DK2847214T3 - Konstruerede iminreduktaser og fremgangsmåder til den reduktive aminering af keton- og aminforbindelser - Google Patents

Konstruerede iminreduktaser og fremgangsmåder til den reduktive aminering af keton- og aminforbindelser Download PDF

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DK2847214T3
DK2847214T3 DK13724127.9T DK13724127T DK2847214T3 DK 2847214 T3 DK2847214 T3 DK 2847214T3 DK 13724127 T DK13724127 T DK 13724127T DK 2847214 T3 DK2847214 T3 DK 2847214T3
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polypeptide
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Jeffrey Moore
Steven J Collier
Jovana Nazor
Gregory Hughes
Jacob Janey
Gjalt Huisman
Scott Novick
Nicholas Agard
Oscar Alvizo
Gregory Cope
Stefanie Ng Minor
Haibin Chen
Derek Smith
Wan Lin Yeo
Joly Sukumaran
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Codexis Inc
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Claims (11)

1. Konstrueret polypeptid med iminreduktaseaktivitet, omfattende en aminosyresekvens med mindst 80% sekvensidentitet til SEQ ID NO: 2 og én eller flere restforskelle som sammenlignet med sekvensen af SEQ ID NO: 2 ved restpositioner valgt blandt X198, Xlll, X136, X156, X197, X201, X259, X280, X292, og X293 der er i stand til at: i) omdanne substratforbindelse (la) pyruvat,
og substratforbindelse (2b) butylamin
til produktforbindelse (3b), N-2-(butylamino)propansyre,
under egnede reaktionsbetingelser hvor polypeptidet har forbedret iminreduktaseaktivitet sammenlignet med polypeptidet af SEQ ID NO: 2; ii) omdanne substratforbindelse (lb) cyclohexanon,
og substratforbindelse (2a) L-norvalin
til produktforbindelse (3c), (S)-2-(cyclohexylamino)pentansyre,
under egnede reaktionsbetingelser hvor polypeptidet har forbedret iminreduktaseaktivitet sammenlignet med polypeptidet af SEQ ID NO: 2; eller iii) omdanne substratforbindelse (lb) cyclohexanon,
og substratforbindelse (2b) butylamin
til produktforbindelse (3d), N-butylcyclohexanamin,
under egnede reaktionsbetingelser hvor polypeptidet har forbedret iminreduktaseaktivitet sammenlignet med polypeptidet af SEQ ID NO: 2. . Konstrueret polypeptid ifølge krav 1 hvor restforskellene er valgt blandt 198E/A/H/P/S, X111M/Q/S, X136G, X156G/I/Q/S/T/V, X197I/P, X201L, 259E/H/I/L/M/S/T, X280L, X292C/G/I/P/S/T/V/Y, og X293H/I/K/IVN/Q/T/V. . Konstrueret polypeptid ifølge krav 1 hvor aminosyresekvensen omfatter mindst n kombination af restforskellene valgt fra: (a) X111M, X156T, X198H, X259M, X280L, X292V, og X293H; (b) X156T, X197P, X198H, X259H, X280L, X292P, og X293H; (C) X111M, X136G, X156S, X197I, X198H, X201L, X259H, X280L, X292V, og X293H; (d) X197I, X198E, X259M, og X280L; (e) X156T, X197I, X198E, X201L, X259H, X280L, X292V, og X293H; (f) X111M, X136G, X198H, X259M, X280L, X292S, og X293H; og (g) X156V, X197P, X198E, X201L, X259M, X280L, og X292T.
4. Konstrueret polypeptid ifølge et hvilket som helst af kravene 1 til 3 der yderligere omfatter én eller flere restforskelle som sammenlignet med sekvensen af SEQ ID NO: 2 ved restpositioner valgt blandt X4, X5, X14, X20, X29, X37, X67, X71, X74, X82, X94, X97, X100, Xlll, X124, X137, X141, X143, X149, X153, X154, X157, X158, X160, X163, X177, X178, X183, X184, X185, X186, X220, X223, X226, X232, X243, X246, X256, X258, X259, X260, X261, X265, X266, X270, X273, X274, X277, X279, X283, X284, X287, X288, X294, X295, X296, X297, X308, X311, X323, X324, X326, X328, X332, X353, og X356.
5. Konstrueret polypeptid ifølge krav 4 hvor: i) restforskellene er valgt blandt X4H/L/R, X5T, X14P, X20T, X29R/T, X37H, X67A/D, X71C/V, X74R, X82P, X94K/R/T, X97P, X100W, X111R, X124IVN, X137N, X141W, X143W, X149L, X153V/Y, X154F/M/Q/Y, X157D/H/L/M/N/R, X158K, X160N, X163T, X177C/H, X178E, X183C, X184K/Q/R, X185V, X186K/R, X220D/H, X223T, X226L, X232A/R, X243G, X246W, X256V, X258D, X259V/W, X260G, X261A/G/I/K/R/S/T, X265G/L/Y, X266T, X270G, X273W, X274M, X277A/I, X279F/L/V/Y, X283V, X284K/L/M/Y, X287S/T, X288G/S, X294A/I/V, X295R/S, X296IVN/V/W, X297A, X308F, X311C/T/V, X323C/I/M/T/V, X324IVT, X326V, X328A/G/E, X332V, X353E, og X356R; ii) aminosyresekvensen omfatter mindst én kombination af restforskellene valgt fra: (a) X29R, X184R, X223T, X261S, X284M, og X287T; (b) X29R, X157R, X184Q, X220H, X223T, X232A, X261I, X284M, X287T, X288S, X324L, X332V, og X353E; (c) X29R, X157R, X184Q, X220H, X223T, X232A, X259V, X261I, X284M, X287T, X288S, X324L, X332V, og X353E; (d) X29R, X94K, XIHR, X137N, X157R, X184Q, X220H, X223T, X232A, X259V, X261I, X279V, X284M, X287T, X288S, X324L, X332V, og X353E; og (e) X29R, X94K, XIHR, X137N, X157R, X184Q, X220H, X223T, X232A, X259V, X261I, X266T, X279V, X284M, X287T, X288S, X295S, X311V, X324L, X328E, X332V, og X353E; iii) aminosyresekvensen omfatter kombinationen af restforskellene X156T, X197I, X198E, X201L, X259H, X280L, X292V, og X293H og én eller flere restforskelle valgt blandt X29R/T, X94K/R/T, XIHR, X137N, X157D/H/L/M/N/R, X184K/Q/R, X220D/H, X223T, X232A/R, X259V/W, X261A/G/I/K/R/S/T, X266T, X279F/L/V/Y, X284K/L/M/Y, X287S/T, X288G/S, X295S, X311V, X324L/T, X328E, X332V, og X353E; eller iv) aminosyresekvensen omfatter kombinationen af restforskellene X156T, X197I, X198E, X201L, X259H, X280L, X292V, og X293H mindst én kombination af restforskellene valgt fra: (a) X29R, X184R, X223T, X261S, X284M, og X287T; (b) X29R, X157R, X184Q, X220H, X223T, X232A, X261I, X284M, X287T, X288S, X324L, X332V, og X353E; (c) X29R, X157R, X184Q, X220H, X223T, X232A, X259V, X261I, X284M, X287T, X288S, X324L, X332V, og X353E; (d) X29R, X94K, XIHR, X137N, X157R, X184Q, X220H, X223T, X232A, X259V, X261I, X279V, X284M, X287T, X288S, X324L, X332V, og X353E; og (e) X29R, X94K, XIHR, X137N, X157R, X184Q, X220H, X223T, X232A, X259V, X261I, X266T, X279V, X284M, X287T, X288S, X295S, X311V, X324L, X328E, X332V, og X353E.
6. Konstrueret polypeptid ifølge krav 4 der er i stand til at: i) omdanne substratforbindelse (li),
og substratforbindelse (2b)
til produktforbindelse (3d),
under egnede reaktionsbetingelser; eller ii) omdanne substratforbindelse (lj),
og substratforbindelse (2b)
til produktforbindelse (3o),
under egnede reaktionsbetingelser.
7. Konstrueret polypeptid ifølge krav 1, hvor aminosyresekvensen omfatter en sekvens valgt fra gruppen bestående af de lige-nummererede sekvensidentifi-katorer af SEQ ID NO: 4 - 100, og 112 - 750.
8. Polynukleotid der koder for et konstrueret polypeptid ifølge et hvilket som helst af kravene 1 til 7, eventuelt hvor polynukleotidet omfatter en nukleinsyresekvens valgt fra gruppen bestående af de ulige-nummererede sekvensidentifikatorer af SEQ ID NO: 3 - 99, og 111 - 749.
9. Ekspressionsvektor omfattende polynukleotidet ifølge krav 8.
10. Værtscelle omfattende polynukleotidet ifølge krav 8 eller ekspressionsvektoren ifølge krav 9.
11. Fremgangsmåde til fremstilling af et konstrueret polypeptid med imin-reduktaseaktivitet, omfattende dyrkning af værtscellen ifølge krav 10 under betingelser der er egnede for ekspression af polypeptidet, eventuelt yderligere omfattende isolering af det konstruerede polypeptid.
12. Fremgangsmåde til fremstilling af en forbindelse med formel (III),
hvor R1- og R2-grupperne er uafhængigt valgt fra et hydrogenatom, og eventuelt substitueret alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, og heteroarylalkyl; og eventuelt R1 og R2 er bundet for at danne en 3-leddet til 10-leddet ring; R3- og R4-grupperne er uafhængigt valgt fra et hydrogenatom, og eventuelt substitueret alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, og heteroarylalkyl, med det forbehold at begge R3 og R4 kan ikke være hydrogen; og eventuelt R3 og R4 er bundet for at danne en 3-leddet til 10-leddet ring; og eventuelt, er carbonatomet og/eller nitrogenet indikeret med * er kiralt; fremgangsmåden omfatter at bringe en forbindelse med formel (I),
hvor R1, og R2 er som defineret ovenfor; og en forbindelse med formel (II),
hvor R3, og R4 er som defineret ovenfor; i kontakt med et konstrueret polypeptid med iminreduktaseaktivitet i tilstedeværelse af en cofaktor under egnede reaktionsbetingelser, hvor det konstruerede polypeptid er et konstrueret polypeptid ifølge et hvilket som helst af kravene 1 til 7.
13. Fremgangsmåden ifølge krav 12 hvor: i) R3 og R4 er bundet for at danne en 3-leddet til 10-leddet ring; ii) substratforbindelsen med formel (II) er valgt fra methylamin, dimethylamin, isopropylamin, butylamin, isobutylaminl, L-norvalin, anilin, (S)-2-aminopent-4-enoensyre, pyrrolidin, og hydroxypyrrolidin; iii) mindst én af R1 og R2 af forbindelsen med formel (I) er bundet til mindst én af R3 og R4 af aminforbindelsen med formel (II), hvorved fremgangsmåden til fremstilling af aminforbindelsen med formel (III) omfatter en intramolekylær reaktion; eller iv) de egnede reaktionsbetingelser omfatter (a) substratpåfyldning ved ca. 10 g/l til 100 g/l; (b) ca. 0,1 g/l til ca. 50 g/l af det konstruerede polypeptid; (c) ca. 0,05 g/l (0,001 M) til ca. 2,5 g/l (0,050 M) NAD(P)H; (d) en pH på ca. 6 til 10; (e) temperatur på ca. 20° til 50°C; og (f) reaktionstid på 2-120 timer.
DK13724127.9T 2012-05-11 2013-05-09 Konstruerede iminreduktaser og fremgangsmåder til den reduktive aminering af keton- og aminforbindelser DK2847214T3 (da)

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