都提高了对葡萄糖-6-磷酸(G6P)和NADP+的亲和力(表2)。通过替换来源于C.glutamicum中解除反馈调节作用的G6PD和6GPD可以解除葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶的反馈调节作用,进而影响胞内G6PD和6GPD酶活力。
表2不同重组菌株中G6PD和6GPD的酶学性质
注:a未检测
NADH浓度测定:通过离心(4℃,6,000rpm和10min)收获细胞,并用冰冷的淬火溶液(60%MeOH和70mM HEPES)洗涤3次以去除残留的细胞外代谢物,具体参照Xu等人方法测定(Xu JZ,et al.,Amino Acids,2014,46(9),2165-2175)。G6PD和6GPD是细胞胞内NADPH再生的关键性酶,而胞内NADPH水平又显著影响控制着L-赖氨酸合成(图1)。经测定,由于不同重组菌株胞内G6PD和6GPD的酶活性不同,导致菌体胞内NADPH水平也不同(图5A)。从图5A可知,胞内G6PD和6GPD酶活力越高,胞内NADPH水平越高。重组菌株B.subtilis XH4胞内NADPH水平最高,从出发菌株B.subtilis XH1的3.51×10-4nmol/(104细胞)增加到5.38×10- 4nmol/(104细胞),胞内NADPH/NADP+提高了43.2%(图5A)。此外,重组菌株B.subtilis XH2、B.subtilis XH3和B.subtilis XH4胞外L-赖氨酸积累量都有显著提升(图5B)。重组菌株B.subtilis XH4摇瓶发酵40h后积累了20.3±1.9g/L的L-赖氨酸,比出发菌株B.subtilisXH1提高了73.5%(图5B)。引入外源的解除反馈调节的G6PD和6GPD可以强化PP途径碳通量,从而有效提高胞内NADPH供给水平,促进L-赖氨酸的合成。
实施例4:重组菌株产L-赖氨酸发酵情况
①种子培养基:葡萄糖5g/L,NaCl 5g/L,牛肉蛋白胨10g/L,酵母膏10g/L,121℃20min。②发酵培养基葡萄糖80g/L、玉米浆35g/L、甜菜糖蜜12g/L、(NH4)2SO4 36g/L、MgSO4·7H2O 1.5g/L、K2HPO4 1g/L、KH2PO4 1g/L、FeSO4 0.02g/L、MnSO4 0.02d/L、甜菜碱0.05d/L、烟酰胺0.008g/L、硫胺素0.00045g/L、生物素0.00085g/L、CaCO3 40g/L。所有培养基用20%(m/v)NaOH调节pH 7.0-7.2,发酵培养基于115℃灭菌10min。
将上述经验证的重组菌株和出发菌株分别进行摇瓶发酵实验。从新鲜活化的斜面培养基中挑取一满环枯草芽孢杆菌(出发菌和重组菌)接种到50mL装液量250mL液体种子培养基,4层纱布封口,30~38℃,50~200r·min-1培养8-10h;以10%接种量将种子培养液转接至50/250mL发酵培养基,30~38℃,50~200r·min-1发酵40h。
摇瓶发酵结果表明,重组菌株B.subtilis XH5(即:XH4 hom::hom59)菌体生长性能表现出与菌株B.subtilis XH4相似的水平(表3)。需要指出的是,尽管重组菌B.subtilisXH5菌体量低于出发菌株B.subtilis XH0和B.subtilis XH1,但其L-赖氨酸产量要远高于出发菌株B.subtilis XH0和B.subtilis XH11(表3)。重组菌株B.subtilis XH5中L-赖氨酸产量达到23.2±1.7g/L,比菌株B.subtilis XH1和B.subtilis XH4分别增加了98.3%和14.3%(表3)。此外,当重组菌种HSD酶(编码基因hom59)活力降低时,促进了L-赖氨酸的合成,同时减少了其他四种天冬氨酸族氨基酸的积累量。重组菌株B.subtilis XH5发酵液中未检测到L-蛋氨酸、L-苏氨酸和L-异亮氨酸,而菌株B.subtilis XH1和B.subtilis XH4在胞外都积累了一定量的副产物(表3)。这一结果表明,替换来源C.glutamicum中渗漏型HSD,可以调节B.subtilis胞内HSD酶活水平,从而影响L-赖氨酸合成和副产物积累。
表3不同菌株生长情况以及L-赖氨酸和副产物含量
且本发明在菌株B.subtilis XH5中引入C.glutamicum中二氨基庚二酸脱氢酶(DapDH,编码基因ddh),获得重组菌株B.subtilis XH6(即:XH5 pksD::ddh)。与出发菌株B.subtilis XH5不同,重组菌B.subtilis XH6表现出延滞的菌体生长,但最终菌体量两者基本一致(图6A)。此外,重组菌B.subtilis XH6中L-赖氨酸产量达到25.6±2.3g/L(图6B),比菌株B.subtilis XH5增加了10.3%。研究表明,来源于C.glutamicum的GapDH可以调节B.subtilis中DAP碳通量,引导部分碳流进入脱氢酶途径,从而途径L-赖氨酸的高效合成。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (11)
1.代谢工程改造枯草芽孢杆菌促进L-赖氨酸的合成,其特征在于,所述的重组菌以益生菌B.subtilis ACCC11025为宿主,将B.subtilis中thrD、zwf和gnd用来源C.glutamicum中lysC311、zwf243和gnd361替换,且B.subtilis中hom替换成来源C.glutamicum中hom59,最后再引入C.glutamicum中DapDH改变二氨基庚二酸途径碳分布而促进L-赖氨酸的合成。
2.根据权利要求1所述的枯草芽孢杆菌重组菌,其特征在于,所述的枯草芽孢杆菌包括B.subtilis XH0、B.subtilis XH1、B.subtilis XH2、B.subtilis XH3、B.subtilis XH4、B.subtilisXH5或B.subtilis XH6。
3.根据权利要求1所述的枯草芽孢杆菌重组菌,其特征在于,所述的编码天冬氨酸激酶的基因lysC311来源于C.glutamicum,核苷酸序列如SEQ ID NO.1所示。
4.根据权利要求1所述的枯草芽孢杆菌重组菌,其特征在于,所述的编码葡萄糖-6-磷酸脱氢酶的基因zwf243来源于C.glutamicum,核苷酸序列如SEQ ID NO.2所示。
5.根据权利要求1所述的枯草芽孢杆菌重组菌,其特征在于,所述的编码6-磷酸葡萄糖酸脱氢酶的基因gnd361来源于C.glutamicum,核苷酸序列如SEQ ID NO.3所示。
6.根据权利要求1所述的枯草芽孢杆菌重组菌,其特征在于,所述编码高丝氨酸脱氢酶的hom59来源于C.glutamicum,核苷酸序列如SEQ ID NO.4所示。
7.根据权利要求1所述的枯草芽孢杆菌重组菌,其特征在于,所述的编码二氨基庚二酸脱氢酶基因ddh来源于C.glutamicum,核苷酸序列如SEQ ID NO.5所示。
8.一种权利要求1~7任一项所述的重组菌的构建方法,其特征在于,包括如下步骤:
(1)天冬氨酸激酶基因lysC、葡萄糖-6-磷酸脱氢酶基因zwf、6-磷酸葡萄糖酸脱氢酶基因gnd和高丝氨酸脱氢酶基因hom的替换,以及异源表达DapDH基因ddh:以枯草芽孢杆菌ACCC11025为宿主,采用CRISPR-Cas9系统将基因thrD、zwf、gnd和hom替换为来源于C.glutamicum的lysC311、zwf243、gnd361和hom59,再异源引入C.glutamicum的DapDH基因ddh。
(2)重组质粒构建:以酿脓链球菌的基因组作为模板,将基因Spcas9进行PCR扩增,随后利用限制性内切酶对质粒pHT01和片段Spcas9进行双酶切并酶连,获得目标重组质粒pHT01-Cas9。再将B.subtilis的thrD、zwf、gnd、hom和pksD基因的sgRNA无缝连接到质粒pBE980b,获得五个带有靶向位点的质粒。再将C.glutamicum的lysC311基因、zwf243基因、gnd361基因、hom59基因和ddh基因与B.subtilis的thrD、zwf、gnd、hom和pksD基因的上下同源臂通过融合PCR进行基因融合,再依次酶切连接至质粒pBE980b后得到重组质粒。
(3)重组菌构建:将重组质粒pHT01-Cas9电转至B.subtilisACCC11025感受态细胞中,通过氨苄抗性筛选得到带有Cas9蛋白的转化子。再将其余重组质粒电转至带有Cas9蛋白的B.subtilis ACCC11025感受态细胞中,筛选得到所述的重组菌。
9.一种权利要求1~6任一项所述的重组菌发酵生产L-赖氨酸的方法,其特征在于,所述方法是将所述重组菌单菌落接种至液体种子培养基,30~38℃,50~200r·min-1培养8-10h;以8~12%接种量将种子培养液转接至发酵培养基培养40h。
10.根据权利要求8所述的方法,其特征在于,所述的种子培养基为:葡萄糖5g/L,NaCl5g/L,牛肉蛋白胨10g/L,酵母膏10g/L;所述的发酵培养基为:葡萄糖80g/L、玉米浆35g/L、甜菜糖蜜12g/L、(NH4)2SO4 36g/L、MgSO4·7H2O 1.5g/L、K2HPO4 1g/L、KH2PO4 1g/L、FeSO40.02g/L、MnSO4 0.02 g/L、甜菜碱0.05g/L、烟酰胺0.008g/L、硫胺素0.00045g/L、生物素0.00085g/L、CaCO3 40g/L。培养基用20%(m/v)NaOH调至pH=7.0~7.2。
11.权利要求1~8任一项所述利用B.subtilis生产L-赖氨酸等饲用氨基酸提供参考,为实现简化饲料生产工艺提供了一定的理论基础。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210183889.6A CN114774336B (zh) | 2022-02-24 | 2022-02-24 | 一种产l-赖氨酸的枯草芽孢杆菌重组菌的构建及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210183889.6A CN114774336B (zh) | 2022-02-24 | 2022-02-24 | 一种产l-赖氨酸的枯草芽孢杆菌重组菌的构建及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114774336A true CN114774336A (zh) | 2022-07-22 |
CN114774336B CN114774336B (zh) | 2024-05-28 |
Family
ID=82423441
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210183889.6A Active CN114774336B (zh) | 2022-02-24 | 2022-02-24 | 一种产l-赖氨酸的枯草芽孢杆菌重组菌的构建及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114774336B (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120107882A1 (en) * | 2008-03-03 | 2012-05-03 | Sheng Yang | Recombinant Microorganism And Method For Producing L-Lysine |
US20130203130A1 (en) * | 2010-06-15 | 2013-08-08 | Paik Kwang Industrial Co., Ltd. | Production process for amino acids of the aspartate family using microorganisms |
CN103555625A (zh) * | 2013-10-28 | 2014-02-05 | 李丽立 | 一种高产赖氨酸的枯草芽孢杆菌pl83及制备方法和应用 |
CN110591996A (zh) * | 2019-09-27 | 2019-12-20 | 天津市畜牧兽医研究所 | 一种高产l-赖氨酸枯草芽孢杆工程菌的构建方法及应用 |
-
2022
- 2022-02-24 CN CN202210183889.6A patent/CN114774336B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120107882A1 (en) * | 2008-03-03 | 2012-05-03 | Sheng Yang | Recombinant Microorganism And Method For Producing L-Lysine |
US20130203130A1 (en) * | 2010-06-15 | 2013-08-08 | Paik Kwang Industrial Co., Ltd. | Production process for amino acids of the aspartate family using microorganisms |
CN103555625A (zh) * | 2013-10-28 | 2014-02-05 | 李丽立 | 一种高产赖氨酸的枯草芽孢杆菌pl83及制备方法和应用 |
CN110591996A (zh) * | 2019-09-27 | 2019-12-20 | 天津市畜牧兽医研究所 | 一种高产l-赖氨酸枯草芽孢杆工程菌的构建方法及应用 |
Non-Patent Citations (1)
Title |
---|
付凌;曾黎明;李新国;: "高产赖氨酸菌的选育与应用", 氨基酸和生物资源, no. 04, pages 18 - 23 * |
Also Published As
Publication number | Publication date |
---|---|
CN114774336B (zh) | 2024-05-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5482846A (en) | Ethanol production in Gram-positive microbes | |
KR100838038B1 (ko) | L-라이신 생산능이 향상된 코리네박테리움 속 미생물 및그를 이용한 l-라이신 생산 방법 | |
EP1360312B1 (en) | Method for l-threonine production | |
KR100838035B1 (ko) | L-라이신 생산능이 향상된 코리네박테리움 속 미생물 및그를 이용한 l-라이신 생산 방법 | |
WO2019085445A1 (zh) | 生产l-赖氨酸的重组菌、其构建方法以及l-赖氨酸的生产方法 | |
CN114717172B (zh) | 合成l-缬氨酸的大肠杆菌及其构建方法与应用 | |
CN100519740C (zh) | 具有改善的l-赖氨酸生产力的棒杆菌属微生物和利用棒杆菌微生物生产l-赖氨酸的方法 | |
CN102165056B (zh) | 生产l-氨基酸的微生物和使用其生产l-氨基酸的方法 | |
CN102906272A (zh) | 生产o-磷酸丝氨酸的微生物和使用该微生物由o-磷酸丝氨酸生产l-半胱氨酸或其衍生物的方法 | |
US20200224233A1 (en) | Method for producing tetrahydropyrimidine by fermenting recombinant corynebacterium glutamicum | |
CN103403147A (zh) | 产腐胺的微生物以及使用此微生物生产腐胺的方法 | |
CN107709540A (zh) | 新型库德里阿兹威氏毕赤酵母菌种ng7及其用途 | |
CN102197135A (zh) | 生产2-脱氧蟹肌醇(doi)的细菌及使用其生产2-脱氧蟹肌醇(doi)的方法 | |
CN113278568B (zh) | 生产l-缬氨酸的重组大肠杆菌及其应用 | |
CN105543156A (zh) | 重组菌株及其制备方法、用途 | |
CN104480058A (zh) | 一株高产l-亮氨酸工程菌及其应用 | |
JPWO2020208842A5 (zh) | ||
US6849434B2 (en) | Ethanol production in recombinant hosts | |
CN113278655A (zh) | 生产l-缬氨酸的重组微生物及构建方法、应用 | |
TW201142032A (en) | Utilization of agro residual substrates for fermentative production of L-arginine | |
CN111607601A (zh) | 谷氨酸棒杆菌转录调控因子IpsA突变体及应用 | |
KR100630819B1 (ko) | 신규 루멘 박테리아 변이균주 및 이를 이용한 숙신산의제조방법 | |
CN114774336A (zh) | 一种产l-赖氨酸的枯草芽孢杆菌重组菌的构建及其应用 | |
CN103476922A (zh) | 用来源于埃希氏菌(Escherichia sp.)的果糖激酶基因转化的棒杆菌(Corynebacterium sp.)及使用该棒杆菌制备L-氨基酸的方法 | |
KR100816472B1 (ko) | 글루타메이트 에이비씨-타입 트랜스포터 활성이 결실된코리네박테리아 및 이를 이용한 엘-라이신 생산방법 |
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 |