DK2444487T3 - Cellulolytiske enzymer, nukleinsyrer, der koder for dem, samt fremgangsmåder til deres fremstilling og anvendelse - Google Patents

Cellulolytiske enzymer, nukleinsyrer, der koder for dem, samt fremgangsmåder til deres fremstilling og anvendelse Download PDF

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DK2444487T3
DK2444487T3 DK11007471.3T DK11007471T DK2444487T3 DK 2444487 T3 DK2444487 T3 DK 2444487T3 DK 11007471 T DK11007471 T DK 11007471T DK 2444487 T3 DK2444487 T3 DK 2444487T3
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nucleic acid
polypeptide
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beta
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Lishan Zhao
Michelle H Cayouette
Kevin A Gray
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Bp Corp North America Inc
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Claims (18)

1. Isoleret, syntetisk eller rekombinant nukleinsyre omfattende (a) en nukleinsyresekvens med mindst 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% eller mere, eller fuldstændig (100%) sekvensidentitet med SEQ ID NO: 521, hvor nukleinsyren koder for et polypeptid med en beta-glucosidaseaktivitet, og eventuelt bestemmes sekvensidentiteterne ved analyse med en sekvenssammenligningsalgoritme eller ved en visuel inspektion; eller (b) en nukleinsyresekvens, der hybridiserer under stringente betingelser til en nukleinsyre omfattende SEQ ID NO: 521, hvor nukleinsyren koder for et polypeptid med en beta-glucosidaseaktivitet og har mindst 80% identitet med SEQ ID NO: 521, og de stringente betingelser indbefatter et vasketrin omfattende en vask i 0,2X SSC ved en temperatur på ca. 65°C i ca. 15 minutter; (c) en nukleinsyresekvens kodende for et polypeptid med mindst 80% sekvensidentitet til sekvensen ifølge SEQ ID NO: 522, hvor polypeptidet har en beta-glucosidaseaktivitet; (d) nukleinsyren fra (a), (b) eller (c) kodende for et polypeptid med en beta-glucosidaseaktivitet, men som mangler en signalsekvens eller et carbohydratbindingsmo-dul; (e) nukleinsyren i (a), (b), (c) eller (d) kodende for et polypeptid, der har en beta-glucosidaseaktivitet, men som har en heterolog sekvens, hvor den heterologe sekvens eventuelt omfatter en heterolog signalsekvens, et car-bohydratbindingsmodul, et katalytisk domæne (CD), eller en kombination deraf, og eventuelt er den heterologe signalsekvens, carbohydratbindingsmodulet eller det katalytiske domæne (CD) afledt fra en oligomerase, en cellulase eller et cellulolytisk enzym, eller en ikke-oligomerase, en cellulase elleret cellulolytisk enzym; eller (f) en nukleinsyresekvens fuldstændig komplementærtil (a), (b), (c), (d) eller (e).
2. Ekspressionskassette, en vektor eller et kloningsvehikel omfattende en nukleinsyre omfattende sekvensen ifølge krav 1, hvor kloningsvehiklet eventuelt omfatter en viral vektor, et plasmid, en fag, et fagemid, et cosmid, et fosmid, en bakteriofag eller et kunstigt kromosom, eller hvor kloningsvehiklet eventuelt omfatter en viral vektor omfattende en adenovirusvektor, en retroviral vektor eller en adenoassocieret viral vektor, eller hvor kloningsvehiklet eventuelt omfatter et bakterielt, kunstigt kromosom (BAC), et plasmid, en bakteriophag Pl-afledt vektor (PAC), et kunstigt gær-kromosom (YAC) eller et kunstigt pattedyrskromosom (MAC).
3. Transformeret celle omfattende en nukleinsyre omfattende sekvensen ifølge krav 1, eller en vektor eller et kloningsvehikel ifølge krav 2, hvor cellen eventuelt er en bakteriecelle, en pattedyrcelle, en svampecelle, en gærcelle, en insektcelle eller en plantecelle, og hvor den transformerede celle producerer et polypeptid kodet af nukleinsyren ifølge krav 1 og har en beta-glucosidaseaktivitet.
4. Transgen(t) ikke-humant dyr, plante, plantedel eller plantefrø omfattende sekvensen ifølge krav 1 eller ekspressionskassetten, vektoren eller kloningsvehiklet ifølge krav 3, hvor det transgene ikke-humane dyr eventuelt er en mus eller en rotte, og hvor den transgene, transformerede ikke-humane dyre-, plante-, plantedels- eller plantefrøcelle producerer et polypeptid kodet af nukleinsyren ifølge krav 1 og har en beta-glucosidaseaktivitet.
5. Isoleret, syntetisk eller rekombinant polypeptid (i) med en aminosyresekvens med mindst 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% eller mere, eller 100% sekvensidentitet med SEQ ID NO: 522, hvor aminosyresekvensen har beta-glucosidaseaktivitet, hvor sekvensidentiteterne eventuelt bestemmes ved analyse med en sekvenssammenligningsalgoritme eller ved en visuel inspektion, og eventuelt er sekvenssammenligningsalgoritmen en BLAST-version 2.2.2-algoritme, hvor en filtreringsindstilling er indstillet til blastall -p blastp -d "nr pataa "-F F, og alle andre indstillinger er sat til standard ("default"); (ii) med en aminosyresekvens kodet af en nukleinsyre ifølge krav 1, hvor polypepti-det har en beta-glucosidaseaktivitet, eller har immunogen aktivitet ved at den er i stand til at danne et antistof, der specifikt binder til et polypeptid med sekvensen af SEQ ID nr: 522; (iii) polypeptidet ifølge (i) eller (ii) har en beta-glucosidaseaktivitet, men mangler en signalsekvens eller et carbohydratbindingsmodul; (iv) polypeptidet ifølge (i), (ii) eller (iii) med en beta-glucosidaseaktivitet, men som har en heterolog sekvens, hvor den heterologe sekvens eventuelt omfatter en heterolog signalsekvens, carbohydratbindingsmodul, katalytisk domæne (CD) eller en kombination deraf, og eventuelt er den heterologe signalsekvens, carbohydratbindingsmodulet eller det katalytisk domæne (CD) afledt fra et andet oligomerase-, cellulase- eller cellulolytisk enzym eller fra et ikke-oligomerase-, -cellulase- eller -cellulolytisk enzym; eller (v) polypeptidet ifølge (i), (ii), (iii) eller (iv), hvor polypeptidet er glycosyleret.
6. Isoleret, syntetisk eller rekombinant polypeptid med en aminosyresekvens ifølge SEQ ID NO: 522.
7. Polypeptid ifølge krav 6, der har en beta-glucosidaseaktivitet, en signalsekvens og et carbohydratbindingsmodul.
8. Fremgangsmåde til fremstilling af et rekombinant polypeptid, omfattende trinnene: (a) tilvejebringelse afen nukleinsyre ifølge krav 1; og (b) ekspression af nukleinsyren fra trin (a) under betingelser, som tillader ekspression af polypeptidet, hvorved der dannes et rekombinant polypeptid, hvorved fremgangsmåden yderligere omfatter omdannelse af en værtscelle med nukleinsyren fra trin (a) efterfulgt af ekspression af nukleinsyren fra trin (a), hvorved der produceres et rekombinant polypeptid i en transformeret celle.
9. Fremgangsmåde til dannelse af en variant af en nukleinsyre kodende for et polypeptid med en beta-glucosidaseaktivitet, omfattende trinnene: (a) tilvejebringelse af en skabelonnukleinsyre omfattende sekvensen ifølge krav 1; og (b) modifikation, deletering eller tilføjelse af en eller flere nukleotider i skabelonsekvensen, eller en kombination deraf, til dannelse af en variant af skabelonnukleinsyren, hvorved fremgangsmåden yderligere omfatter ekspression af variantnukleinsyren til dannelse af et variant- oligomerase- og/eller cellulasepolypeptid, og eventuelt introduceres modifikationerne, tilføjelserne eller deletionerne ved hjælp af en fremgangsmåde omfattende fejlbehæftet PCR, shuffling, oligonukleotid-rettet muta-genese, samlings-PCR, seksuel PCR-mutagenese, in v/Vo-mutagenese, kassette-mutagenese, rekursiv ensemble-mutagenese, eksponentiel ensemble-mutagenese, stedspecifik mutagenese, genreassembling, GSSM ("Gene Site Saturation Mutagenesis", SLR ("synthetic ligation reassembly"), rekombination, rekursiv sekvensrekombination, phosphothioat-modificeret DNA-mutagenese, uracil-indeholdende skabelonmutagenese, gapped duplex mutagenese, punkt mismatch reparationsmutagenese, reparationsmangelfuld værtsstammemutagenese, kemisk mutagenese, radiogen mutagenese, deleti-onsmutagenese, restriktionsselektionsmutagenese, restriktion-oprensning mutagenese, kunstig gensyntese, ensemble-mutagenese, kimærnukleinsyre multimerdannelse, og en kombination deraf; og hvor fremgangsmåden gentages iterativt, indtil en oligomerase og/eller en cellulase har en ændret eller forskellig aktivitet, en ændret eller forskellig stabilitet, en ændret codonanvendelse, eller fremkommer et højere eller lavere niveau af meddelelsesekspression sammenlignet med det fra et polypeptid kodet af skabelonnukleinsyren.
10. Fremgangsmåde til modifikation af codoner i en nukleinsyre kodende for et beta-glucosidase polypeptid, hvilken fremgangsmåde omfatter følgende trin: (a) tilvejebringelse af en nukleinsyre kodende for et polypeptid ifølge krav 5; og, (b) identifikation af et kodon i nukleinsyren fra trin (a) og erstatning af den med en anden kodon kodende for den samme aminosyre som det erstattede kodon, for der ved at modificere kodoner i en nukleinsyre kodende for en cellulase.
11. Kimært polypeptid omfattende et første domæne og mindst et andet domæne, hvor det første domæne omfatter et enzym ifølge krav 5, og det andet domæne omfatter et heterologt eller modificeret carbohydratbindende domæne eller et heterologt eller modificeret dockerin-domæne, og eventuelt er carbohydratbindingsdomænet et cellulosebindende modul (CBM) eller et ligninbindende domæne, og det andet domæne er eventuelt fastgjort ved siden af enzymets katalytiske domæne, og det andet domæne er eventuelt fastgjort nær C-terminalen af enzymets katalytiske domæne.
12. Sammensætning eller fremstillet produkt omfattende (a) en blanding (eller "cocktail") af hemicellulose- og cellulosehydrolyserende enzymer, hvor de cellulosehydrolyserende enzymer omfatter mindst én af hver af en endoglu-canase, celleobiohydrolase I (CBH I), celleobiohydrolase II (CBH II) og beta-glucosidase; og de hemicellulosehydrolyserende enzymer omfatter mindst én af hver af en xylanase, beta-xylosidase og arabinofuranosidase, og hvor mindst én af beta-glucosidaserne er et polypeptid ifølge krav 5 eller et polypeptid kodet af en nukleinsyre ifølge krav 1; (b) en blanding (eller "cocktail") af hemicellulose- og cellulosehydrolyserende enzymer omfattende mindst én af hver af en endoglucanase, oligomerase, celleobiohydrolase I (CBH I), celleobiohydrolase II (CBH II), arabinofuranosidase og xylanase, hvor mindst én oligomerase er en beta-glucosidase, og eventuelt en yderligere oligomerase, der, hvis den er tilstede, er en oligomerase-1, oligomerase-2 eller beta-xylosidase, hvor mindst én af beta-glucosidaserne er et polypeptid ifølge krav 5 eller en polypeptid kodet af en nukleinsyre ifølge krav 1; (c) en blanding (eller "cocktail") af hemicellulose- og cellulosehydrolyserende enzymer omfattende mindst én af hver af en endoglucanase; en celleobiohydrolase I (CBH I); en celleobiohydrolase II (CBH II); en arabinofuranosidase; en xylanase; en beta-glucosidase; og en oligomerase-2 eller beta-xylosidase, hvor mindst én af beta-glucosidaserne er et polypeptid ifølge krav 5 eller et polypeptid kodet af en nukleinsyre ifølge krav 1; eller (d) en blanding (eller "cocktail") af enzymer omfattende (1) en endoglucanase, som spalter interne p-l,4-bindinger, hvilket resulterer i kortere glucooligosaccharider, (2) en celleobiohydrolase, som virker på en "exo" måde, der processivt frigiver cellobioseenheder (β-1,4 glucose-glucosedisaccharid), og (3) en beta-glucosidase til frigivelse af glucosemo-nomer fra korte cellooligosaccharider (f.eks. cellobiose), hvor mindst én af beta-glucosidaserne er et polypeptid ifølge krav 5 eller et polypeptid kodet af en nukleinsyre ifølge krav 1; hvor eventuelt, (i) endoglucanasen omfatter SEQ ID NO: 106, cellobiohydrolase I'eren omfatter SEQ ID NO: 34 eller SEQ ID NO: 46, cellobiohydrolase H'eren omfatter SEQ ID NO: 98, en yderligere beta-glucosidase, hvis den er til stede, omfatter SEQ ID NO: 94, xylanasen omfatter SEQ ID NO: 100, SEQ ID NO: 102 eller SEQ ID NO: 524, beta-xylosidasen omfatter SEQ ID NO: 96, arabinofuranosidasen omfatter SEQ ID NO: 92 eller SEQ ID NO: 104 , eller en hvilken som helst kombination deraf, hvor SEQ ID NO: 106 eventuelt omfatter et yderligere carbohydratbindende domæne; (ii) blandingen omfatter en endoglucanase omfattende SEQ ID NO: 106, en celleobiohydrolase I (CBH I) omfattende SEQ ID NO: 34 eller SEQ ID NO: 46, en celleobiohydrolase II (CBH II) omfattende SEQ ID NO: 98, en arabinofuranosidase omfattende SEQ ID NO: 92, en oligomerase-2 (en beta-xylosidase) omfattende SEQ ID NO: 520, og en xylana- se omfattende SEQ ID NO: 102, SEQ ID NO: 524 og/eller SEQ ID NO: 100; eller (iii) sammensætningen eller det fremstillede produkt yderligere omfatter et biomassemateriale, hvor biomassematerialet eventuelt omfatter et lignocellulosemateriale afledt af en landbrugsafgrøde, eller biomassematerialet er et biprodukt af en fødevare- eller en foderstofproduktion, eller biomassematerialet er et lignocelluloseaffaldsprodukt, eller biomassematerialet er en planterest eller et papiraffald eller et papiraffaldsprodukt, eller biomassematerialet omfatter en planterest, og eventuelt omfatter planteresten stængler, blade, bælge, skaller, majskolber, majsstængler, strå, træ, træflis, træmasse og/eller savsmuld, og eventuelt omfatter papiraffaldet kasseret eller brugt fotokopipapir, computer-printerpapir, notebook-papir, notepad-papir, skrivemaskinepapir, aviser, blade, pap og papirbaserede emballagematerialer.
13. Enzym-ensemble eller -"cocktail" til depolymerisering af cellulose- og hemicellu-losepolymerer til metabolisérbare carbondele omfattende et polypeptid ifølge krav 5, eller et polypeptid kodet af en nukleinsyre ifølge krav 1.
14. Sammensætning omfattende et polypeptid ifølge krav 5, eller et polypeptid kodet af en nukleinsyre ifølge krav 1, eller en sammensætning eller fremstillet produkt ifølge krav 12 eller et enzym-ensemble ifølge krav 13, hvor sammensætningen eventuelt er en farmaceutisk sammensætning, en detergentsammensætning, en kontaktlinseopløsning, en affaldsbehandlingssammensætning, et desinfektionsmiddel, et bio-forsvars- eller bioafgiftningsmiddel, eller hvor sammensætningen eventuelt er et brændsel, hvor sammensætningen eventuelt er en alkohol, hvor alkoholen eventuelt er ethanol, eller hvor sammensætningen eventuelt er en biomasse eller et biomassemateriale, et papir, papiraffald, genbrugspapirprodukt, papirmasse, papirprodukt, træ, træprodukt, træmasse, tekstil, stof, garn, eller fiber, eller hvor sammensætningen eventuelt er en cellulose- eller en cellulose-derivat-sammensætning, eller hvor sammensætningen eventuelt er en drikkevare, en fødevare, et foder, et mejeriprodukt eller et kosttilskud, eller hvor fødevaren eventuelt er en dej, et brød eller et bagt produkt, eller hvor drikkevaren, fødevaren, foderet eller kosttil-skudet eventuelt er til et dyr, eller hvor biomassematerialet eventuelt omfatter et lignocellulosemateriale afledt af en landbrugsafgrøde, eller biomassematerialet er et biprodukt af en fødevare- eller en foderproduktion, eller biomassematerialet er et lignocelluloaffalds-produkt, eller biomassematerialet er en planterest eller et papiraffald eller papiraffaldsprodukt, eller biomassematerialet omfatter en planterest, og eventuelt omfatter planteresten stængler, blade, bælge, skaller, majskolber, majsstængler, strå, træ, træflis, træmasse og/eller savsmuld, og eventuelt omfatter papiraffaldet kasseret eller brugt fotokopipapir, computerprinterpapir, notebook-papir, notepad-papir, skrivemaskinepapir, aviser, blade, pap og papirbaserede emballagematerialer.
15. Fremgangsmåde til behandling eller modifikation af en sammensætning omfattende, kontakte en sammensætning med et polypeptid ifølge krav 5, eller et polypeptid kodet af en nukleinsyre ifølge krav 1, eller en sammensætning eller fremstillet produkt ifølge krav 12 eller et enzym-ensemble ifølge krav 13, hvor sammensætningen eventuelt er en farmaceutisk sammensætning, en detergentsammensætning, en kontaktlinseopløsning, en affaldssammensætning, et desinfektionsmiddel, bio-forsvars- eller bio-afgiftende middel, eller hvor sammensætningen eventuelt er et brændsel, hvor sammensætningen eventuelt er en alkohol, hvor alkoholen eventuelt er ethanol, eller hvor sammensætningen eventuelt er en biomasse eller et biomassemateriale, et papir, papiraffald, genbrugspapirprodukt, papirmasse, papirprodukt, træ, træprodukt, træmasse, tekstil, stof, garn eller fiber, eller hvor sammensætningen eventuelt er en cellulose- eller en cellulosederivat-sammensætning, eller hvor sammensætningen eventuelt er en drikkevare, en fødevare, et foder, et mejeriprodukt eller et kosttilskud, eller hvor fødevaren eventuelt er en dej, et brød eller et bagt produkt, eller hvor drikkevaren, fødevaren, foderet eller kosttilskuddet eventuelt er til et dyr, eller hvor biomassematerialet eventuelt omfatter et lignocellulose-materiale afledt fra en landbrugsafgrøde, eller biomassematerialet er et biprodukt af en fødevare- eller en foderstofproduktion, eller biomassematerialet er et lignocellulosaffalds-produkt, eller biomassematerialet er en planterest eller et papiraffald eller papiraffaldsprodukt, eller biomassematerialet omfatter en planterest, og eventuelt omfatter planteresten stængler, blade, bælge, skaller, majskolber, majsstængler, strå, træ, træflis, træmasse og/eller savsmuld, og eventuelt omfatter papiraffaldet kasseret eller brugt fotokopipapir, computerprinterpapir, notebook-papir, notepad-papir, skrivemaskinepapir, aviser, blade, pap og papirbaserede emballagematerialer.
16. Fremgangsmåde til hydrolyse, opbrydning eller nedbrydning af en celluloseomfattende sammensætning omfattende følgende trin: (a) tilvejebringe et polypeptid med en beta-glucosidaseaktivitet ifølge krav 5, eller et polypeptid kodet af en nukleinsyre ifølge krav 1, eller en sammensætning eller et fremstillet produkt ifølge krav 12 eller et enzym-ensemble ifølge krav 13; (b) tilvejebringe en sammensætning omfattende en cellulose; og (c) bringe polypeptidet fra trin (a) i kontakt med sammensætningen fra trin (b) under betingelser, hvor polypeptidet hydrolyserer, opbryder eller nedbryder den celluloseomfattende sammensætning.
17. Fremgangsmåde til fremstilling af et brændsel, omfattende at kontakte en sammensætning omfattende en cellulose eller et fermenterbart sukker med et polypeptid ifølge krav 5, eller et polypeptid kodet af en nukleinsyre ifølge krav 1, eller en sammensætning eller et fremstillet produkt ifølge krav 12 eller et enzym-ensemble ifølge krav 13, hvor sammensætningen, der omfatter en cellulose eller et fermenterbart sukker, eventuelt omfatter en plante, et planteprodukt eller plantederivat, og eventuelt omfatter planten eller planteproduktet sukkerplanter eller planteprodukter, roer eller sukkerroer, hvede, majs, sojabønner, kartoffel, ris eller byg, og eventuelt omfatter brændstoffet en bioethanol eller en benzin-ethanol-blanding.
18. Fremgangsmåde til forarbejdning af et biomassemateriale, omfattende at kon- takte et biomassemateriale med et polypeptid ifølge krav 5 eller et polypeptid kodet af en nukleinsyre ifølge krav 1 eller en sammensætning eller et fremstillet produkt ifølge krav 12 eller et enzym-ensemble ifølge krav 13, hvor biomassematerialet omfattende lignocellulose eventuelt er afledt fra en landbrugsafgrøde, er et biprodukt af en fødevare- eller en foderstofproduktion, er et lignocellu-loseaffaldsprodukt eller er en planterest eller et affaldspapir eller affaldspapirprodukt, og planteresten omfatter stængler, blade, bælge, skaller, majskolber, majsstængler, strå, træ, træflis, træmasse og savsmuld, og papiraffaldet omfatter eventuelt kasseret eller brugt fotokopipapir, computerprinterpapir, notebook-papir, notepad-papir, skrivemaskinepapir, aviser, blade, karton og papirbaserede emballagematerialer, og eventuelt frembringer behandlingen af biomassematerialet en bioethanol.
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