DK2803727T3 - Synteseregulerende srna og fremgangsmåde til fremstilling deraf - Google Patents

Synteseregulerende srna og fremgangsmåde til fremstilling deraf Download PDF

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DK2803727T3
DK2803727T3 DK13735942.8T DK13735942T DK2803727T3 DK 2803727 T3 DK2803727 T3 DK 2803727T3 DK 13735942 T DK13735942 T DK 13735942T DK 2803727 T3 DK2803727 T3 DK 2803727T3
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Sang Yup Lee
Dokyun Na
Seung Min Yoo
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Korea Advanced Inst Sci & Tech
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Claims (14)

1. sRNA til inhibering af genekspression i prokaryoter, hvilken sRNA omfatter: (i) et Hfq-bindingssted fra sRNA'en af MicC; og (ii) et område, der baseparrer med target-gen-mRNA'en hvor området, der baseparrer med target-gen-mRNA'en, er konstrueret således, at energien ved binding til target-gen-mRNA'en ligger i området fra -20 kcal/mol til -40 kcal/mol; og hvor området, der baseparrer med target-gen-mRNA'en, baseparrer med en del af ribosombindingsstedet af target-gen-mRNA'en, og området har en længde på 19-37 nukleotider.
2. sRNA ifølge krav 1, hvor et sted, der ikke binder til mRNA'en af target-genet, dannes ved insertion, deletion eller substitution af en eller flere nukleotider i det område, der baseparrer med target-gen-mRNA'en.
3. sRNA ifølge krav 1, hvor prokaryoterne er udvalgt fra gruppen bestående af E. coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Entero-bacter, Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomo-nas, Vibrio, Pseudomonas, Azotobacter, Acinetobacter, Ralstonia, Agrobacterium, Rhizobium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Strepto-myces, Bifidobacterium og Cyclobacterium.
4. sRNA ifølge krav 1, hvor target-gen-mRNA'en er en mRNA af et gen udvalgt fra gruppen bestående af kanamycinresistensgen, tyrosinregulator, phospho-enolpyruvat-carboxylase, carbonlagringsregulator, glucose-6-phosphatisome-rase, citratsyntase, acetyl-CoA-carboxyltransferase, alfa-underenhed, biotiny-leret biotin-carboxyl-bæreprotein, acetyl-CoA-carboxylase, acetyl-CoA-carbo-xyltransferase, beta-underenhed, pyruvatdehydrogenase, propionatkinase / acetatkinase-aktivitet, adiY, acetylglutamatkinase, N-acetylgluta-mylphosphatreduktase, argininosuccinatsyntase, argininosuccinat-lyase, aspartat-ammoniak-lyase, phosphopentomutase, deoxyribose-phosphat-al-dolase, KASHI, -ketoacyl-ACP-syntaser, fedtacyl-CoA-syntetase, fructose-1,6-bisphosphatase, NADP+-afhængig succinatsemialdehyd-dehydrogenase, APC-transporter, 4-aminobutyrat-aminotransferase, glutamat-decarboxylase- A-underenhed, glutamat-decarboxylase-B-underenhed, GABA-APC-transpor-ter, glycerolkinase, sn-Glycerol-3-phosphat-repressor, fructose-1,6-bisphosphatase II, citratsyntase, lysogeniseringsregulator, acetohydroxybuta-noatsyntase I acetolactatsyntase, acetohydroxysyre-isomeroreduktase, dihy-droxysyredehydratase, acetolactatsyntase I acetohydroxybutanoatsyntase, acetohydroxybutanoatsyntase I acetolactatsyntase, UDP-3-O-acyl-N-acetyl-glucosamin-deacetylase, L-aspartatoxidase, nitrat/nitrit-responsregulator, phosphoenolpyruvat-carboxykinase, phosphoenolpyruvatsyntetase, phosphatacetyltransferase, adenylosuccinatsyntetase, adenylosuccinat-lyase, 4-aminobutyrat-aminotransferase, ribose-ABC-transporter, ribosepyra-nase, ribokinase, alfa-ketoglutarat-reduktase I D-3-phosphoglycerat-dehydro-genase, phosphohydroxythreonin-aminotransferase I 3-phosphoserinaminot-ransferase, glucose-6-phosphat-1-dehydrogenase, asparaginsyntetase A, carbamoylphosphatsyntetase, D-alanin-D-alanin-ligase B, thymidin-phosphorylase I uracilphosphorylase, L-2-hydroxyglutarat-oxidase, Lipoamid-dehydrogenase, O-succinylhomoserin-lyase I O-succinylhomoserin(thiol)-lyase, aspartatkinase I homoserin-dehydrogenase, phospho-N-acetyl-muramoyl-pentapeptidtransferase, UDP-N-acetylmuramoylalanyl-D-glutamat-2,6-diaminopimelatligase, D-alanyl-D-alanin-tilsætningsenzym, N-acetylglu-cosaminyltransferase, quinolinatsyntase , pantothenatsyntetase, aspartat 1-1-decarboxylase, 6-phosphogluconolactonase, dihydroorotase, ri-bulosphosphat-3-epimerase, transaldolase A, aspartatkinase I homoserin-dehydrogenase, homoserinkinase, threoninsyntase, transketolase I og transke-tolase II.
5. Fremgangsmåde til inhibering af et target-gen, hvilken fremgangsmåde omfatter trinnene: at indføre eller eksprimere sRNA'en ifølge et hvilket som helst af kravene 1 til 4 i en prokaryot; og inhibere ekspressionen af mRNA'en i tar-get-genet.
6. Fremgangsmåde til screening af et gen, der skal deleteres til produktion af et anvendeligt stof, hvilken fremgangsmåde omfatter trinnene: (a) at inhibere ekspressionen af mindst et gen blandt gener, som er til stede i en stamme, til produktion af det anvendelige stof og medvirken i biosyntesevejen af det anvendelige stof under anvendelse af sRNA'en ifølge et hvilket som helst af kravene 1 til 4, som er rettet mod genet; og (b) at udvælge det gen, hvis ekspression blev inhiberet, som det gen, der skal deleteres til produktion af det anvendelige stof, hvis produktionsudbyttet af det anvendelige stof øges ved at inhibere ekspressionen af genet.
7. Fremgangsmåde til forbedring af en stamme til produktion af et anvendeligt stof, hvilken fremgangsmåde omfatter trinnene: (a) at inhibere ekspressionen af mindst et gen blandt gener, som er til stede i en stamme, til produktion af det anvendelige stof og medvirken i biosyntesevejen af det anvendelige stof under anvendelse af sRNA'en ifølge et hvilket som helst af kravene 1 til 4, som er rettet mod genet; (b) at udvælge det gen, hvis ekspression blev inhiberet, som det gen, der skal deleteres til produktion af det anvendelige stof, hvis produktionsudbyttet af det anvendelige stof øges ved at inhibere ekspressionen af genet. (c) at konstruere en rekombinant stamme med en deletion af det udvalgte gen eller en kombination af de udvalgte gener.
8. Rekombinant mikroorganisme med en forbedret evne til at producere tyrosin, hvilken rekombinant mikroorganisme opnås ved at inaktivere funktionerne af tyrR- og csrA-gener i en værtscelle med tyrosinbiosyntesevejen, hvor funktionerne af tyrR- og csrA-gener inaktiveres ved at indføre eller eksprimere en syntetisk sRNA ifølge et hvilket som helst af kravene 1 til 4 i en værtscelle med tyrosinbiosyntesevejen til inhibering af ekspressionen af tyrR- og csrA-ge-nerne, og hvor den rekombinante mikroorganisme omfatter den syntetiske sRNA.
9. Rekombinant mikroorganisme ifølge krav 8, hvor mikroorganismen har et eller flere gener udvalgt fra gruppen bestående af phenolpthiocerol-syntese-type-l-polyketid-syntase A, transketolase, 3-deoxy-7-phosphoheptulonat-syn-tase, shikimatkinase I og prephenatdehydrogenase, der indføres eller amplifi-ceres deri.
10. Rekombinant mikroorganisme ifølge krav 8, hvor phenolpthiocerol-syn-tese-type-l-polyketid-syntase A eller transketolase eksprimeres af en stærk promoter, og 3-deoxy-7-phosphoheptulonat-syntase, shikimatkinase I eller prephenatdehydrogenase eksprimeres af en svag promoter.
11. Rekombinant mikroorganisme ifølge krav 8, hvor værtscellen er udvalgt fra gruppen bestående af E. coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Pasteurella, Mannheimia, Actinobacillus, Aggre-gatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter, Acinetobacter, Ralstonia, Agrobacterium, Rhizobium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium og Cyclobacterium.
12. Rekombinant mikroorganisme, som kan producere phenol, hvilken rekombinant mikroorganisme opnås ved indføring eller amplificering af et tpl-gen i den rekombinante mikroorganisme ifølge et hvilket som helst af kravene 8 til 11.
13. Rekombinant mikroorganisme med en forbedret evne til at producere cadaverin, hvilken rekombinant mikroorganisme opnås ved at inaktivere funktionen af mindst et gen i en værtscelle med cadaverinbiosyntesevejen, hvor funktionerne af genet inaktiveres ved at indføre eller eksprimere en syntetisk sRNA ifølge et hvilket som helst af kravene 1 til 4 i en værtscelle med tyrosinbiosyntesevejen til inhibering af ekspressionen af mindst et gen udvalgt fra gruppen bestående afmurE, metB, thrL og ackA, og hvor den rekombinante mikroorganisme omfatter den syntetiske sRNA.
14. Rekombinant mikroorganisme ifølge krav 13, hvor værtscellen er udvalgt fra gruppen bestående af E. coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Pasteurella, Mannheimia, Actinobacillus, Ag-gregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter, Acinetobacter, Ralstonia, Agrobacterium, Rhizobium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium og Cyclobacterium.
DK13735942.8T 2012-01-11 2013-01-11 Synteseregulerende srna og fremgangsmåde til fremstilling deraf DK2803727T3 (da)

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KR101690780B1 (ko) * 2014-06-11 2016-12-29 한국과학기술원 합성 조절 sRNA를 이용한 클로스트리듐 속 미생물 유전자의 발현 조절 방법
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CN107208106A (zh) * 2014-12-09 2017-09-26 韩国科学技术院 通过降低rnpA基因表达制备重组蛋白的方法
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