CN113969288A - 一种高产法尼醇基因工程菌及其构建方法与应用 - Google Patents
一种高产法尼醇基因工程菌及其构建方法与应用 Download PDFInfo
- Publication number
- CN113969288A CN113969288A CN202111273441.5A CN202111273441A CN113969288A CN 113969288 A CN113969288 A CN 113969288A CN 202111273441 A CN202111273441 A CN 202111273441A CN 113969288 A CN113969288 A CN 113969288A
- Authority
- CN
- China
- Prior art keywords
- farnesol
- yield
- saccharomyces cerevisiae
- linker
- genetic engineering
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000260 (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-ol Substances 0.000 title claims abstract description 80
- 229930002886 farnesol Natural products 0.000 title claims abstract description 80
- 229940043259 farnesol Drugs 0.000 title claims abstract description 80
- CRDAMVZIKSXKFV-UHFFFAOYSA-N trans-Farnesol Natural products CC(C)=CCCC(C)=CCCC(C)=CCO CRDAMVZIKSXKFV-UHFFFAOYSA-N 0.000 title claims abstract description 80
- 241000894006 Bacteria Species 0.000 title claims abstract description 58
- 238000010276 construction Methods 0.000 title claims abstract description 32
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 26
- CRDAMVZIKSXKFV-FBXUGWQNSA-N (2-cis,6-cis)-farnesol Chemical compound CC(C)=CCC\C(C)=C/CC\C(C)=C/CO CRDAMVZIKSXKFV-FBXUGWQNSA-N 0.000 title claims abstract 21
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 65
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 65
- 230000014509 gene expression Effects 0.000 claims abstract description 45
- 238000010353 genetic engineering Methods 0.000 claims abstract description 32
- 238000000855 fermentation Methods 0.000 claims abstract description 25
- 230000004151 fermentation Effects 0.000 claims abstract description 25
- 230000037361 pathway Effects 0.000 claims abstract description 25
- 108010022535 Farnesyl-Diphosphate Farnesyltransferase Proteins 0.000 claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 claims abstract description 17
- 102100037997 Squalene synthase Human genes 0.000 claims abstract description 15
- 101150082238 PAH1 gene Proteins 0.000 claims abstract description 14
- 101150116391 erg9 gene Proteins 0.000 claims abstract description 14
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims abstract description 11
- 101100390535 Mus musculus Fdft1 gene Proteins 0.000 claims abstract description 11
- 101100390536 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) erg-6 gene Proteins 0.000 claims abstract description 11
- 229910001431 copper ion Inorganic materials 0.000 claims abstract description 11
- 101710165761 (2E,6E)-farnesyl diphosphate synthase Proteins 0.000 claims abstract description 6
- 101710156207 Farnesyl diphosphate synthase Proteins 0.000 claims abstract description 6
- 101710125754 Farnesyl pyrophosphate synthase Proteins 0.000 claims abstract description 6
- 102100035111 Farnesyl pyrophosphate synthase Human genes 0.000 claims abstract description 6
- 101710089428 Farnesyl pyrophosphate synthase erg20 Proteins 0.000 claims abstract description 6
- 101710150389 Probable farnesyl diphosphate synthase Proteins 0.000 claims abstract description 6
- 108020004414 DNA Proteins 0.000 claims description 67
- 239000012634 fragment Substances 0.000 claims description 64
- 238000006243 chemical reaction Methods 0.000 claims description 34
- 229930182830 galactose Natural products 0.000 claims description 31
- 102000034356 gene-regulatory proteins Human genes 0.000 claims description 31
- 108091006104 gene-regulatory proteins Proteins 0.000 claims description 31
- 101150103317 GAL80 gene Proteins 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 24
- 239000002773 nucleotide Substances 0.000 claims description 20
- 125000003729 nucleotide group Chemical group 0.000 claims description 20
- 239000013600 plasmid vector Substances 0.000 claims description 18
- 239000000463 material Substances 0.000 claims description 13
- 239000013613 expression plasmid Substances 0.000 claims description 11
- 238000007857 nested PCR Methods 0.000 claims description 11
- 239000013612 plasmid Substances 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 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 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 108091008146 restriction endonucleases Proteins 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 7
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 claims description 7
- 230000001105 regulatory effect Effects 0.000 claims description 7
- 238000011426 transformation method Methods 0.000 claims description 7
- 238000012795 verification 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
- 230000004927 fusion Effects 0.000 claims description 6
- 150000007523 nucleic acids Chemical group 0.000 claims description 6
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 claims description 6
- 230000001131 transforming effect Effects 0.000 claims description 6
- 230000002457 bidirectional effect Effects 0.000 claims description 5
- 239000013598 vector Substances 0.000 claims description 5
- 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 claims description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- 230000003115 biocidal effect Effects 0.000 claims description 4
- 230000003828 downregulation Effects 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 101100190268 Caenorhabditis elegans pah-1 gene Proteins 0.000 claims description 3
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 3
- 102000043276 Oncogene Human genes 0.000 claims description 3
- 108700020796 Oncogene Proteins 0.000 claims description 3
- 108091081024 Start codon 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
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 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
- 229960000367 inositol Drugs 0.000 claims description 3
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 235000008160 pyridoxine Nutrition 0.000 claims description 3
- 239000011677 pyridoxine Substances 0.000 claims description 3
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 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
- 235000019157 thiamine Nutrition 0.000 claims description 3
- 239000011721 thiamine Substances 0.000 claims description 3
- 229940011671 vitamin b6 Drugs 0.000 claims description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 2
- 239000004471 Glycine Substances 0.000 claims description 2
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 claims description 2
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 claims description 2
- 241000793189 Saccharomyces cerevisiae BY4741 Species 0.000 claims description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims description 2
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 claims description 2
- 229960002079 calcium pantothenate Drugs 0.000 claims description 2
- 230000002018 overexpression Effects 0.000 claims description 2
- 101150084072 ERG20 gene Proteins 0.000 claims 2
- 230000008021 deposition Effects 0.000 claims 1
- 239000012533 medium component Substances 0.000 claims 1
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 abstract description 5
- RQOCXCFLRBRBCS-UHFFFAOYSA-N (22E)-cholesta-5,7,22-trien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CCC(C)C)CCC33)C)C3=CC=C21 RQOCXCFLRBRBCS-UHFFFAOYSA-N 0.000 abstract description 5
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 abstract description 5
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 abstract description 5
- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 0.000 abstract description 5
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 abstract description 4
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 abstract description 4
- 230000001965 increasing effect Effects 0.000 abstract description 4
- 230000002503 metabolic effect Effects 0.000 abstract description 4
- 230000006801 homologous recombination Effects 0.000 abstract description 3
- 238000002744 homologous recombination Methods 0.000 abstract description 3
- 230000002860 competitive effect Effects 0.000 abstract description 2
- 102000004286 Hydroxymethylglutaryl CoA Reductases Human genes 0.000 abstract 1
- 108090000895 Hydroxymethylglutaryl CoA Reductases Proteins 0.000 abstract 1
- CRDAMVZIKSXKFV-YFVJMOTDSA-N (2-trans,6-trans)-farnesol Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CO CRDAMVZIKSXKFV-YFVJMOTDSA-N 0.000 description 59
- ZJPGOXWRFNKIQL-JYJNAYRXSA-N Phe-Pro-Pro Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=CC=C1 ZJPGOXWRFNKIQL-JYJNAYRXSA-N 0.000 description 8
- 238000012258 culturing Methods 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- KJTLQQUUPVSXIM-ZCFIWIBFSA-N (R)-mevalonic acid Chemical compound OCC[C@](O)(C)CC(O)=O KJTLQQUUPVSXIM-ZCFIWIBFSA-N 0.000 description 7
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 101150008021 80 gene Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 239000000306 component Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000009776 industrial production Methods 0.000 description 4
- 230000010354 integration Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 239000007222 ypd medium Substances 0.000 description 4
- HIQIXEFWDLTDED-UHFFFAOYSA-N 4-hydroxy-1-piperidin-4-ylpyrrolidin-2-one Chemical compound O=C1CC(O)CN1C1CCNCC1 HIQIXEFWDLTDED-UHFFFAOYSA-N 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000012137 tryptone Substances 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
- 229930006000 Sucrose Natural products 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- -1 allo-albizium Chemical compound 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- NUHSROFQTUXZQQ-UHFFFAOYSA-N isopentenyl diphosphate Chemical compound CC(=C)CCO[P@](O)(=O)OP(O)(O)=O NUHSROFQTUXZQQ-UHFFFAOYSA-N 0.000 description 2
- CDOSHBSSFJOMGT-UHFFFAOYSA-N linalool Chemical compound CC(C)=CCCC(C)(O)C=C CDOSHBSSFJOMGT-UHFFFAOYSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- MGSRCZKZVOBKFT-UHFFFAOYSA-N thymol Chemical compound CC(C)C1=CC=C(C)C=C1O MGSRCZKZVOBKFT-UHFFFAOYSA-N 0.000 description 2
- 239000001490 (3R)-3,7-dimethylocta-1,6-dien-3-ol Substances 0.000 description 1
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 1
- CDOSHBSSFJOMGT-JTQLQIEISA-N (R)-linalool Natural products CC(C)=CCC[C@@](C)(O)C=C CDOSHBSSFJOMGT-JTQLQIEISA-N 0.000 description 1
- 102100029077 3-hydroxy-3-methylglutaryl-coenzyme A reductase Human genes 0.000 description 1
- 101710158485 3-hydroxy-3-methylglutaryl-coenzyme A reductase Proteins 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000795928 Pseudomonas zeshuii Species 0.000 description 1
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 1
- 239000005844 Thymol Substances 0.000 description 1
- 229930000062 acyclic sesquiterpene Natural products 0.000 description 1
- 150000000553 acyclic sesquiterpene derivatives Chemical class 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 125000003118 aryl group Chemical group 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
- 230000001851 biosynthetic effect Effects 0.000 description 1
- FAPWYRCQGJNNSJ-CTWWJBIBSA-L calcium;3-[[(2s)-2,4-dihydroxy-3,3-dimethylbutanoyl]amino]propanoate Chemical compound [Ca+2].OCC(C)(C)[C@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-CTWWJBIBSA-L 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000012824 chemical production Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000002418 insect attractant Substances 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229930007744 linalool Natural products 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000018612 quorum sensing Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 229940031439 squalene Drugs 0.000 description 1
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229960000790 thymol Drugs 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 239000002699 waste material Substances 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- 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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
- C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
- C12Y205/0101—(2E,6E)-Farnesyl diphosphate synthase (2.5.1.10), i.e. geranyltranstransferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
- C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
- C12Y205/01021—Squalene synthase (2.5.1.21), i.e. farnesyl-disphosphate farnesyltransferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03004—Phosphatidate phosphatase (3.1.3.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03047—[Hydroxymethylglutaryl-CoA reductase (NADPH)]-phosphatase (3.1.3.47)
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
- C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron
- C12N2830/205—Vector systems having a special element relevant for transcription transcription of more than one cistron bidirectional
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Botany (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明提供了一种高产法尼醇基因工程菌的构建方法,是利用同源重组的方式在酿酒酵母中整合表达MVA途径限速酶‑截短形式的HMG‑CoA还原酶编码基因(tHMG1)和整合融合表达FPP合酶编码基因(ERG20)与酿酒酵母内源性磷酸酶编码基因(PAH1),以增强MVA途径代谢强度,增强法尼醇的表达。同时使用铜离子诱导启动子pCUP1替换角鲨烯合酶编码基因ERG9启动子,下调麦角固醇竞争途径,提高法尼醇产量;最终获得高产法尼醇的基因工程菌株。该基因工程菌的法尼醇的摇瓶发酵产量可达到约573mg/L,发酵罐产量可达到约21g/L,完全具备商业化生产水平,具有良好的工业应用前景。
Description
技术领域
本发明涉及微生物发酵技术领域,尤其涉及一种高产法尼醇基因工程菌及其构建方法与应用。
背景技术
法尼醇(Farnesol)分子式:C15H26O,又名法呢醇,合金欢醇,里哪醇,菌绿烯醇,麝子油醇,是一种具有芳香气味的非环状倍半帖醇。广泛分布于许多植物精油中,也存在于动物和微生物中,在信号转导、群体感应和细胞凋亡诱导等过程中发挥重要作用,可以作为杀菌清洁剂、杀虫剂、昆虫引诱剂。它是重要化工生产原料,被广泛应用于化妆品和医学药物的工业化生产,也可作为航空燃料的理想替代品。
法尼醇可以从植物中分离提取,但提取步骤繁琐,且质量与原料息息相关,而经化学方法合成的法尼醇,往往存在异构体混杂、毒性大等问题。除此之外,微生物发酵也是法尼醇的一个来源,微生物发酵法具有条件温和,不受地理和气候影响和容易进行大规模生产等优势,但目前能用于发酵生产法尼醇的菌株的产量都普遍偏低,且菌株稳定性很差,很难用于工业化生产。因此,构建高产稳定的法尼醇生产菌株,对其生产和应用具有重要意义。
酿酒酵母细胞能够合成内源性法尼醇,但其产量很低,无法满足工业生产的需要。
发明内容
本发明的目的在于,针对现有技术的上述不足,提出一种高产法尼醇基因工程菌及其构建方法与应用。
为实现上述目的,本发明采用如下的技术方案:
本发明提供的一种高产法尼醇基因工程菌的构建方法,包括以下步骤:
步骤S1,构建酿酒酵母MVA途径相关基因表达模块:过表达tHMG1模块,包括诱导型双向强启动子pGAL1-10和MVA途径限速酶编码基因tHMG1,所述pGAL1-10的核苷酸序列如SEQ NO.1所示,所述tHMG1的核苷酸序列如 SEQ NO.2所示;
步骤S2,构建法尼醇生产相关基因表达模块:ERG20-Linker-PAH1模块,包括FPP合酶的编码基因ERG20和酿酒酵母内源性磷酸酶的编码基因PAH1,所述ERG20的核苷酸序列如SEQ NO.3所示,所述PAH1的核苷酸序列如SEQ NO.4所示;
步骤S3,构建敲除半乳糖调节蛋白GAL80基因表达的工程菌:将步骤S1 所述酿酒酵母MVA途径相关基因表达模块和步骤S2所述法尼醇生产相关基因表达模块整合至半乳糖调节蛋白GAL80基因位点,同时敲除半乳糖调节蛋白 GAL80基因,得到敲除半乳糖调节蛋白GAL80基因表达模块,将所述敲除半乳糖调节蛋白GAL80基因表达模块转化至构建的酿酒酵母基因工程菌GS-A3中,经多次基因工程操作,获得敲除半乳糖调节蛋白GAL80基因表达的基因工程菌;
步骤S4,构建角鲨烯合酶途径下调表达质粒,包括将铜离子诱导启动子 pCUP1替换为角鲨烯合酶基因ERG9启动子,所述pCUP1的核苷酸序列如SEQ NO.5所示;所述角鲨烯合酶基因ERG9启动子取至ERG9基因起始密码子前 450bp,其核苷酸序列如SEQ NO.6所示;
步骤S5,将步骤S5所述角鲨烯合酶途径下调表达质粒转化至步骤S3所述的敲除半乳糖调节蛋白GAL80基因表达的基因工程菌中,依次经抗生素抗性筛选,然后通过菌落PCR验证,获得高产法尼醇基因工程菌。
进一步的,在步骤S1中,所述过表达tHMG1模块的构建方法包括以下步骤:
步骤S1,以酿酒酵母3000B基因组DNA为模板,分别用tCYC1-F和 tCYC1-R、tHMG1-F和tHMG1-R、pGAL1pGAL10-F和pGAL1pGAL10-R引物进行PCR反应,获得DNA片段tCYC1、tHMG1和pGAL10pGAL1;
步骤S2,将步骤S1获得的三个DNA片段tCYC1、tHMG1和pGAL10pGAL1,通过用引物tCYC1-F和pGAL1pGAL10-R进行重叠延伸PCR反应,连接在一起,获得过表达tHMG1模块,即tCYC1_tHMG1_pGAL10pGAL1模块。
进一步的,步骤S2中,所述ERG20-Linker-PAH1模块的构建方法包括以下步骤:
步骤S1,以酿酒酵母3000B基因组DNA为模板,分别用ERG20-F和ERG20- Linker-R引物进行PCR反应,扩增得到DNA片段ERG20_Linker;
步骤S2,用引物Linker-PAH1-F和PAH1-R进行PCR反应,扩增得到DNA 片段Linker_PAH1;
步骤S3,用引物tERG20-F和tERG20-R进行PCR反应,扩增得到DNA片段tERG20;
步骤S4,将步骤S1获得的DNA片段ERG20_Linker、步骤S2获得的DNA 片段Linker_PAH1和步骤S3获得的DNA片段tERG20,通过用引物ERG20-F 和tERG20-R进行重叠延伸PCR反应,连接在一起,获得融合表达 ERG20-Linker-PAH1模块,即ERG20_Linker_PAH1_tERG20。
进一步的,所述Linker包括甘氨酸和丝氨酸,其组合结构包括GSG、GGGS 和GSGGSG中的任一种,其中G对应核酸序列GGT,S对应核酸序列TCT。
进一步的,在步骤S2中,所述ERG20-Linker-PAH1模块中的Linker为GSG。
进一步的,在步骤S3中,所述构建敲除半乳糖调节蛋白GAL80基因表达模块的构建方法包括以下步骤:
步骤S1,以酿酒酵母3000B基因组DNA为模板,分别用GAL80left-F和 GAL80left-R、GAL80right-F和GAL80right-R引物进行PCR反应,扩增得到DNA 片段GAL80的左右同源臂GAL80left和GAL80right;
步骤S2,以质粒载体pFZ201为模板,用引物Hyg-F和Hyg-R进行PCR反应,获得潮霉素表达盒Hyg;
步骤S3,将所述过表达tHMG1模块和所述ERG20-Linker-PAH1模块、 GAL80left,GAL80right和Hyg五个DNA片段,通过用引物GAL80left-F和 GAL80right-R进行重叠延伸PCR反应,连接在一起,获得DNA片段为敲除半乳糖调节蛋白GAL80基因表达模块的;
步骤S4,将步骤S3获得的DNA片段与质粒pMD19-T连接,获得重组质粒载体,用限制性内切酶PmeI线性化所述重组质粒,回收带有目的基因的片段,将片段用酵母醋酸锂转化法转化至宿主菌酿酒酵母中,依次经抗生素抗性筛选,然后通过菌落PCR获取阳性菌落,获得敲除半乳糖调节蛋白GAL80基因表达的工程菌。
进一步的,在步骤S3中,所述宿主菌酿酒酵母包括酿酒酵母30000B、酿酒酵母S.cerevisiae CEN.PK2-1D、酿酒酵母BY4741和酿酒酵母GS-A3中的任一种,所述酿酒酵母GS-A3于2021年9月17日保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:M20211191。
进一步的,在步骤S4中,所述角鲨烯合酶途径下调表达质粒的构建方法包括以下步骤:
步骤S4,将步骤S3获得的DNA片段G418_pCUP1/ΔpEGR9与质粒
pMD19-T连接,获得角鲨烯合酶途径下调表达质粒载体,记为pCZ101。
本发明还提供了一种高产法尼醇基因工程菌,采用上述构建方法得到的。
本发明还提供了利用上述高产法尼醇基因工程菌的生产法尼醇,所述高产法尼醇基因工程菌的液体发酵培养基组分包括葡萄糖20-50g/L,酵母提取物5-10g/L,硫酸铵6-15g/L,磷酸二氢钾3-8g/L,七水硫酸镁5-10g/L,硫胺素 100-500mg/L,吡哆素100-500mg/L,肌醇400-800mg/L,生物素20-100mg/L和泛酸钙100-500mg/L。
本发明提供的技术方案带来的有益效果是:
(1)本发明的构建方法是利用同源重组的方式将双向强启动子pGAL1-10 驱动的酿酒酵母菌来源的甲羟戊酸途径限速酶编码基因tHMG1和FPP合酶 (ERG20)与酿酒酵母内源性磷酸酶(PAH1)融合蛋白质的基因元件 (ERG20-Linker-PAH1)整合入出发菌株酿酒酵母基因组,整合位点为半乳糖调节蛋白80基因(GAL80),整合目的基因的同时敲除半乳糖调节蛋白80基因,以增强甲羟戊酸代谢途径代谢强度,进一步增强法尼醇的表达。该构建方法具有简单快速高效的优点,2~3周即可获得大片段整合工程菌株,明显缩短工程菌构建周期。
(2)本发明以(1)中所获工程菌为基础,使用铜离子诱导启动子pCUP1替换角鲨烯合酶编码基因ERG9启动子下调其表达水平,可以减少FPP流向麦角固醇竞争途径,进一步提高法尼醇产量;最终获得高产法尼醇的基因工程菌株。按照本发明的方法获得的基因工程菌的法尼醇的摇瓶发酵产量可达到约 573mg/L,发酵罐产量可达到约21g/L,完全具备商业化生产水平,具有良好的工业应用前景。
(3)本发明可利用简单培养基发酵生产法尼醇,可实现多基因一次性高效转化与整合,可明显缩短工程菌构建时间,所得工程菌可利用葡萄糖、蔗糖等简单碳源进行法尼醇的发酵生产,有较好的应用前景。
附图说明
图1为本发明的酿酒酵母基因工程菌的生产法尼醇的生物代谢原理示意图;
图2为本发明实施例1构建的敲除半乳糖调节蛋白GAL80基因表达模块的重组质粒pCZ100的结构示意图;
图3为本发明实施例1构建的角鲨烯合酶途径下调表达的重组质粒pCZ101 的结构示意图;
图4为不同Linke连接对法尼醇产量的影响比较图;
图5为不同基因工程菌GS-A3-C4、G30000B-C2、CEN.PK2-1D-C2和 BY474-C2生产法尼醇的比较图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图和实施例对本发明实施方式作进一步地描述。
本发明在研究的过程中使用的材料和方法如下:
本发明中的全基因合成、引物合成及测序皆由武汉天一华煜基因科技有限公司完成,用到的高保真酶(PrimeSTAR GXL DNA Polymerase),普通Taq 酶(Premix Taq),pMD19-T Vector等购买于武汉友名生物技术有限公司,限制性内切酶购买于湖北晶茂生物技术有限公司;酿酒酵母30000B为市售。
本发明中的分子生物学实验包括质粒构建、酶切、感受态细胞制备﹑转化等主要参照《分子克隆实验指南》(第三版),J.萨姆布鲁克,D.W.拉塞尔(美)编著,黄培堂等译,科学出版社,2002)进行。
LB固体培养基:10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠,20g/L 琼脂粉;
YPD培养基:10g/L酵母提取物,20g/L胰蛋白胨,20g/L葡萄糖;发酵时添加10%的肉豆蔻酸异丙酯,防止产物挥发;
YPD固体培养基:10g/L酵母提取物,20g/L胰蛋白胨,20g/L葡萄糖,20g/L 琼脂粉。
下列实施例中还有未注明具体实验条件的试验方法,通常按照常规实验条件或按照制造厂所建议的实验条件。所使用的材料、试剂等,如无特殊说明,为从商业途径得到的试剂和材料。
如图1所示为生物合成法尼醇的代谢原理图,本发明构建的高产法尼醇基因工程菌,是利用同源重组的方式将双向强启动子pGAL1-10驱动的酿酒酵母菌来源的甲羟戊酸途径限速酶编码基因tHMG1;FPP合酶(ERG20)与酿酒酵母内源性磷酸酶(PAH1)融合蛋白质的基因元件(ERG20-Linker-PAH1)整合入出发菌株酿酒酵母基因组,整合位点为半乳糖调节蛋白80基因(GAL80),整合目的基因的同时敲除半乳糖调节蛋白80基因;然后用铜离子诱导启动子 pCUP1替换上述重组菌体内角鲨烯合酶基因ERG9的启动子下调其表达水平。
其中,诱导型双向强启动子pGAL1-10的核苷酸序列如SEQ NO.1所示;甲羟戊酸途径限速酶为截短的3-羟基-3-甲基戊二酰辅酶A还原酶,编码基因 tHMG1的核苷酸序列如SEQ NO.2所示,FPP合酶(ERG20)编码基因的苷酸序列如SEQ NO.3所示,酿酒酵母内源性磷酸酶(PAH1)编码基因的苷酸序列如SEQ NO.4所示,将SEQ ID NO.3所示序列的3’端终止密码子TAG去除,与 SEQ ID NO.4所示序列的5'端用编码Linker的碱基序列连接;构建出ERG20-Linker-PAH1融合蛋白质的基因元件;ERG20的催化产物FPP,是PAH1 的底物,两个酶通过linker序列融合表达,在空间构象上拉近它们的距离,可以提高由IPP到FPP再到法尼醇的反应效率,避免FPP的浪费和减少FPP去往其他代谢支路,从而提高法尼醇的产量。
又因为角鲨烯合酶竞争底物的能力较强,大多数FPP都流向了麦角固醇,导致其他萜类物质产量较低。因此,要想实现目的产物的高效合成,阻断或下调麦角固醇竞争途径是十分必要的。但ERG9是生长必须基因,不能敲除,必须动态调控。因此本发明用铜离子诱导启动子pCUP1替换角鲨烯合酶基因ERG9 的启动子下调其表达水平;铜离子诱导启动子pCUP1的苷酸序列如SEQ NO.5 所示;选用角鲨烯合酶基因ERG9启动子取ERG9基因起始密码子前450bp,其核苷酸序列如SEQ NO.6所示。
为了更准确的指构建的各个模块的正确结合,构建过程中还使用了两个终止子tCYC1和tERG20,分别对应的核苷酸序列为SEQ NO.7和SEQ NO.8。
实施例1-3和对比例1-3中使用的引物序列信息如表1所示:
表1:引物序列
实施例1
高产法尼醇基因工程菌GS-A3-C4的构建:
步骤S1,过表达tHMG1模块的构建
tCYC1_tHMG1_pGAL10pGAL1
1)以酿酒酵母3000B基因组DNA为模板,分别用tCYC1-F和tCYC1-R、 tHMG1-F和tHMG1-R、pGAL1pGAL10-F和pGAL1pGAL10-R引物进行PCR反应,获得DNA片段tCYC1、tHMG1和pGAL10pGAL1;
2)将步骤S1获得的三个DNA片段tCYC1、tHMG1和pGAL10pGAL1,通过用引物tCYC1-F和pGAL1pGAL10-R进行重叠延伸PCR反应,连接在一起,获得过表达tHMG1模块,即tCYC1_tHMG1_pGAL10pGAL1模块。
步骤S2,融合表达ERG20-Linker-PAH1模块的构建
ERG20_Linker_PAH1_tERG20
1)以酿酒酵母3000B基因组DNA为模板,分别用ERG20-F和ERG20-Linker -R引物进行PCR反应,扩增得到DNA片段ERG20_Linker。
2)用引物Linker-PAH1-F和PAH1-R进行PCR反应,扩增得到DNA片段 Linker_PAH1。
3)用引物tERG20-F和tERG20-R进行PCR反应,扩增得到DNA片段 tERG20。
4)将上述获得的DNA片段ERG20_Linker、步骤S2获得的DNA片段 Linker_PAH1和步骤S3获得的DNA片段tERG20,通过用引物ERG20-F和 tERG20-R进行重叠延伸PCR反应,连接在一起,获得融合表达 ERG20-Linker-PAH1模块,即ERG20_Linker_PAH1_tERG20。
其中,Linker包括Linker1、Linker2和Linker3,分别对应的氨基酸序列为 GSG、GGGS和GSGGSG,G对应核酸序列GGT,S对应核酸序列TCT。
步骤S3,构建敲除半乳糖调节蛋白GAL80基因表达的工程菌
1)以酿酒酵母3000B基因组DNA为模板,分别用GAL80left-F和 GAL80left-R、GAL80right-F和GAL80right-R引物进行PCR反应,扩增得到DNA 片段GAL80的左右同源臂GAL80left和GAL80right。
2)以质粒载体pFZ201为模板,用引物Hyg-F和Hyg-R进行PCR反应,获得潮霉素表达盒Hyg。
3)将所述过表达tHMG1模块和所述ERG20-Linker-PAH1模块、GAL80left,GAL80right和Hyg五个DNA片段,通过用引物GAL80left-F和GAL80right-R 进行重叠延伸PCR反应,连接在一起,获得DNA片段为敲除半乳糖调节蛋白 GAL80基因表达模块的。
选用Linker1连接,可获得模块:
GAL80left_Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker1_PAH1_tERG20_GAL80right。
4)将上述获得的模块连接pMD19-T载体,转入大肠内扩增,酶切和测序验证正确后,获得重组质粒载体pCZ100:
pCZ100ΔGAL80::Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker1_PAH1_tERG20;
质粒载体pCZ100的结构图如图2所示。
5)将上述构建的重组质粒载体pCZ100,用限制性内切酶PmeI分别线性化,回收带有目的基因的片段,将片段用酵母醋酸锂转化法转化至酿酒酵母GS-A3,涂布到含500μg/mL潮霉素的YPD平板上,挑取转化子并提基因组PCR验证,获得对应正确的转化菌株GS-A3-C1。
步骤S4,角鲨烯合酶途径下调表达质粒的构建
pCZ101ΔpEGR9::G418_pCUP1
1)以酿酒酵母3000B基因组DNA为模板,分别用pERG9-left-F和pERG9-left-R、pCUP1-F和pCUP1-R、pERG9-right-F和pERG9-right-R引物进行PCR反应,扩增得到DNA片段pERG9-left、pCUP1和pERG9-righ。
2)以质粒载体pFZ202为模板,用引物G418-F和G418-R进行PCR反应,获得G418表达盒G418。
3)将上述获得的DNA片段pERG9-left、pCUP1和pERG9-righ和步骤S1 获得的表达盒G418五个DNA片段,通过用引物pERG9-left-F和pERG9-right-R 进行重叠延伸PCR反应,连接在一起,获得DNA片段G418_pCUP1/ΔpEGR9。
4)将上述获得的DNA片段G418_pCUP1/ΔpEGR9与质粒pMD19-T连接,获得角鲨烯合酶途径下调表达质粒载体,记为pCZ101,其结构图如图3所示。
步骤S5,用限制性内切酶PmeI线性化质粒pCZ101,回收带有目的基因的片段,将片段用酵母醋酸锂转化法转化至上述酿酒酵母GS-A3-C1菌株中,涂布到含500μg/mL G418和200μmol/L铜离子的YPD平板上,挑取转化子并提基因组PCR验证,获得对应正确的高产法尼醇的转化菌株GS-A3-C4。
实施例2
基因工程菌GS-A3-C5的构建:
构建方法同实施例1,其中步骤S3中选用的Linker连接为Linker2,构建的敲除半乳糖调节蛋白GAL80基因表达DNA片段为:
GAL80left_Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker2_PAH1_tERG20_GAL80right;
构建的重组质粒载体pCZ200为:
pCZ200ΔGAL80::Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker2_PAH1_tERG20。
构建的敲除半乳糖调节蛋白GAL80基因表达的工程菌为GS-A3-C2。
实施例3
基因工程菌GS-A3-C6的构建:
构建方法同实施例1,其中步骤S3中选用的Linker连接为Linker3,构建的敲除半乳糖调节蛋白GAL80基因表达DNA片段为:
GAL80left_Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker3_PAH1_tERG20_GAL80righ;
构建的重组质粒载体pCZ300为:
pCZ300ΔGAL80::Hyg_tCYC1_tHMG1_pGAL10pGAL1_ERG20_Linker3_PAH1_tERG20。
构建的敲除半乳糖调节蛋白GAL80基因表达的工程菌为GS-A3-C3。
对比例1
基因工程菌30000B-C2的构建
构建方法同实施例1,将步骤S3获得的质粒载体pCZ100和步骤S4获得质粒载体pCZ101,用限制性内切酶PmeI线性化,回收带有目的基因的片段,分两步将片段用酵母醋酸锂转化法转化至酿酒酵母底盘菌株30000B中,挑取转化子并提基因组PCR验证,获得对应正确的高产法尼醇的转化菌株30000B-C2。
对比例2
基因工程菌CEN.PK2-1D-C2的构建
构建方法同实施例1,将步骤S3获得的质粒载体pCZ100和步骤S4获得质粒载体pCZ101,用限制性内切酶PmeI线性化,回收带有目的基因的片段,分两步将片段用酵母醋酸锂转化法转化至酿酒酵母底盘菌株S.cerevisiae CEN.PK2-1D中,挑取转化子并提基因组PCR验证,获得对应正确的高产法尼醇的转化菌株CEN.PK2-1D-C2。
对比例3
基因工程菌BY474-C2的构建
构建方法同实施例1,将步骤S3获得的质粒载体pCZ100和步骤S4获得质粒载体pCZ101,用限制性内切酶PmeI线性化,回收带有目的基因的片段,分两步将片段用酵母醋酸锂转化法转化至酿酒酵母底盘菌株BY474中,挑取转化子并提基因组PCR验证,获得对应正确的高产法尼醇的转化菌株BY474-C2。
为了说明本发明构建的高产法尼醇基因工程菌的高产法尼醇的机理和应用能力,通过对不同Linker连接对法尼醇产量的影响,改造不同酿酒酵母底盘菌株的对法尼醇产量的影响进行了对比研究。
测定发酵液中法尼醇浓度的方法:
检测流程:
将45mL发酵液置于50mL离心管中,于10000r/min离心10min,小心吸取上层肉豆蔻酸异丙酯液体,用色谱级正己烷稀释至合适浓度检测。
仪器:使用安捷伦7890A气相色谱仪,色谱柱为Agilent HP-5(30mx0.32 mmx0.25μm),柱箱温度:初始温度100℃,保留2min,以10℃/min升至280℃, 保留3min,进样口温度280℃,进样量1μL,柱流量1mL/min,注入分流比1:50,检测器(FID)温度280℃,氮气作为载气,入口压力是12-18psi,模式为恒流模式,标准品:法尼醇(SIGMA)。
1.比较不同Linke连接对法尼醇产量的影响
分别取10μL保存在甘油管的菌株GS-A3-C1、菌株菌株GS-A3-C2和菌株GS-A3-C3到含有5mLYPD(含铜离子终浓度为200μmol/L)培养基的PA瓶中,30℃摇床220rpm培养16h,获得一级种子;将一级种子以1%的转接量转到含有200mL YPD培养基的摇瓶中,30℃摇床220rpm培养五天,测定法尼醇的含量。
如图4所示,结果显示菌株GS-A3-C1的产量高于GS-A3-C2和GS-A3-C3 为183.24±16.23mg/L。可见,Llinker-GSG更有利于ERG20和PAH1融合表达,提高由IPP到FPP再到法尼醇的反应效率,从而提高法尼醇的产量。
2.比较用启动子pCUP1替换pERG9对法尼醇产量的影响
取10μL保存在甘油管的菌株GS-A3-C4到含有5mLYPD(含铜离子终浓度为200μmol/L)培养基的PA瓶中,30℃摇床220rpm培养16h,获得一级种子;将一级种子以1%的转接量转到含有200mL YPD培养基的摇瓶中,30℃摇床220rpm培养五天,测定法尼醇的含量。
结果显示,GS-A3-C4的产量相较于于GS-A3-C1的183.24±16.23mg/L提高到573.62±43.82mg/L。可见,用启动子pCUP1替换pERG9,有利于减少FPP 流向麦角固醇,更多流向法尼醇途径,进而提高法尼醇的产量。
3.比较改造不同酿酒酵母底盘菌株的法尼醇产量
分别取10μL保存在甘油管的菌株GS-A3-C4、菌株30000B-C2、菌株 CEN.PK2-1D-C2和菌株BY474-C2到含有5mLYPD(含铜离子终浓度为 200μmol/L)培养基的PA瓶中,30℃摇床220rpm培养16h,获得一级种子;将一级种子以1%的转接量转到含有200mL YPD培养基的摇瓶中,30℃摇床 220rpm培养五天,测定法尼醇的含量。
如图5所示,结果显示菌株GS-A3-C4的法尼醇产量明显高于菌株 G30000B-C2、菌株CEN.PK2-1D-C2和菌株BY474-C2。可见,GS-A3是本申请人筛选进化的一株高产角鲨烯菌株,能提供更充足的前体物质,从而提高法尼醇的产量。
3.利用菌株GS-A3-C4发酵生产法尼醇
取10μL保存在甘油管的菌株到含有5mLYPD(含铜离子终浓度为 200μmol/L)培养基的PA瓶中,30℃摇床220rpm培养16h,获得一级种子;将一级种子以1%的转接量转到含有200mL YPD(含铜离子终浓度为200μmol/L)培养基的摇瓶中,30℃摇床220rpm培养12h,获得二级级种子;向5L生物发酵罐中加入2.5L发酵培养基,以10%的转接量接入活化的二级种子液。发酵过程中,温度控制在30℃,pH用浓氨水全程控制在5.0,发酵罐起始转速为200rpm,通气量为1L/min,溶氧100%,随着发酵的进行,OD不断增大,溶氧会不断下降,待降到20%左右,就通过逐步提升转速和通气量控制溶氧在 20%左右,直到最大转速和通气;前期通过调整补料一把发酵罐中葡萄糖浓度控制在1g/L以下;发酵到30小时时,加入发酵体积15%肉豆蔻酸异丙酯作为有机相,并改用补料二,将发酵罐中乙醇浓度维持在1g/L以下,发酵5天。
发酵培养基中包含如下组分:葡萄糖40g/L,酵母提取物5g/L,硫酸铵12g/L,磷酸二氢钾8g/L,七水硫酸镁6.2g/L,硫胺素200mg/L,吡哆素200mg/L,肌醇500mg/L,生物素50mg/L,泛酸钙200mg/L。
补料一组分为:葡萄糖800g/L,酵母提取物50g/L。
补料二组分为:蔗糖800g/L,酵母提取物50g/L。
最终发酵罐生产的法尼醇产量高达到21g/L,完全具备工业化生产能力,本发明构建的高产法尼醇基因工程菌可应用于工业生产法尼醇。
在不冲突的情况下,本文中上述实施例及实施例中的特征可以相互结合。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 湖北冠众通科技有限公司
<120> 一种高产法尼醇基因工程菌及其制备方法与应用
<141> 2021-10-22
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 668
<212> DNA
<213> 酿酒酵母 pGAL1-10
<400> 1
ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60
atcatattac atggcaatac caccatatac atatccatat ctaatcttac ttatatgttg 120
tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag 180
taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg 240
acagccctcc gacggaagac tctcctccgt gcgtcctggt cttcaccggt cgcgttcctg 300
aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct 360
tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaatcaac 420
gaatcaaatt aacaaccata ggataataat gcgattagtt ttttagcctt atttctgggg 480
taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat gcaaaagctg 540
cataaccact ttaactaata ctttcaacat tttcggtttg tattacttct tattcaaatg 600
tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa 660
aaactata 668
<210> 2
<211> 1509
<212> DNA
<213> 酿酒酵母 tHMG1
<400> 2
ttaggattta atgcaggtga cggacccatc tttcaaacga tttatatcag tggcgtccaa 60
attgttaggt tttgttggtt cagcaggttt cctgttgtgg gtcatatgac tttgaaccaa 120
atggccggct gctagggcag cacataagga taattcacct gccaagacgg cacaggcaac 180
tattcttgct aattgacgtg cgttggtacc aggagcggta gcatgcgggc ctcttacacc 240
taataagtcc aacatggcac cttgtggttc tagaacagta ccaccaccga tggtacctac 300
ttcgatggat ggcatggata cggaaattct caaatcaccg tccacttctt tcatcaatgt 360
tatacagttg gaactttcaa cattttgtgc aggatcttgt cctaatgcca agaaaacagc 420
tgtcactaaa ttagctgcat gtgcgttaaa tccaccaaca gacccagcca ttgcagatcc 480
aaccaaattc ttagcaatgt tcaactcaac caatgcggaa acatcacttt ttaacacttt 540
tctgacaaca tcaccaggaa tagtagcttc tgcgacgaca ctcttaccac gaccttcgat 600
ccagttgatg gcagctggtt ttttgtcggt acagtagtta ccagaaacgg agacaacctc 660
catatcttcc cagccatact cttctaccat ttgctttaat gagtattcga cacctttaga 720
aatcatattc atacccattg cgtcaccagt agttgttcta aatctcatga agagtaaatc 780
tcctgctaga caagtttgaa tatgttgcag acgtgcaaat cttgatgtag agttaaaagc 840
ttttttaatt gcgttttgtc cctcttctga gtctaaccat atcttacagg caccagatct 900
tttcaaagtt gggaaacgga ctactgggcc tcttgtcata ccatccttag ttaaaacagt 960
tgttgcacca ccgccagcat tgattgcctt acagccacgc atggcagaag ctaccaaaca 1020
accctctgta gttgccattg gtatatgata agatgtacca tcgataacca aggggcctat 1080
aacaccaacg ggcaaaggca tgtaacctat aacattttca caacaagcgc caaatacgcg 1140
gtcgtagtca taatttttat atggtaaacg atcagatgct aatacaggag cttctgccaa 1200
aattgaaaga gccttcctac gtaccgcaac cgctctcgta gtatcaccta attttttctc 1260
caaagcgtac aaaggtaact taccgtgaat aaccaaggca gcgacctctt tgttcttcaa 1320
ttgttttgta tttccactac ttaataatgc ttctaattct tctaaaggac gtattttctt 1380
atccaagctt tcaatatcgc gggaatcatc ttcctcacta gatgatgaag gtcctgatga 1440
gctcgattgc gcagatgata aacttttgac tttcgatcca gaaatgactg ttttattggt 1500
taaaaccat 1509
<210> 3
<211> 1059
<212> DNA
<213> 酿酒酵母 ERG20
<400> 3
atggcttcag aaaaagaaat taggagagag agattcttga acgttttccc taaattagta 60
gaggaattga acgcatcgct tttggcttac ggtatgccta aggaagcatg tgactggtat 120
gcccactcat tgaactacaa cactccaggc ggtaagctaa atagaggttt gtccgttgtg 180
gacacgtatg ctattctctc caacaagacc gttgaacaat tggggcaaga agaatacgaa 240
aaggttgcca ttctaggttg gtgcattgag ttgttgcagg cttacttctt ggtcgccgat 300
gatatgatgg acaagtccat taccagaaga ggccaaccat gttggtacaa ggttcctgaa 360
gttggggaaa ttgccatcaa tgacgcattc atgttagagg ctgctatcta caagcttttg 420
aaatctcact tcagaaacga aaaatactac atagatatca ccgaattgtt ccatgaggtc 480
accttccaaa ccgaattggg ccaattgatg gacttaatca ctgcacctga agacaaagtc 540
gacttgagta agttctccct aaagaagcac tccttcatag ttactttcaa gactgcttac 600
tattctttct acttgcctgt cgcattggcc atgtacgttg ccggtatcac ggatgaaaag 660
gatttgaaac aagccagaga tgtcttgatt ccattgggtg aatacttcca aattcaagat 720
gactacttag actgcttcgg taccccagaa cagatcggta agatcggtac agatatccaa 780
gataacaaat gttcttgggt aatcaacaag gcattggaac ttgcttccgc agaacaaaga 840
aagactttag acgaaaatta cggtaagaag gactcagtcg cagaagccaa atgcaaaaag 900
attttcaatg acttgaaaat tgaacagcta taccacgaat atgaagagtc tattgccaag 960
gatttgaagg ccaaaatttc tcaggtcgat gagtctcgtg gcttcaaagc tgatgtctta 1020
actgcgttct tgaacaaagt ttacaagaga agcaaatag 1059
<210> 4
<211> 2589
<212> DNA
<213> 酿酒酵母 PAH1
<400> 4
atgcagtacg taggcagagc tcttgggtct gtgtctaaaa catggtcttc tatcaatccg 60
gctacgctat caggtgctat agatgtcatt gtagtggagc atccagacgg aaggctatca 120
tgttctccct ttcatgtgag gttcggcaaa tttcaaattc taaagccatc tcaaaagaaa 180
gtccaagtgt ttataaatga gaaactgagt aatatgccaa tgaaactgag tgattctgga 240
gaagcctatt tcgttttcga gatgggtgac caggtcactg atgtccctga cgaattgctt 300
gtgtcgcccg tgatgagcgc cacatcaagc ccccctcaat cacctgaaac atccatctta 360
gaaggaggaa ccgagggtga aggtgaaggt gaaaatgaaa ataagaagaa ggaaaagaaa 420
gtgctagagg aaccagattt tttagatatc aatgacactg gagattcagg cagtaaaaat 480
agtgaaacta cagggtcgct ttctcctact gaatcctcta caacgacacc accagattca 540
gttgaagaga ggaagcttgt tgagcagcgt acaaagaact ttcagcaaaa actaaacaaa 600
aaactcactg aaatccatat acccagtaaa cttgataaca atggcgactt actactagac 660
actgaaggtt acaagccaaa caagaatatg atgcatgaca cagacataca actgaagcag 720
ttgttaaagg acgaattcgg taatgattca gatatttcca gttttatcaa ggaggacaaa 780
aatggcaaca tcaagatcgt aaatccttac gagcacctta ctgatttatc tcctccaggt 840
acgcctccaa caatggccac aagcggatca gttttaggct tagatgcaat ggaatcagga 900
agtactttga attcgttatc ttcttcacct tctggttccg atactgagga cgaaacatca 960
tttagcaaag aacaaagcag taaaagtgaa aaaactagca agaaaggaac agcagggagc 1020
ggtgagaccg agaaaagata catacgaacg ataagattga ctaatgacca gttaaagtgc 1080
ctaaatttaa cttatggtga aaatgatctg aaattttccg tagatcacgg aaaagctatt 1140
gttacgtcaa aattattcgt ttggaggtgg gatgttccaa ttgttatcag tgatattgat 1200
ggcaccatca caaaatcgga cgctttaggc catgttctgg caatgatagg aaaagactgg 1260
acgcacttgg gtgtagccaa gttatttagc gagatctcca ggaatggcta taatatactc 1320
tatctaactg caagaagtgc tggacaagct gattccacga ggagttattt gcgatcaatt 1380
gaacagaatg gcagcaaact accaaatggg cctgtgattt tatcacccga tagaacgatg 1440
gctgcgttaa ggcgggaagt aatactaaaa aaacctgaag tctttaaaat cgcgtgtcta 1500
aacgacataa gatccttgta ttttgaagac agtgataacg aagtggatac agaggaaaaa 1560
tcaacaccat tttttgccgg ctttggtaat aggattactg atgctttatc ttacagaact 1620
gtggggatac ctagttcaag aattttcaca ataaatacag agggtgaggt tcatatggaa 1680
ttattggagt tagcaggtta cagaagctcc tatattcata tcaatgagct tgtcgatcat 1740
ttctttccac cagtcagcct tgatagtgtc gatctaagaa ctaatacttc catggttcct 1800
ggctcccccc ctaatagaac gttggataac tttgactcag aaattacttc aggtcgcaaa 1860
acgctattta gaggcaatca ggaagagaaa ttcacagacg taaatttttg gagagacccg 1920
ttagtcgaca tcgacaactt atcggatatt agcaatgatg attctgataa catcgatgaa 1980
gatactgacg tatcacaaca aagcaacatt agtagaaata gggcaaattc agtcaaaacc 2040
gccaaggtca ctaaagcccc gcaaagaaat gtgagcggca gcacaaataa caacgaagtt 2100
ttagccgctt cgtctgatgt agaaaatgcg tctgacctgg tgagttccca tagtagctca 2160
ggatccacgc ccaataaatc tacaatgtcc aaaggggaca ttggaaaaca aatatatttg 2220
gagctaggtt ctccacttgc atcgccaaaa ctaagatatt tagacgatat ggatgatgaa 2280
gactccaatt acaatagaac taaatcaagg agagcatctt ctgcagccgc gactagtatc 2340
gataaagagt tcaaaaagct ctctgtgtca aaggccggcg ctccaacaag aattgtttca 2400
aagatcaacg tttcaaatga cgtacattca cttgggaatt cagataccga atcacgaagg 2460
gagcaaagtg ttaatgaaac agggcgcaat cagctacccc acaactcaat ggacgataaa 2520
gatttggatt caagagtaag cgatgaattc gatgacgatg aattcgacga agatgaattc 2580
gaagattaa 2589
<210> 5
<211> 459
<212> DNA
<213> 酿酒酵母 pCUP1
<400> 5
cgatcccatt accgacattt gggcgctata cgtgcatatg ttcatgtatg tatctgtatt 60
taaaacactt ttgtattatt tttcctcata tatgtgtata ggtttatacg gatgatttaa 120
ttattacttc accacccttt atttcaggct gatatcttag ccttgttact agttagaaaa 180
agacattttt gctgtcagtc actgtcaaga gattcttttg ctggcatttc ttctagaagc 240
aaaaagagcg atgcgtcttt tccgctgaac cgttccagca aaaaagacta ccaacgcaat 300
atggattgtc agaatcatat aaaagagaag caaataactc cttgtcttgt atcaattgca 360
ttataatatc ttcttgttag tgcaatatca tatagaagtc atcgaaatag atattaagaa 420
aaacaaactg tacaatcaat caatcaatca tcacataaa 459
<210> 6
<211> 450
<212> DNA
<213> 酿酒酵母 pERG9
<400> 6
tgcgaagcct gctaaaatgc agtggaggcc gtgtaccctt tgccaaattg gctattggaa 60
tcggcagaga acctgggtcc cgttctagag accctgcgag cgtgtcccgg tgggttctgg 120
gagctctaac tccgcaggaa ctacaaacct tgcttacaca gagtgaacct gctgcctggc 180
gtgctctgac tcagtacatt tcatagccca tcttcaacaa caataccgac ttaccatcct 240
atttgctttg ccctttttct tttccactgc actttgcatc ggaaggcgtt atcggttttg 300
ggtttagtgc ctaaacgagc agcgagaaca cgaccacggg ctatataaat ggaaagttag 360
gacaggggca aagaataaga gcacagaaga agagaaaaga cgaagagcag aagcggaaaa 420
cgtatacacg tcacatatca cacacacaca 450
<210> 7
<211> 273
<212> DNA
<213> 酿酒酵母 tCYC1
<400> 7
gcaaattaaa gccttcgagc gtcccaaaac cttctcaagc aaggttttca gtataatgtt 60
acatgcgtac acgcgtttgt acagaaaaaa aagaaaaatt tgaaatataa ataacgttct 120
taatactaac ataactataa aaaaataaat agggacctag acttcaggtt gtctaactcc 180
ttccttttcg gttagagcgg atgtgggggg agggcgtgaa tgtaagcgtg acataactaa 240
ttacatgata tcgacaaagg aaaaggggcc tgt 273
<210> 8
<211> 149
<212> DNA
<213> 酿酒酵母 tERG20
<400> 8
aactaacgct aatcgataaa acattagatt tcaaactaga taaggaccat gtataagaac 60
tatatacttc caatataata tagtataagc tttaagatag tatctctcga tctaccgttc 120
cacgtgacta gtccaaggat tttttttaa 149
Claims (10)
1.一种高产法尼醇基因工程菌的构建方法,其特征在于:包括以下步骤:
S1、构建酿酒酵母MVA途径相关基因表达模块:过表达tHMG1模块,包括诱导型双向强启动子pGAL1-10和MVA途径限速酶编码基因tHMG1,所述pGAL1-10的核苷酸序列如SEQ NO.1所示,所述tHMG1的核苷酸序列如SEQ NO.2所示;
S2、构建法尼醇生产相关基因表达模块:ERG20-Linker-PAH1模块,包括FPP合酶的编码基因ERG20和酿酒酵母内源性磷酸酶的编码基因PAH1,所述ERG20的核苷酸序列如SEQ NO.3所示,所述PAH1的核苷酸序列如SEQ NO.4所示;
S3、构建敲除半乳糖调节蛋白GAL80基因表达的工程菌:将步骤S1所述酿酒酵母MVA途径相关基因表达模块和步骤S2所述法尼醇生产相关基因表达模块整合至半乳糖调节蛋白GAL80基因位点,同时敲除半乳糖调节蛋白GAL80基因,得到敲除半乳糖调节蛋白GAL80基因表达模块,将所述敲除半乳糖调节蛋白GAL80基因表达模块转化至构建的酿酒酵母基因工程菌GS-A3中,经多次基因工程操作,获得敲除半乳糖调节蛋白GAL80基因表达的基因工程菌;
S4、构建角鲨烯合酶途径下调表达质粒,包括将铜离子诱导启动子pCUP1替换为角鲨烯合酶基因ERG9启动子,所述pCUP1的核苷酸序列如SEQ NO.5所示;所述角鲨烯合酶基因ERG9启动子取至ERG9基因起始密码子前450bp,其核苷酸序列如SEQ NO.6所示;
S5、将步骤S5所述角鲨烯合酶途径下调表达质粒转化至步骤S3所述的敲除半乳糖调节蛋白GAL80基因表达的基因工程菌中,依次经抗生素抗性筛选,然后通过菌落PCR验证,获得高产法尼醇基因工程菌;
所述构建方法还包括两个终止子tCYC1和tERG20,所述终止子tCYC1的核苷酸序列如SEQ NO.7所示,所述终止子tERG20的核苷酸序列如SEQ NO.8所示。
2.如权利要求1所述的一种高产法尼醇基因工程菌的构建方法,其特征在于:步骤S1中,所述过表达tHMG1模块的构建方法包括以下步骤:
S1、以酿酒酵母3000B基因组DNA为模板,分别用tCYC1-F和tCYC1-R、tHMG1-F和tHMG1-R、pGAL1pGAL10-F和pGAL1pGAL10-R引物进行PCR反应,获得DNA片段tCYC1、tHMG1和pGAL10pGAL1;
S2、将步骤S1获得的三个DNA片段tCYC1、tHMG1和pGAL10pGAL1,通过用引物tCYC1-F和pGAL1pGAL10-R进行重叠延伸PCR反应,连接在一起,获得过表达tHMG1模块,即tCYC1_tHMG1_pGAL10pGAL1模块。
3.如权利要求1所述的一种高产法尼醇基因工程菌的构建方法,其特征在于:步骤S2中,所述ERG20-Linker-PAH1模块的构建方法包括以下步骤:
S1、以酿酒酵母3000B基因组DNA为模板,分别用ERG20-F和ERG20-Linker-R引物进行PCR反应,扩增得到DNA片段ERG20_Linker;
S2、用引物Linker-PAH1-F和PAH1-R进行PCR反应,扩增得到DNA片段Linker_PAH1;
S3、用引物tERG20-F和tERG20-R进行PCR反应,扩增得到DNA片段tERG20;
S4、将步骤S1获得的DNA片段ERG20_Linker、步骤S2获得的DNA片段Linker_PAH1和步骤S3获得的DNA片段tERG20,通过用引物ERG20-F和tERG20-R进行重叠延伸PCR反应,连接在一起,获得融合表达ERG20-Linker-PAH1模块,即ERG20_Linker_PAH1_tERG20。
4.如权利要求3所述的一种高产法尼醇基因工程菌的构建方法,其特征在于:所述Linker包括甘氨酸和丝氨酸,其组合结构包括GSG、GGGS和GSGGSG中的任一种,其中G对应核酸序列GGT,S对应核酸序列TCT。
5.如权利要求4所述的一种高产法尼醇基因工程菌的构建方法,其特征在于:步骤S2中,所述ERG20-Linker-PAH1模块中的Linker为GSG。
6.如权利要求1所述的一种高产法尼醇基因工程菌的构建方法,其特征在于:步骤S3中,所述构建敲除半乳糖调节蛋白GAL80基因表达模块的构建方法包括以下步骤:
S1、以酿酒酵母3000B基因组DNA为模板,分别用GAL80left-F和GAL80left-R、GAL80right-F和GAL80right-R引物进行PCR反应,扩增得到DNA片段GAL80的左右同源臂GAL80left和GAL80right;
S2、以质粒载体pFZ201为模板,用引物Hyg-F和Hyg-R进行PCR反应,获得潮霉素表达盒Hyg;
S3、将所述过表达tHMG1模块和所述ERG20-Linker-PAH1模块、GAL80left,GAL80right和Hyg五个DNA片段,通过用引物GAL80left-F和GAL80right-R进行重叠延伸PCR反应,连接在一起,获得DNA片段为敲除半乳糖调节蛋白GAL80基因表达模块的;
S4、将步骤S3获得的DNA片段与质粒pMD19-T连接,获得重组质粒载体,用限制性内切酶PmeI线性化所述重组质粒,回收带有目的基因的片段,将片段用酵母醋酸锂转化法转化至宿主菌酿酒酵母中,依次经抗生素抗性筛选,然后通过菌落PCR获取阳性菌落,获得敲除半乳糖调节蛋白GAL80基因表达的工程菌。
7.如权利要求6所述的一种高产法尼醇基因工程菌的构建方法,其特征在于:步骤S3中,所述宿主菌酿酒酵母包括酿酒酵母30000B、酿酒酵母S.cerevisiae CEN.PK2-1D、酿酒酵母BY4741和酿酒酵母GS-A3中的任一种,所述酿酒酵母GS-A3于2021年9月17日保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:M20211191。
8.如权利要求1所述的一种高产法尼醇基因工程菌的构建方法,其特征在于:步骤S4中,所述角鲨烯合酶途径下调表达质粒的构建方法包括以下步骤:
S1、以酿酒酵母3000B基因组DNA为模板,分别用pERG9-left-F和pERG9-left-R、pCUP1-F和pCUP1-R、pERG9-right-F和pERG9-right-R引物进行PCR反应,扩增得到DNA片段pERG9-left、pCUP1和pERG9-righ;
S2、以质粒载体pFZ202为模板,用引物G418-F和G418-R进行PCR反应,获得G418表达盒G418;
S3、将步骤S1获得的DNA片段pERG9-left、pCUP1和pERG9-righ和步骤S1获得的表达盒G418五个DNA片段,通过用引物pERG9-left-F和pERG9-right-R进行重叠延伸PCR反应,连接在一起,获得DNA片段G418_pCUP1/ΔpEGR9;
S4、将步骤S3获得的DNA片段G418_pCUP1/ΔpEGR9与质粒pMD19-T连接,获得角鲨烯合酶途径下调表达质粒载体,记为pCZ101。
9.一种高产法尼醇基因工程菌,其特征在于:采用如权利要求1-8任一项所述的构建方法得到的。
10.一种如权利要求9所述的高产法尼醇基因工程菌的应用,其特征在于:.利用所述的基因工程菌发酵生产法尼醇,所述高产法尼醇基因工程菌的液体发酵培养基组分包括葡萄糖20-50g/L,酵母提取物5-10g/L,硫酸铵6-15g/L,磷酸二氢钾3-8g/L,七水硫酸镁5-10g/L,硫胺素100-500mg/L,吡哆素100-500mg/L,肌醇400-800mg/L,生物素20-100mg/L和泛酸钙100-500mg/L。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111273441.5A CN113969288A (zh) | 2021-10-29 | 2021-10-29 | 一种高产法尼醇基因工程菌及其构建方法与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111273441.5A CN113969288A (zh) | 2021-10-29 | 2021-10-29 | 一种高产法尼醇基因工程菌及其构建方法与应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113969288A true CN113969288A (zh) | 2022-01-25 |
Family
ID=79588985
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111273441.5A Pending CN113969288A (zh) | 2021-10-29 | 2021-10-29 | 一种高产法尼醇基因工程菌及其构建方法与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113969288A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112063647A (zh) * | 2020-09-17 | 2020-12-11 | 云南农业大学 | 酿酒酵母重组菌Cuol01的构建方法、酿酒酵母重组菌Cuol02及应用 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000001650A1 (en) * | 1998-07-06 | 2000-01-13 | Dcv, Inc. Doing Business As Bio-Technical Resources | Method of vitamin production |
US20040063182A1 (en) * | 2000-12-28 | 2004-04-01 | Chikara Ohto | Process for producing prenyl alcohol |
JP2006055052A (ja) * | 2004-08-19 | 2006-03-02 | Hakutsuru Shuzo Kk | ファルネソール生産性酵母 |
US20080213847A1 (en) * | 2000-12-28 | 2008-09-04 | Chikara Ohto | Process for producing prenyl alcohols |
KR20160047870A (ko) * | 2014-10-23 | 2016-05-03 | 한국생명공학연구원 | 연속 대사경로 효소 집합체 구축을 통한 이소프레노이드 고효율 생산 방법 |
CN112094765A (zh) * | 2020-09-24 | 2020-12-18 | 江南大学 | 一种生产法尼烯的酿酒酵母工程菌的构建和应用 |
-
2021
- 2021-10-29 CN CN202111273441.5A patent/CN113969288A/zh active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000001650A1 (en) * | 1998-07-06 | 2000-01-13 | Dcv, Inc. Doing Business As Bio-Technical Resources | Method of vitamin production |
US20040063182A1 (en) * | 2000-12-28 | 2004-04-01 | Chikara Ohto | Process for producing prenyl alcohol |
US20080213847A1 (en) * | 2000-12-28 | 2008-09-04 | Chikara Ohto | Process for producing prenyl alcohols |
JP2006055052A (ja) * | 2004-08-19 | 2006-03-02 | Hakutsuru Shuzo Kk | ファルネソール生産性酵母 |
KR20160047870A (ko) * | 2014-10-23 | 2016-05-03 | 한국생명공학연구원 | 연속 대사경로 효소 집합체 구축을 통한 이소프레노이드 고효율 생산 방법 |
CN112094765A (zh) * | 2020-09-24 | 2020-12-18 | 江南大学 | 一种生产法尼烯的酿酒酵母工程菌的构建和应用 |
Non-Patent Citations (1)
Title |
---|
吴伟鹏;于洪巍;叶丽丹;周萍萍;姚震;边旗;: "基于关键节点代谢流重排提高酿酒酵母的香叶基香叶醇产量", 高校化学工程学报, no. 04 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112063647A (zh) * | 2020-09-17 | 2020-12-11 | 云南农业大学 | 酿酒酵母重组菌Cuol01的构建方法、酿酒酵母重组菌Cuol02及应用 |
CN112063647B (zh) * | 2020-09-17 | 2023-05-02 | 云南农业大学 | 酿酒酵母重组菌Cuol01的构建方法、酿酒酵母重组菌Cuol02及应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN114058525A (zh) | 一种高产角鲨烯基因工程菌及其构建方法与应用 | |
CN110607247B (zh) | 一种提高酿酒酵母合成角鲨烯能力的方法 | |
CN112695003B (zh) | 一种高产西柏三烯一醇的基因工程菌及其构建方法与应用 | |
CN113621631A (zh) | 一种甲羟戊酸激酶基因rkmk及其应用 | |
CN114621968A (zh) | 四氢嘧啶生物合成基因簇、突变体及制备四氢嘧啶的方法 | |
CN114107078A (zh) | 一种高产瓦伦烯基因工程菌及其构建方法与应用 | |
CN110804561B (zh) | 一种高产c6-c10乙基酯的酿酒酵母及其构建方法与用途 | |
CN114107152B (zh) | 一种高产3-岩藻糖基乳糖微生物的构建方法及应用 | |
CN113969288A (zh) | 一种高产法尼醇基因工程菌及其构建方法与应用 | |
CN111088175A (zh) | 一种产红没药烯的解脂耶氏酵母及其构建方法与用途 | |
CN107034150A (zh) | 一种重组耶氏解脂酵母菌株及其构建方法与应用 | |
CN111607546B (zh) | 一种高产法尼烯的基因工程菌及其构建方法与应用 | |
CN111154665B (zh) | 一株重组解脂耶罗维亚酵母及其构建方法和应用 | |
CN112608936A (zh) | 调控酵母外源基因表达的启动子,调控方法及其应用 | |
CN115873836B (zh) | 一种橙花叔醇合成酶及应用 | |
CN110305855A (zh) | 天麻GeCPR基因及其应用 | |
CN113817757B (zh) | 一种生产樱桃苷的重组酵母工程菌株及应用 | |
CN114277068B (zh) | 一种r-3-羟基丁酸乙酯微生物发酵制备方法 | |
CN114774297B (zh) | 产t-杜松醇的重组酿酒酵母及其应用 | |
CN110004099A (zh) | 一种红景天苷的发酵生产方法 | |
CN114517164A (zh) | 一种脂肪酸延长酶基因在酵母合成神经酸中的应用 | |
CN112646834A (zh) | 一种羽扇豆醇衍生物及其合成方法和应用 | |
CN114806914B (zh) | 一种高产β-胡萝卜素的解脂耶氏酵母菌及其应用 | |
CN115305254B (zh) | 一种萜类底盘微生物与工程菌及其构建方法和应用 | |
CN114606146B (zh) | 一种生产d-柠檬烯的酵母及其应用 |
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 |