CN114574516B - 一种基于CRISPR/Cas9的酵母基因组稳定整合方法 - Google Patents
一种基于CRISPR/Cas9的酵母基因组稳定整合方法 Download PDFInfo
- Publication number
- CN114574516B CN114574516B CN202210053387.1A CN202210053387A CN114574516B CN 114574516 B CN114574516 B CN 114574516B CN 202210053387 A CN202210053387 A CN 202210053387A CN 114574516 B CN114574516 B CN 114574516B
- Authority
- CN
- China
- Prior art keywords
- genbank accession
- accession number
- artificial sequence
- integration
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000010354 integration Effects 0.000 title claims abstract description 83
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 55
- 108091033409 CRISPR Proteins 0.000 title claims abstract description 25
- 238000010453 CRISPR/Cas method Methods 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 60
- 230000006696 biosynthetic metabolic pathway Effects 0.000 claims abstract description 16
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 50
- 230000037361 pathway Effects 0.000 claims description 17
- IXQKXEUSCPEQRD-DKRGWESNSA-N cucurbitacin B Chemical compound C([C@H]1[C@]2(C)C[C@@H](O)[C@@H]([C@]2(CC(=O)[C@]11C)C)[C@@](C)(O)C(=O)/C=C/C(C)(C)OC(=O)C)C=C2[C@H]1C[C@H](O)C(=O)C2(C)C IXQKXEUSCPEQRD-DKRGWESNSA-N 0.000 claims description 15
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 claims description 10
- 235000013793 astaxanthin Nutrition 0.000 claims description 10
- 239000001168 astaxanthin Substances 0.000 claims description 10
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 claims description 10
- 229940022405 astaxanthin Drugs 0.000 claims description 10
- 108090000201 Carboxypeptidase B2 Proteins 0.000 claims description 7
- 101000783542 Catharanthus roseus 7-deoxyloganetic acid glucosyl transferase Proteins 0.000 claims description 7
- 101100454340 Catharanthus roseus LAMT gene Proteins 0.000 claims description 7
- 102100039556 Galectin-4 Human genes 0.000 claims description 7
- 101000608765 Homo sapiens Galectin-4 Proteins 0.000 claims description 7
- 101150021948 SAM2 gene Proteins 0.000 claims description 7
- 101150085516 ZWF1 gene Proteins 0.000 claims description 7
- 102100034044 All-trans-retinol dehydrogenase [NAD(+)] ADH1B Human genes 0.000 claims description 5
- 101710193111 All-trans-retinol dehydrogenase [NAD(+)] ADH4 Proteins 0.000 claims description 5
- 101001059200 Arabidopsis thaliana Heterodimeric geranylgeranyl pyrophosphate synthase large subunit 2 Proteins 0.000 claims description 5
- 102100031655 Cytochrome b5 Human genes 0.000 claims description 5
- 101000922386 Homo sapiens Cytochrome b5 Proteins 0.000 claims description 5
- 101710130324 NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 claims description 5
- 101150028535 CRTZ gene Proteins 0.000 claims description 4
- 101100127715 Phaffia rhodozyma crtYB gene Proteins 0.000 claims description 4
- 101100061456 Streptomyces griseus crtB gene Proteins 0.000 claims description 4
- 101150011633 crtI gene Proteins 0.000 claims description 4
- 101100114901 Streptomyces griseus crtI gene Proteins 0.000 claims description 3
- 101150000046 crtE gene Proteins 0.000 claims description 3
- -1 7-DLH Proteins 0.000 claims description 2
- 101150078509 ADH2 gene Proteins 0.000 claims description 2
- 101150026777 ADH5 gene Proteins 0.000 claims description 2
- 101100460671 Aspergillus flavus (strain ATCC 200026 / FGSC A1120 / IAM 13836 / NRRL 3357 / JCM 12722 / SRRC 167) norA gene Proteins 0.000 claims description 2
- 238000010354 CRISPR gene editing Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 70
- 230000014509 gene expression Effects 0.000 description 21
- 238000000855 fermentation Methods 0.000 description 10
- 230000004151 fermentation Effects 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 9
- 108700039887 Essential Genes Proteins 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 240000001592 Amaranthus caudatus Species 0.000 description 6
- 235000009328 Amaranthus caudatus Nutrition 0.000 description 6
- 235000012735 amaranth Nutrition 0.000 description 6
- 239000004178 amaranth Substances 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000010362 genome editing Methods 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- SEHFUALWMUWDKS-UHFFFAOYSA-N 5-fluoroorotic acid Chemical compound OC(=O)C=1NC(=O)NC(=O)C=1F SEHFUALWMUWDKS-UHFFFAOYSA-N 0.000 description 4
- 101100246753 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) pyrF gene Proteins 0.000 description 4
- 101100317378 Mus musculus Wnt3 gene Proteins 0.000 description 4
- 101150050575 URA3 gene Proteins 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 101100011399 Danio rerio eif3ea gene Proteins 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 102000008016 Eukaryotic Initiation Factor-3 Human genes 0.000 description 3
- 101150008815 INT6 gene Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 230000008707 rearrangement Effects 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 102100037944 Integrator complex subunit 12 Human genes 0.000 description 2
- 101710149803 Integrator complex subunit 12 Proteins 0.000 description 2
- 102100030147 Integrator complex subunit 7 Human genes 0.000 description 2
- 101710092890 Integrator complex subunit 7 Proteins 0.000 description 2
- 101000767160 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Intracellular protein transport protein USO1 Proteins 0.000 description 2
- AEQDJSLRWYMAQI-UHFFFAOYSA-N Tetrahydropalmatine Natural products C1CN2CC(C(=C(OC)C=C3)OC)=C3CC2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- AEQDJSLRWYMAQI-KRWDZBQOSA-N tetrahydropalmatine Chemical compound C1CN2CC(C(=C(OC)C=C3)OC)=C3C[C@H]2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-KRWDZBQOSA-N 0.000 description 2
- APJYDQYYACXCRM-UHFFFAOYSA-N tryptamine Chemical compound C1=CC=C2C(CCN)=CNC2=C1 APJYDQYYACXCRM-UHFFFAOYSA-N 0.000 description 2
- FQVLRGLGWNWPSS-BXBUPLCLSA-N (4r,7s,10s,13s,16r)-16-acetamido-13-(1h-imidazol-5-ylmethyl)-10-methyl-6,9,12,15-tetraoxo-7-propan-2-yl-1,2-dithia-5,8,11,14-tetrazacycloheptadecane-4-carboxamide Chemical compound N1C(=O)[C@@H](NC(C)=O)CSSC[C@@H](C(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@@H]1CC1=CN=CN1 FQVLRGLGWNWPSS-BXBUPLCLSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- CJDRUOGAGYHKKD-RQBLFBSQSA-N 1pon08459r Chemical compound CN([C@H]1[C@@]2(C[C@@]3([H])[C@@H]([C@@H](O)N42)CC)[H])C2=CC=CC=C2[C@]11C[C@@]4([H])[C@H]3[C@H]1O CJDRUOGAGYHKKD-RQBLFBSQSA-N 0.000 description 1
- 101150066797 ARP7 gene Proteins 0.000 description 1
- 101710190443 Acetyl-CoA carboxylase 1 Proteins 0.000 description 1
- ATSKDYKYMQVTGH-DNCUWRPASA-N Amaranthin Natural products O=C(O)[C@@H]1[C@@H](O)[C@H](O)[C@H](O)[C@H](O[C@@H]2[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]2Oc2c(O)cc3/[N+](=C\C=C\4/C=C(C(=O)O)N[C@@H](C(=O)O)C/4)/[C@@H](C(=O)[O-])Cc3c2)O1 ATSKDYKYMQVTGH-DNCUWRPASA-N 0.000 description 1
- 102100021334 Bcl-2-related protein A1 Human genes 0.000 description 1
- 101100042788 Caenorhabditis elegans him-1 gene Proteins 0.000 description 1
- 101100410043 Caenorhabditis elegans rpn-12 gene Proteins 0.000 description 1
- 101100521421 Caenorhabditis elegans rpt-5 gene Proteins 0.000 description 1
- 102100032254 DNA-directed RNA polymerases I, II, and III subunit RPABC1 Human genes 0.000 description 1
- 102100025734 Dual specificity protein phosphatase CDC14A Human genes 0.000 description 1
- 102100035493 E3 ubiquitin-protein ligase NEDD4-like Human genes 0.000 description 1
- 102100028138 F-box/WD repeat-containing protein 7 Human genes 0.000 description 1
- 102100039111 FAD-linked sulfhydryl oxidase ALR Human genes 0.000 description 1
- 101150038242 GAL10 gene Proteins 0.000 description 1
- 102100024637 Galectin-10 Human genes 0.000 description 1
- 102100028652 Gamma-enolase Human genes 0.000 description 1
- 208000031448 Genomic Instability Diseases 0.000 description 1
- 108020005004 Guide RNA Proteins 0.000 description 1
- 101001088179 Homo sapiens DNA-directed RNA polymerases I, II, and III subunit RPABC1 Proteins 0.000 description 1
- 101000932600 Homo sapiens Dual specificity protein phosphatase CDC14A Proteins 0.000 description 1
- 101001023703 Homo sapiens E3 ubiquitin-protein ligase NEDD4-like Proteins 0.000 description 1
- 101001060231 Homo sapiens F-box/WD repeat-containing protein 7 Proteins 0.000 description 1
- 101000959079 Homo sapiens FAD-linked sulfhydryl oxidase ALR Proteins 0.000 description 1
- 101001058231 Homo sapiens Gamma-enolase Proteins 0.000 description 1
- 101000973510 Homo sapiens Melanoma-derived growth regulatory protein Proteins 0.000 description 1
- 101000579123 Homo sapiens Phosphoglycerate kinase 1 Proteins 0.000 description 1
- 101000721172 Homo sapiens Protein DBF4 homolog A Proteins 0.000 description 1
- 101000873111 Homo sapiens Vesicle transport protein SEC20 Proteins 0.000 description 1
- 101000615747 Homo sapiens tRNA-splicing endonuclease subunit Sen2 Proteins 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 108091029795 Intergenic region Proteins 0.000 description 1
- CJDRUOGAGYHKKD-UHFFFAOYSA-N Iso-ajmalin Natural products CN1C2=CC=CC=C2C2(C(C34)O)C1C1CC3C(CC)C(O)N1C4C2 CJDRUOGAGYHKKD-UHFFFAOYSA-N 0.000 description 1
- 102100022185 Melanoma-derived growth regulatory protein Human genes 0.000 description 1
- 101001088317 Methanothermobacter thermautotrophicus (strain ATCC 29096 / DSM 1053 / JCM 10044 / NBRC 100330 / Delta H) DNA-directed RNA polymerase subunit Rpo5 Proteins 0.000 description 1
- 101100446506 Mus musculus Fgf3 gene Proteins 0.000 description 1
- 101100348848 Mus musculus Notch4 gene Proteins 0.000 description 1
- 101100068676 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) gln-1 gene Proteins 0.000 description 1
- 101100407828 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) ptr-3 gene Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- KJWZYMMLVHIVSU-IYCNHOCDSA-N PGK1 Chemical compound CCCCC[C@H](O)\C=C\[C@@H]1[C@@H](CCCCCCC(O)=O)C(=O)CC1=O KJWZYMMLVHIVSU-IYCNHOCDSA-N 0.000 description 1
- 102100028251 Phosphoglycerate kinase 1 Human genes 0.000 description 1
- 102100025198 Protein DBF4 homolog A Human genes 0.000 description 1
- 101150087675 RIB5 gene Proteins 0.000 description 1
- 244000061121 Rauvolfia serpentina Species 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 101100010928 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) tuf gene Proteins 0.000 description 1
- 101100473113 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) RPO31 gene Proteins 0.000 description 1
- 241000793189 Saccharomyces cerevisiae BY4741 Species 0.000 description 1
- 101000832889 Scheffersomyces stipitis (strain ATCC 58785 / CBS 6054 / NBRC 10063 / NRRL Y-11545) Alcohol dehydrogenase 2 Proteins 0.000 description 1
- 108091027544 Subgenomic mRNA Proteins 0.000 description 1
- 101150001810 TEAD1 gene Proteins 0.000 description 1
- 101150074253 TEF1 gene Proteins 0.000 description 1
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 description 1
- 101000642183 Trypanosoma brucei brucei Terminal uridylyltransferase 2 Proteins 0.000 description 1
- 102100030441 Ubiquitin-conjugating enzyme E2 Z Human genes 0.000 description 1
- 101710192875 Ubiquitin-conjugating enzyme E2 Z Proteins 0.000 description 1
- 102100035030 Vesicle transport protein SEC20 Human genes 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 229960004332 ajmaline Drugs 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- OENHQHLEOONYIE-UKMVMLAPSA-N all-trans beta-carotene Natural products CC=1CCCC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C OENHQHLEOONYIE-UKMVMLAPSA-N 0.000 description 1
- ATSKDYKYMQVTGH-POBNKHOBSA-N amaranthin Chemical compound [N+]1([C@H](C([O-])=O)CC=2C=C(C(=CC=21)O)O[C@@H]1O[C@@H]([C@H]([C@H](O)[C@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@H](O1)C(O)=O)O)O)CO)=C\C=C1/C[C@@H](C(O)=O)NC(C(O)=O)=C1 ATSKDYKYMQVTGH-POBNKHOBSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000006793 arrhythmia Effects 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 235000013734 beta-carotene Nutrition 0.000 description 1
- 239000011648 beta-carotene Substances 0.000 description 1
- TUPZEYHYWIEDIH-WAIFQNFQSA-N beta-carotene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2=CCCCC2(C)C TUPZEYHYWIEDIH-WAIFQNFQSA-N 0.000 description 1
- 229960002747 betacarotene Drugs 0.000 description 1
- 230000008238 biochemical pathway Effects 0.000 description 1
- 230000008236 biological pathway Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000003198 gene knock in Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229930000223 plant secondary metabolite Natural products 0.000 description 1
- 230000003234 polygenic effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 101150077823 rpc17 gene Proteins 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 102100021774 tRNA-splicing endonuclease subunit Sen2 Human genes 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
- BLGXFZZNTVWLAY-SCYLSFHTSA-N yohimbine Chemical class C1=CC=C2C(CCN3C[C@@H]4CC[C@H](O)[C@@H]([C@H]4C[C@H]33)C(=O)OC)=C3NC2=C1 BLGXFZZNTVWLAY-SCYLSFHTSA-N 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- 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
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in 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
- 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)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种基于CRISPR/Cas9的酵母基因组稳定整合方法。本发明通过筛选在酵母基因组中筛选到了17个稳定整合的位点，可以用于外源基因的稳定整合，特别是对于需要整个多个基因的化合物生物合成途径的整合，具有很好的效果。本发明开发的基于CRISPR/Cas9的酵母基因组稳定整合方法，能够实现外源基因的稳定整合，为外源基因在酵母体内的稳定组装提供了有力工具。
Description
技术领域
本发明涉及基因编辑领域，具体涉及一种基于CRISPR/Cas9的酵母基因组稳定整合方法。
背景技术
酿酒酵母(Saccharomyces cerevisiae)是第一个进行全基因组测序的真核微生物，其遗传操作简便、基因表达调控机理清楚且高密度发酵技术成熟，特别是近年来一系列适用于酿酒酵母途径组装工具的开发，使得酿酒酵母成为合成生物学研究的理想底盘生物。酵母中多基因通路的组装常被认为是构建细胞工厂的关键步骤。目前，已公开了多种用于多基因或多拷贝整合的基于CRISPR的多重基因组整合系统，但这些研究更多关注多重基因组的整合效率，而对整合的异源基因和途径的稳定性研究较少。目前，一些强组成型(如TDH3p和TEF1p)或诱导型(例如GAL1p和GAL10p)启动子常重复用于驱动异源途径基因的表达。考虑到酿酒酵母的高同源重组效率和多个重复序列的存在，基因组的重排和不稳定性存在相当大的隐患，特别是涉及较长的生物合成途径。
自从在酵母中建立成簇规则间隔短回文重复序列相关的CRISPR/Cas系统以来，该系统已广泛应用于精确的基因组编辑领域。该系统发挥作用是建立在single-guide RNA(sgRNA)与Cas9蛋白形成的蛋白核酸复合体上，sgRNA负责引导Cas9蛋白结合到目标基因靶位点区域，随即触发Cas9蛋白上的两个核酸酶对DNA双链进行切割产生双链切口。再利用酵母的同源重组修复机制，提供一个与切口位置具有同源序列的修复模板，从而进行包括基因敲除、敲入和点突变在内的精确修饰。
自从在酵母中建立成簇规则间隔短回文重复(CRISPR)/CRISPR相关(Cas)系统(DiCarlo et al.,Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems,Nucleic Acids Res.2013Apr；41(7):4336-43.doi:10.1093/nar/gkt135.Epub 2013 Mar 4.)以来，它已被广泛用作精确基因组编辑的必备技术。目前，已经开发了各种基于CRISPR的多重基因组整合系统，用于多基因或多拷贝的整合。例如，CrEdit(CRISPR/Cas9介导的基因组编辑)系统能够同时整合参与β-胡萝卜素生物合成的三个途径基因(84％的整合效率与约500bp的同源臂)(Ronda et al.,CrEdit:CRISPR mediatedmulti-loci gene integration in Saccharomyces cerevisiae,Microbial cellfactories,2015,DOI:10.1186/s12934-015-0288-3)。同样，开发了Di-CRISPR(Delta整合CRISPR/Cas9)以在一个步骤中实现从8kb到24kb的DNA构建体的多拷贝和无标记整合，并且效率很高(Shi et al.,A highly efficient single-step,markerless strategy formulti-copy chromosomal integration of large biochemical pathways inSaccharomyces cerevisiae,Metab Eng.2016 Jan；33:19-27.doi:10.1016/j.ymben.2015.10.011.Epub 2015 Nov 4.)。最近，构建了一个Landing Pad系统，以将异源基因表达盒整合到酵母基因组中，并具有精确控制的拷贝数(14个拷贝)(Bourgeois etal.,A Highly Characterized Synthetic Landing Pad System for Precise MulticopyGene Integration in Yeast,ACS Synth.Biol.2018,7,11,2675–2685)。然而，这些研究中的大多数主要关注多重基因组整合的效率，而对整合的异源基因和途径的稳定性研究较少。
现有技术中，寻找合适的整合位点是必要的，特别是对于需要在酵母中建立整条合成途径的情况，往往需要外源整合多个基因，合适整合位点的选择对外源基因的稳定高效表达至关重要。
发明内容
针对现有技术中存在的上述不足，本发明开发了一种基于CRISPR/Cas9的酵母基因组稳定整合方法，用于构建和稳定维持多基因生物合成途径。
本发明首先提供了一种基于CRISPR/Cas9的酵母基因组稳定整合方法，在酵母基因组中整合位点选自以下17个，其中，整合位点对应基因组上的位置为相邻两个基因的基因间区域，整合位点int1位于RPB5和CNS1之间；int2位于RIB5和POP4之间；int4位于DBF4和CDC34之间；int5位于SEC20和LCD1之间；int6位于RSP5和NSA2之间；int7位于RET2和RPN12之间；int9位于CES10和RPS31之间；int10位于IRI3和PBR1之间；int11位于ACC1和TIM23之间；int12位于RPO31和RPT5之间；int14位于ARP7和GLN1之间；int15位于CDC4和SMC1之间；int16位于CDC14和PTR3之间；int17位于USE1和SRM1之间；int18位于ERV1和POP6之间；int19位于RPC17和NOP9之间；int21位于SEN2和MDN1之间。本申请中整合位点int1～int22对应使用的sgRNA对应结合到酵母基因组的靶点序列如SEQ ID No.3～24所示。
所述基于CRISPR/Cas9的酵母基因组稳定整合方法中使用的酵母菌株可以是常用的用于基因工程的酵母，比如酵母为酿酒酵母。
优选的，待整合的为虾青素生物合成途径，整合进酵母基因组中的基因为虾青素生物合成途径基因：CrtE、CrtYB、CrtI、CrtZ和CrtW，各基因整合位点为：CrtE基因为Int12；CrtYB基因为Int17；CrtI基因为nt19；CrtZ基因为Int7；CrtW基因为Int18。进一步优选的，CrtE的Genbank登录号为CDZ97186，CrtYB的Genbank登录号为AII26676，CrtI的Genbank登录号为ATB19150，CrtZ的Genbank登录号为CRH37458，CrtW的Genbank登录号为WP_127898117。一种用于生产虾青素的酵母基因工程菌，使用所述基于CRISPR/Cas9的酵母基因组稳定整合方法制备得到。
优选的，待整合的为阿玛碱生物合成途径，整合进酵母基因组中的基因为阿玛碱生物合成途径基因：GES、G8H、GOR、ISY、IO、7-DLGT、7-DLH、LAMT、SLS、STR、SGD和HYS，以及辅助途径基因CPR、CYB5、SAM2、ZWF1、GAPN、ADH2、GAL4和GGPS2，各基因整合位点为：CPR和CYB5为int16；GES为int9；G8H为int11；ISY和GOR为int14；ADH2为int12；GGPS2为int4；GAL4为int10；SLS和STR为int17；LAMT和7-DLGT为int19；IO和7-DLH为int7；SGD和HYS为int18；ZWF1和GAPN为int5；SAM2为int6。进一步优选的，GES的Genbank登录号为AFD64744，G8H是Genbank登录号为AGX93050.1，GOR的Genbank登录号为KF302069，ISY的Genbank登录号为KY882236，IO的Genbank登录号为KF302067，7-DLGT的Genbank登录号为KF302068，7-DLH的Genbank登录号为KF302067，LAMT的Genbank登录号为B2KPR3，SLS的Genbank登录号为L10081.1，STR的Genbank登录号为X61932，SGD的Genbank登录号为AJ302044，HYS的Genbank登录号为KU865325，CPR的Genbank登录号为NP_001190823，CYB5的Genbank登录号为KP411012，SAM2的Genbank登录号为NP_010790，ZWF1的Genbank登录号为NP_014158，GAPN的Genbank登录号为WP_002262986，ADH2的Genbank登录号为KP411010，GAL4的Genbank登录号为NP_015076，GGPS2的Genbank登录号为AF513112。一种用于生产阿玛碱的酵母基因工程菌，使用所述基于CRISPR/Cas9的酵母基因组稳定整合方法制备得到。以简单碳源发酵生产植物次级代谢产物阿玛碱(ajmalicine)的酿酒酵母工程菌，生产阿玛碱的生物合成途径涉及20个异源基因，多轮的异源基因整合和代谢水平的调控，对基因组编辑工具包的效率有较为严格的要求。同时，由于多基因表达往往由相同的启动子和终止子驱动，对整合位点的稳定性也是较为严峻的考验，以保证异源合成途径菌株的稳定性。经多次传代和发酵，利用本发明开发的稳定整合工具包，成功获得了以简单碳源发酵稳定生产阿玛碱的酿酒酵母工程菌。
本发明开发的基于CRISPR/Cas9的酵母基因组稳定整合方法，能够实现外源基因的稳定整合，为外源基因在酵母体内的稳定组装提供了有力工具。
附图说明
图1为Cas9表达载体的质粒图谱。
图2为pRS426-SpSgH-intx的质粒图谱。
图3为pRS426-mVenus(A)及pRS426-URA3(B)的质粒图谱。
图4为不同整合位点荧光表达水平。
图5为荧光蛋白基因整合到对照组位点及int11的荧光表达水平。
图6为pRS415-ENO2p-CrtYB/I/E/W/Z-PGK1t的质粒图谱。
图7为虾青素合成途径基因检测结果图，每个泳道对应一个位点整合的基因，其中每组5个泳道(Int12-17-19-7-18)对应平板上的一个合成虾青素的单菌落。
图8为阿玛碱生物合成途径。
图9为pESC双基因表达盒的质粒图谱
图10为阿玛碱标准品和样品AJM7的色谱图。
图11为AJM7样品合成的阿玛碱的质谱图。
具体实施方式
实施例1：稳定整合位点的全基因组分析
高附加值化合物细胞工厂的构建需要在酵母基因组中插入多个受强启动子控制的基因。如果由相同的启动子驱动，同源重组介导的等位基因替换或基因组重排可能在异源基因之间发生，导致多基因生物合成途径的稳定性低。基于侧翼区域很可能受等位基因替换或基因组重排影响的假设，本发明将异源基因和途径整合到两个必需基因之间的区域中。在这种情况下，任何异源基因表达盒之间的重组都会导致细胞致死。因此，本发明可以稳定地维持酵母染色体中的异源基因和通路。总得来说，理想的整合位点应具备以下特点：1)在两个必需基因之间；2)大于700bp的基因间区长度；3)整合效率高；4)异源基因的高表达水平；5)对细胞适应度的影响最小。
本发明在全基因规模上，对必需基因两侧的基因间区域分析潜在的稳定整合位点。根据功能基因组研究，酿酒酵母中约6000个基因中有1156个对富培养基中的细胞生长至关重要。在这些必需基因中，本发明确定了约210个区域，其中两个或多个必需基因连续定位，这意味着两者之间不存在任何非必需基因。为了尽量减少对转录元件(例如启动子和终止子)的影响以及相应的细胞适应性，本发明过滤掉了那些基因间区域短于700bp的基因，并将潜在整合位点列表缩小到22个以进行实验验证，详细位置信息如表1所示。
表1本发明确定的22个稳定整合位点
本发明采用Cas9表达载体的质粒图谱如图1所示。插入各整合位点对应sgRNA的N20后的质粒图谱如图2所示。其中，Cas9基因序列如SEQ ID No.1所示，Cas9对应的启动子序列如SEQ No.2所示。各整合位点对应的sgRNA对应结合到酵母基因组的靶点序列如SEQID No.3～24所示。各整合位点对应的上游通用同源修复引物序列如SEQ ID No.25～46所示；下游通用同源修复引物序列如SEQ ID No.47～68所示。
实施例2：选定整合位点的表征和稳定性验证
使用CRISPR/Cas9系统，以mVenus表达盒作为报告基因，如图3所示，将mVenus分别整合到本发明初步确定的整合位点中。每个位点挑取32个整合后的单菌落，用SCD-Leu-Ura液体培养基培养(整合位点为int3、int13时，生长缓慢)，生长至饱和后转接50μL至新的培养基，24h后测荧光值，mVenus激发/发射波长498/533；判断插入位点sgRNA整合效率(X/32)，以及各位点荧光表达强度。
观察转化子的生长状况(如快慢及疏密程度)，整合位点为int8、int20、int22的转化子个数极少，说明对菌株自身代谢有影响，因此排除上述三个整合位点。如图4和5所示，除上述三个位点外，其余17个位点挑取的32个单菌落均有荧光，由此可得100％的整合效率。因此，选择其余17个整合位点进行后续研究。
此外，表达水平彼此相当接近(Int14达到最高)，并且与先前研究中报道的充分表征的整合位点相当。更重要的是，我们发现整合位点在不同的酿酒酵母菌株中高度保守。例如，尽管酿酒酵母BY4741和CEN.PK2具有相对多样化的基因组序列，但gRNA靶向序列完全相同，这可能是由于必需基因和基因间区域的高度保守性。
进一步，本发明验证了新确定的整合位点的稳定性，之前报告的整合位点作为对照。如图3所示，当我们整合mVenus和URA3表达盒时，它们都在启动子TDH3和终止子CYC1的控制下，同源重组介导的等位基因替换会导致URA3表达盒的丢失，得到的菌株能够在添加5-氟乳清酸(5-FOA)琼脂板上生长。因此，我们可以通过在SCD/5-FOA琼脂板(每100mL的SCD培养基中加入0.1g 5-FOA)上生长的能力来评估整合到基因组中的异源基因表达盒的稳定性。实验组和对照组URA3的丢失率如表2所示。没有任何菌落表明整合到两侧为必需基因(Int4和Int11)的基因组基因座中的异源基因表达盒是高度稳定的。相反，当整合到三个前人报道过的基因组位点(YPRCδ15c、XII-1和XII-4)时，可以检测到不少数量的克隆，说明存在基因组不稳定导致URA3表达盒丢失的情况。根据实验估算，前人报道的基因组位点(YPRCδ15c、XII-1和XII-4)均存在不稳定的情况，平均丢失率大约为10-5到10-4，而本发明的所表征的位点，没有存在表达盒丢失的情况，进一步证明了本发明开发的位点的稳定性。
表2本发明开发的整合位点和以前报道的整合位点的稳定性测试
实施例3：稳定整合工具包在多基因虾青素合成途径中的应用
为了进一步证明这些新发现的整合位点在构建多基因生物合成途径中的应用，虾青素生物合成途径基因CrtE(Genbank登录号为CDZ97186)、CrtYB(Genbank登录号为AII26676)、CrtI(Genbank登录号为ATB19150)、CrtZ(Genbank登录号为CRH37458)和CrtW(Genbank登录号为WP_127898117)，如图6所示，均受启动子ENO2和终止子PGK1控制，利用所构建的整合工具包，将上述基因整合到本申请所开发的位点上，具体的整合信息如表3所示。
表3虾青素合成途径基因整合到基因组上对应的位点信息
| 基因 | 整合位点 |
| CrtE | Int12 |
| CrtYB | Int17 |
| CrtI | int19 |
| CrtZ | Int7 |
| CrtW | Int18 |
能够将其高效整合到酵母基因组中，如图7所示。在非选择性培养基中连续转移5次后，所有菌落均出现橙色至红色和阳性诊断PCR条带(图7)，这证实了5个异源基因表达盒虽然都在相同的启动子和终止子的控制下，但稳定维持在酵母基因组中。
实施例4：利用稳定整合工具包构建从头合成阿玛碱的酵母工程菌
阿玛碱临床上被广泛用于治疗高血压、心率不齐、哮喘等疾病，其生物合成途径为牻牛儿基焦磷酸(GPP)在GES(Genbank登录号为AFD64744)、G8H(Genbank登录号为AGX93050.1)、GOR(Genbank登录号为KF302069)、ISY(Genbank登录号为KY882236)、IO(Genbank登录号为KF302067)、7-DLGT(Genbank登录号为KF302068)、7-DLH(Genbank登录号为KF302067)、LAMT(Genbank登录号为B2KPR3)、SLS(Genbank登录号为L10081.1)作用下生产断马钱子苷，断马钱子苷与色胺在STR(Genbank登录号为X61932)作用下生产异胡豆苷(strictosidine)，异胡豆苷在SGD(Genbank登录号为AJ302044)和HYS(Genbank登录号为KU865325)两个酶作用下生成阿玛碱(生物合成途径如图8所示)。阿玛碱具有重要应用价值的MIAs类天然产物，其工程菌构建存在的主要问题是生物合成途径长导致工程菌不稳定。将上述生物合成途径对应的基因整合到本发明开发的稳定整合位点中，包括辅助途径基因CPR(Genbank登录号为NP_001190823)、CYB5(Genbank登录号为KP411012)、SAM2(Genbank登录号为NP_010790)、ZWF1(Genbank登录号为NP_014158)、GAPN(Genbank登录号为WP_002262986)、ADH2(Genbank登录号为KP411010)、GAL4(Genbank登录号为NP_015076)、GGPS2(Genbank登录号为AF513112)，详细的整合信息如表4所示。
其中int16、int14、int17、int19、int7、int18、int5位点利用pESC质粒的双基因表达盒(启动子GAL1-目的基因-终止子CYC1和启动子GAL10-目的基因-终止子ADH1)，如图9所示，实现一个位点分别整合两个基因。
表4途径基因对应的整合位点
| 基因 | 整合位点 |
| CPR和CYB5 | int16 |
| GES | int9 |
| G8H | int11 |
| ISY和GOR | int14 |
| ADH2 | int12 |
| GGPS2 | int4 |
| GAL4 | int10 |
| SLS和STR | int17 |
| LAMT和7-DLGT | int19 |
| IO和7-DLH | int7 |
| SGD和HYS | int18 |
| ZWF1和GAPN | int5 |
| SAM2 | int6 |
实施例5：阿玛碱底盘菌发酵及产物检测
挑取单菌落(AJM7)，接种到含3mL YPD培养基的试管中，在250rpm、30℃的摇床中培养24h左右；接种到含有20mL YPD+2％葡萄糖培养基的250mL摇瓶中，30℃、250rpm的摇床上培养24h左右，离心后将菌体用YPD+2％半乳糖培养基重悬后继续24℃、250rpm的摇床上继续培养7天，每组样品3个重复。
样品处理及LCMS检测发酵产物：发酵结束后离心收集发酵液，吸取10mL发酵冷冻干燥后，1mL蒸馏水复溶后加入1mL乙酸乙酯萃取2次，0.22μm有机微孔滤膜过滤后置于进样瓶中。采用HPLC-MS-ESI离子源对阿玛碱进行定量检测，其中色谱条件：Thermo ScientificHyPURITYTM C18 HPLC column(150mm×4.6mm,3μm)；岛津LC-MS 8045：按色谱条件进行含量测定流动相为0.1％甲酸水(A)，甲醇(B)，梯度洗脱：0～20分钟，60％A～2％A；20～40分钟，2％A～60％A，流速为0.5mL/min，柱温为30℃，进样量为4μL检测波长420nm。MS条件为：Scan m/z 50-800；阿玛碱标准品在LC-MS中MRM检测条件为[M-H]+m/z：354/144.20，CE＝-25V(DL温度250℃，喷雾电压为1.8kV，雾化气体流量为6L/min。AJM7的色谱和质谱结果分别如图10和图11所示。AJM7经多次传代发酵液中成功检测到目标产物，阿玛碱、表阿玛碱和四氢鸭脚木碱三种物质统称为异育亨宾生物碱，总产量达715.2mg/L；其中，阿玛碱的产量为368.4mg/L，表阿玛碱的产量为179.2mg/L，四氢鸭脚木碱的产量为167.6mg/L。
序列表
<110> 浙江大学杭州国际科创中心
<120> 一种基于CRISPR/Cas9的酵母基因组稳定整合方法
<160> 68
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4179
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggattata aagatgacga tgacaaacct ccaaaaaaga agagaaaggt cgataagaaa 60
tactcaatag gcttagatat cggcacaaat agcgtcggat gggcggtgat cactgatgaa 120
tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa 180
aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc 240
aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag 300
attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct 360
tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat 420
gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat 480
tctacttata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt 540
cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta 600
tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt 660
ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat 720
ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg 780
tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta 840
cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat 900
caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat 960
atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc 1020
tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca 1080
gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat 1140
gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat 1200
ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc 1260
tttgacaacg gctctattac ccatcaaatt cacttgggtg agctgcatgc tattttgaga 1320
agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg 1380
acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg 1440
atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa 1500
ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat 1560
gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg 1620
acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag 1680
aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta 1740
aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa 1800
gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa 1860
gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc 1920
ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat 1980
gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga 2040
aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa 2100
tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt 2160
aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt 2220
gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt 2280
gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca 2340
cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc 2400
gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact 2460
caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg 2520
gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatcacat tgttccacaa 2580
agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt 2640
ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga 2700
caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa 2760
cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact 2820
cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat 2880
gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct 2940
gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc 3000
catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt 3060
gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag 3120
tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac 3180
ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa 3240
actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc 3300
aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga 3360
ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa 3420
gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta 3480
gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta 3540
gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct 3600
aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt 3660
gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat 3720
gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag 3780
ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat 3840
tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat 3900
gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa 3960
caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt 4020
aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat 4080
gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag 4140
ctaggaggtg accctccaaa aaagaagaga aaggtctga 4179
<210> 2
<211> 407
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
catagcttca aaatgtttct actccttttt tactcttcca gattttctcg gactccgcgc 60
atcgccgtac cacttcaaaa cacccaagca cagcatacta aatttcccct ctttcttcct 120
ctagggtgtc gttaattacc cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt 180
ttctttttct tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt ttcttgaaaa 240
tttttttttt gatttttttc tctttcgatg acctcccatt gatatttaag ttaataaacg 300
gtcttcaatt tctcaagttt cagtttcatt tttcttgttc tattacaact ttttttactt 360
cttgctcatt agaaagaaag catagcaatc taatctaagt tttctag 407
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tcttggtaat gctcggtagg 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gattcaaaaa aaatgaatat 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
aacattaatt gctctcacag 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
acacgtttgt ggttataagg 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gctaataaag aggtaacggt 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
acgtatggtt gtaaaaagca 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gtcgaatact acttgcacac 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tagcagccgt cgggaaaacg 20
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tatattaatt tgcaaccgca 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
catgagcagc cactgtatcg 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gaacaagaac aacaaactcc 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
cgttggaatc tcaatgccaa 20
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
attctgagac cctccgatag 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
acccgcatac ggttctcccg 20
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ttctacgctc aacctcccgt 20
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
aagtcacgta tataagctag 20
<210> 19
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gtaaaacacc tatagcactg 20
<210> 20
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
aaattgtaga acgcaggaca 20
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gttgaaatat aagtaaccct 20
<210> 22
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ccccgcgaat tcgttcaagt 20
<210> 23
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
acggactaaa taacaccagg 20
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
agagtttcaa aattcaacca 20
<210> 25
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
acttgagaac acagactctc tacatataaa gtgccagata 40
<210> 26
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
aacattgtgc aatttttctt gtatccaaaa attttacatc 40
<210> 27
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ttcaccgcca gctttgtatc gtcctttaga gtgcagcaag 40
<210> 28
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
gttttcttat ttctttcttt ttaaaaaact ttcttaatat 40
<210> 29
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
acattagatt ggaattagag cttaagtggt acaaactagg 40
<210> 30
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ctattcagtt ttaaaatgca agaaaattaa aaaacaaaaa 40
<210> 31
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
cggactattg ctgtcttctc gtggtaaatg cgtgttccag 40
<210> 32
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tgataggaat gggattcttc tatttttcct ttttccattc 40
<210> 33
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gagagaaatt aaacttggtt ggggttaatt atttgatggg 40
<210> 34
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
tgtacgctat acatttacgt gctgagctcc taggaaagct 40
<210> 35
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gactagtatc atccgtcaag aagaacaaga acaagaacaa 40
<210> 36
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
cagtaactaa tcgcaaacaa atcaggcatc tgtgtatatt 40
<210> 37
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
taagaggaga cataagaaac tcgaaacagt ataagatgta 40
<210> 38
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
acactcgcga gaaccaaaac aaggatcccc taaacccagt 40
<210> 39
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
gtttttcaaa aagatcgata ttctcttgag aattaggaag 40
<210> 40
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
tattctacta aaaacacatc agtagtcaca gaagtcacag 40
<210> 41
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
ttacgtgtca tttattatgg gttcagaaat tatgtgttaa 40
<210> 42
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
aatggcactg agagacgttt ttgccaaaca agtactaaat 40
<210> 43
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
tgaaaattgt gccggatatt caagactaag agatgtacaa 40
<210> 44
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
tgtacattaa actacgatgt aaacatcaag gttattgcta 40
<210> 45
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
agagctgttt gagagctcgt aggctttggt tgttaagaga 40
<210> 46
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
accaccgacc taaaaagcag aagaaatgtt ttggtaagaa 40
<210> 47
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
tacggtttct agtctcggtt gatgataaga gtacgttaat 40
<210> 48
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
ataacaggtg tatttatagc agtatgaata gttttactag 40
<210> 49
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
atgatttcag ttaatgaacg aatcggatgt tcttatgata 40
<210> 50
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
gaattgagaa aaaaagtgta tatcattaca ttactttaca 40
<210> 51
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
cttaacatcg gtgccacaca atacgaacct tagtagagaa 40
<210> 52
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
ttttattaat tttttttttt atttatatac atataatgtg 40
<210> 53
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
aacttaacaa atcggcaaca cttttatggg gccccgctcg 40
<210> 54
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
cacggcagcg tgactccaat tgagcccgaa agagaggatg 40
<210> 55
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
cactattgat aaaggttttg tagaatattt attatcgata 40
<210> 56
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
cctctatgtg acgctgtgta ttctttgttg tagttatgct 40
<210> 57
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
tacaggcaat gagcgaaagc gactgaagcc gagagatgtg 40
<210> 58
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
tagtatgtat tttgcagctt ctactttctg caatgtgatg 40
<210> 59
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
aactttacaa gaacggcata tgatcagatc gtatccttgc 40
<210> 60
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
tatccgggta acactcatcg tccggcctcc gccccctttt 40
<210> 61
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
ttattctaac acaattgtat tagcttttgt atttcttctg 40
<210> 62
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
ctttattttt tcattaatac acctgtgtta gttatgattg 40
<210> 63
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
cacacaattt tggtggcgtt gaaattgatg ccggaatttg 40
<210> 64
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
cttatagcat actattttag ttatgaattg tgaaaatcct 40
<210> 65
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
aaatcgacat gttaatgatc ttacgacaga gtagtttatg 40
<210> 66
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
gaactgccca ttcagctttt ccctttgcaa ttggtgcact 40
<210> 67
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
atagcacgag aaaaaactcg ttcaactaag tctagacaca 40
<210> 68
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
acgattaaga aagaatatac atttaaatgt tttaaataca 40
Claims (2)
1.一种基于CRISPR/Cas9的酵母基因组稳定整合方法，其特征在于，在酵母基因组中整合位点选自以下17个，其中，整合位点对应基因组上的位置为相邻两个基因的基因间区域，整合位点信息如下：
待整合的为虾青素生物合成途径，整合进酵母基因组中的基因为虾青素生物合成途径基因：CrtE、CrtYB、CrtI、CrtZ和CrtW，各基因整合位点为：
CrtE的Genbank登录号为CDZ97186，CrtYB的Genbank登录号为AII26676，CrtI的Genbank登录号为ATB19150，CrtZ的Genbank登录号为CRH37458，CrtW的Genbank登录号为WP_127898117；
或者，待整合的为阿玛碱生物合成途径，整合进酵母基因组中的基因为阿玛碱生物合成途径基因：GES、G8H、GOR、ISY、IO、7-DLGT、7-DLH、LAMT、SLS、STR、SGD和HYS，以及辅助途径基因CPR、CYB5、SAM2、ZWF1、GAPN、ADH2、GAL4和GGPS2，各基因整合位点为：
GES的Genbank登录号为AFD64744，G8H是Genbank登录号为AGX93050.1，GOR的Genbank登录号为KF302069，ISY的Genbank登录号为KY882236，IO的Genbank登录号为KF302067，7-DLGT的Genbank登录号为KF302068，7-DLH的Genbank登录号为KF302067，LAMT的Genbank登录号为B2KPR3，SLS的Genbank登录号为L10081.1，STR的Genbank登录号为X61932，SGD的Genbank登录号为AJ302044，HYS的Genbank登录号为KU865325，CPR的Genbank登录号为NP_001190823，CYB5的Genbank登录号为KP411012，SAM2的Genbank登录号为NP_010790，ZWF1的Genbank登录号为NP_014158，GAPN的Genbank登录号为WP_002262986，ADH2的Genbank登录号为KP411010，GAL4的Genbank登录号为NP_015076，GGPS2的Genbank登录号为AF513112。
2.根据权利要求1所述基于CRISPR/Cas9的酵母基因组稳定整合方法，其特征在于，酵母为酿酒酵母。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210053387.1A CN114574516B (zh) | 2022-01-18 | 2022-01-18 | 一种基于CRISPR/Cas9的酵母基因组稳定整合方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210053387.1A CN114574516B (zh) | 2022-01-18 | 2022-01-18 | 一种基于CRISPR/Cas9的酵母基因组稳定整合方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114574516A CN114574516A (zh) | 2022-06-03 |
| CN114574516B true CN114574516B (zh) | 2023-10-27 |
Family
ID=81772550
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210053387.1A Active CN114574516B (zh) | 2022-01-18 | 2022-01-18 | 一种基于CRISPR/Cas9的酵母基因组稳定整合方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114574516B (zh) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4910138A (en) * | 1985-05-02 | 1990-03-20 | Yoshiharu Miura | Use of an organ culture of Catharanthus roseus to produce vincristine and vinblastine |
| WO2007099231A1 (fr) * | 2006-03-01 | 2007-09-07 | V. Mane Fils | Systeme d'expression d'un gene d'interet chez la levure |
| CN103865818A (zh) * | 2012-12-07 | 2014-06-18 | 上海来益生物药物研究开发中心有限责任公司 | 一种产虾青素基因工程菌的构建方法 |
| CN112251458A (zh) * | 2020-10-13 | 2021-01-22 | 天津大学 | 一种基于非同源末端连接机制的解脂耶氏酵母基因组整合的方法 |
| CN112458092A (zh) * | 2020-12-11 | 2021-03-09 | 浙江工业大学 | 一种基于CRISPR/Cas9的酵母基因组编辑方法 |
| CN113403334A (zh) * | 2021-06-11 | 2021-09-17 | 江南大学 | 一组用于酿酒酵母多拷贝整合的质粒工具包 |
| CN113699053A (zh) * | 2020-05-20 | 2021-11-26 | 万华化学(四川)有限公司 | 一株生产虾青素的重组酿酒酵母及其应用 |
-
2022
- 2022-01-18 CN CN202210053387.1A patent/CN114574516B/zh active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4910138A (en) * | 1985-05-02 | 1990-03-20 | Yoshiharu Miura | Use of an organ culture of Catharanthus roseus to produce vincristine and vinblastine |
| WO2007099231A1 (fr) * | 2006-03-01 | 2007-09-07 | V. Mane Fils | Systeme d'expression d'un gene d'interet chez la levure |
| CN103865818A (zh) * | 2012-12-07 | 2014-06-18 | 上海来益生物药物研究开发中心有限责任公司 | 一种产虾青素基因工程菌的构建方法 |
| CN113699053A (zh) * | 2020-05-20 | 2021-11-26 | 万华化学(四川)有限公司 | 一株生产虾青素的重组酿酒酵母及其应用 |
| CN112251458A (zh) * | 2020-10-13 | 2021-01-22 | 天津大学 | 一种基于非同源末端连接机制的解脂耶氏酵母基因组整合的方法 |
| CN112458092A (zh) * | 2020-12-11 | 2021-03-09 | 浙江工业大学 | 一种基于CRISPR/Cas9的酵母基因组编辑方法 |
| CN113403334A (zh) * | 2021-06-11 | 2021-09-17 | 江南大学 | 一组用于酿酒酵母多拷贝整合的质粒工具包 |
Non-Patent Citations (2)
| Title |
|---|
| 萝芙木阿玛碱生物合成基因对乙酰水杨酸的响应;马丽利;秦白富;常凯;廖志华;;西南大学学报(自然科学版)(04);参见说明书第2段 * |
| 长春花吲哚生物碱合成途径的基因工程研究进展;向蓓蓓;朱晔荣;王勇;;生物学通报(10);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114574516A (zh) | 2022-06-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11643648B2 (en) | Method for constructing chimeric plasmid library | |
| Muñoz et al. | Stable transformation of the green algae Acutodesmus obliquus and Neochloris oleoabundans based on E. coli conjugation | |
| US20160289690A1 (en) | Mortierella alpina recombinant gene expression system and construction method and use thereof | |
| Jeong et al. | Genetic engineering system for syngas-utilizing acetogen, Eubacterium limosum KIST612 | |
| CN110484572A (zh) | 一种提高酿酒酵母橙花叔醇产量的方法 | |
| CN112063646B (zh) | 目的基因多拷贝整合的方法、重组菌以及重组人血清白蛋白的制备方法 | |
| CN109609396A (zh) | 一种基因工程菌及其构建方法与应用 | |
| WO2016029802A1 (zh) | 虫草素的合成基因簇的鉴定和应用 | |
| EP3750989A1 (en) | Production of plant-based active substances (e.g. cannabinoids) by recombinant microorganisms | |
| CN114574516B (zh) | 一种基于CRISPR/Cas9的酵母基因组稳定整合方法 | |
| US11286504B2 (en) | Method to produce protein in Penicillium amagasakiense's sleeping spores by transformation of ssRNA | |
| Aliu et al. | Generation of thymidine auxotrophic Agrobacterium tumefaciens strains for plant transformation | |
| TWI643951B (zh) | 細長聚球藻ｐｃｃ ７９４２之基因編輯系統及其應用 | |
| CN111850050B (zh) | 一种基因编辑工具及其制备方法与多轮基因编辑的方法 | |
| CN114085831A (zh) | 一种基于双链dna重组工程的细菌基因组多重编辑方法及其应用 | |
| Chen et al. | Multiple-copy-gene integration on chromosome of Escherichia coli for beta-galactosidase production | |
| Papp et al. | Integration of a bacterial β-carotene ketolase gene into the Mucor circinelloides genome by the Agrobacterium tumefaciens-mediated transformation method | |
| CN114606146B (zh) | 一种生产d-柠檬烯的酵母及其应用 | |
| JP6979484B2 (ja) | ２，３−ブタンジオール生産用の組換え微生物および２，３−ブタンジオールの生産方法 | |
| TWI629358B (zh) | 細長聚球藻ｐｃｃ ７９４２之基因表現干擾系統以及抑制細長聚球藻ｐｃｃ ７９４２基因表現之方法 | |
| WO2022061417A1 (en) | Standardised cyanobacterial strain engineering | |
| CN112553239A (zh) | 基于非天然氨基酸的基因组重排调控系统和方法 | |
| WO2022189976A1 (en) | Genetic alterations in microalgae organisms, and methods and compositions | |
| CN116064265A (zh) | 一种高产白藜芦醇的酵母基因工程菌及其构建方法和应用 | |
| CN112538496A (zh) | 一种适用于露湿漆斑菌A553的CRISPR/Cas9载体及其构建方法和应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |