US 200901 63376A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0163376 A1 Li et al. (43) Pub. Date: Jun. 25, 2009 (54) KETOL-ACID REDUCTOISOMERASE USING Related U.S. Application Data NADH (60) Provisional application No. 61/015.346, filed on Dec. (75) Inventors: Yougen Li, Lawrenceville, NJ (US); 20, 2007, provisional application No. 61/109,297, Der-Ing Liao, Wilmington, DE filed on Oct. 29, 2008. (US); Mark J. Nelson, Newark, DE Publication Classification (US); Daniel P. Okeefe, Ridley Park, PA (US) (51) Int. Cl. C40B 30/04 (2006.01) Correspondence Address: CI2N 9/90 (2006.01) E DUPONT DE NEMOURS AND COMPANY CI2N IS/II (2006.01) LEGAL PATENT RECORDS CENTER CI2N 5/06 (2006.01) BARLEY MILL PLAZA 25/1122B, 4417 LAN CI2P 7/16 (2006.01) CASTER PIKE (52) U.S. Cl. ............ 506/9: 435/233: 536/23.2; 435/325: WILMINGTON, DE 19805 (US) 435/160 (57) ABSTRACT (73) Assignee: E. DUPONT DE NEMOURS AND COMPANY Methods for the evolution of NADPH binding ketol-acid reductoisomerase enzymes to acquire NADH binding func (21) Appl. No.: 12/337,736 tionality are provided. Specific mutant ketol-acid reductoi Somerase enzymes isolated from Pseudomonas that have (22) Filed: Dec. 18, 2008 undergone co-factor switching to bind NADH are described. Glucose O Y O O Pyruvate CO-acetolate syn thase NXbhOH O 5 valine S-Acetolactate O decarboxylase f p NADPH + H ch isobutylarine NADP Aerof acid reductoisamerase (KAA) valine , co, NH 8 onega transanitase a 2. NH; valine NH3. -- OH 22 Hi / "h"gehydrogenase or 2 e, 2 OH frtisatinase O O OHO OH branched chain keto acid 2,3-Dihydroxy isowalerate acetohydroxy acid dehydratase aecarboxylase H "c" O "d" ) H2 O ci-Ketoisowalerate CO isobutyraldehyde "f 2e. 2 h" "e" 2 e, 2 HK ( , NADH + H branched chain HS-CoA 9 NAD" E.eyenase eranched chain ketoacid HS-CoA yarog. dehydrogenase aidehyde2E. dehydrogenase OH S-CoA u-r"k" ~s. itssobutyryla rice sobutanol isobutyryl CoA butyryl CoA Patent Application Publication Jun. 25, 2009 Sheet 1 of 15 US 2009/O163376 A1 Figure 1 Glucose O Y O N's OH -- Noh O Pyruvate O "a" co- synthase OH OH O O valine S-Acetolactate O decarboxylase t" NADPH + H b c isobutylamine Aerof acid reaucroisonerase (KAAI) ware co. NH NADP Nh H28 omega --a transititase 2 NH, valine NH3. -- OH 22 Hi / "h" dehydrogenase or 2 e, 2h OH e transaintinase ><r O O OHO OH branched chain keto acid 2,3-Dihydroxyisovalerate aceton-aroxytoh-oh aciddidehydratas dehydrarase decarboxylase H "c" S. O "d" H2O ci-Ketoisowalerate CO2 Isobutyraldehyde "f"V 2.e., 2 H" "e" 2 e, 2 HR NADH + H branched chain HS-CoA 9 afcohol NAD" dehydrogenase branched chain Aketoacid HS-CoA dehydrogenase O aldehydean acylatin 3 hydrogenase OH c k' S-A N isobutyryloa -- SCAus figy Isobutanol isobutyryl CoA butyryl CoA Patent Application Publication Jun. 25, 2009 Sheet 2 of 15 US 2009/0163376 A1 Figure 2 a PF5-KARI (44) VGLRKGSATVAKA PAO-KARI (44) VGLRSGSATVAKA Spinach KARI (162) IGLRKGSNSFAEA b 59 Si'8w 72 MMC5 (44) VGLRKNGASWENAK MMS2 (44) VGLRKNGASWNNAK MVSB (44) VGLRKNGASWENAK PF5-i VC (44) VGLRKGSATVAKAE KARI-S1 (44) VGLRKNGASWNKAV ill V5 (59) IGVRKDGASWKAAI KARI-D1 (48) VGLEREGKSWELAK KARI-D2 (45) IGLRRGGKSWELAT C On Sen Slus (59) VGLRKN AK Patent Application Publication Jun. 25, 2009 Sheet 3 of 15 US 2009/O163376 A1 Figure 3 Patent Application Publication Jun. 25, 2009 Sheet 4 of 15 US 2009/O163376 A1 Figure 4 C3A1 O C3B11 C3C8 C4D12 Wt Negative OOO &SS EEE ES:ax -0.05 -0.10 -0.15 -0.20 8 -0.25 -0.30 -O-35 -0.40 f e -0.45 ENADH.W Substrate O NADH W/o Substrate -0.50 NADPH W/ Substrate & NADPH W/o Substrate Patent Application Publication Jun. 25, 2009 Sheet 5 of 15 US 2009/O163376 A1 Figure 5 am s {d - S. sa -0.1 {d 3s -0.1 * { e e - (d : as -0.2 s 0.2 d:: 4 s 4 3 -0.3 - 4d s 9. (d : 5 -0.3 d s 4 -0.4 s 3 NADH NADPH NADH NADPH -04 -O-5 NADH, NADPH -0.5 Patent Application Publication Jun. 25, 2009 Sheet 7 of 15 US 2009/0163376 A1 Figure 7 O5O 2 20 40 60 8O Temperature C Patent Application Publication Jun. 25, 2009 Sheet 9 of 15 US 2009/0163376 A1 Figure 9 10 20 30 40 50 60 gi70732562 ref YP 262325.1/1 gil 28868203 refNP 790822.1/1 gil 26991362 refNP 746787.1/1 gil 15599888 |ref INP 253382.1/1 gil 114319705 ref|YP 741388.1/ gii 83648555 ref YP 436990.1/1 gil 67159493 refize 004 20011, 1 gil 71065099 ref YP 263826.1/1 gil 1754.6794 refinP 520196.1/1 gil 74318.007 ref YP315747. 1/1 gil 66044103 ref YP 233944.1/1 gil 1205538161 ref YP 9581.67. 1/ gil 146306044 ref YP 001186509. gil 56552037 ref YP 162876.1/1 gil 57240359 ref ZP_003.683.08.1 gil 6225553 spo32414 IILvc REOM gil 15897495 refNP 342100.1 1/1 gil 76801743 ref|YP 326751. gi 18313972 ref|NP 560639. gi 19552493 ref INP 600495. gil 1607.9881 ref|NP 390707. gil 42780593 ref|NP 977840. gi 42781005 ref INP 978252. gi 266346 spoo 1292. ILV5 SPIoL. MAATAATTFSLSSSSSTsAAASKALKQSPKPSALNLGFLGSSSTIKACRSLKAARVLPSG 70 90 100 110 120 a s a . gil 70732562 ref Yp 262325.1/1 --KVFYDKDCDLS-----II gi 28868203 refNP 790822. 1/1 ------------- KVFYDKDCDS- - - - - IIQG---------- gi 26991362 refNP 746787.1/1 ----------------- KVFYDKDCDLS-- - - - IIQG---------- gil 15599888 |refNP 253382.1/1 ----------------- RVFYDKDCDLS-- - - - IIQG---------- gill 14319705 ref YP 741388.1 / QVYYDKDADLS----- IIQG---------- gi 183648555 ref YP 436990.1/1 ----------------- QVYYDKDCDLS----- IIQG---------- gil 67159493 refizp 0.0420011. 1 ---------------- KWYYDKDCDLS-- - - - IIQs---------- gi 71065099 ref YP 263826.1/1 NVYYDKDCDLS-- - - - IvoG---------- gi 1754.6794 ref INP 520196.1/1 ---------------- KVFYokdAdLS- or - - - LIKG---------- gil 7431.8007 ref YP315747. 1/1 KWYDKDADS- - - - - LIKQ---------- gil 6604.4103 ref YP 233944.1/1 KVFYDKdCdLS-- - - - IIQG---------- gil 120553816 ref YP 9581.67.1 / ----------------- QVYYDKDCDLS-- - - - IIQG---------- gil 146306044 refi YP 001186509. ----------------- KVFYOKDCDS- - - - - IIQG---------- gi 56552O37 ref YP 162876.1/1 -------------- KWYYDSDADLG- - - - - LIKS---------- gi57240359 refizP_00368308.1 -------------A- -WSYYDKDCDIN- - - - - LIKs---------- gi 16225553 spo32414 ILVC RHOM RVYYORDAOWN- - - - - LIKS---------- gi 15897495 refNP 342100.1/1 KCTSKYTDNDAND- - - - - LIKG---------- gi 76801743 ref YP 326751. Tid-ATIYYDDDAEST- - - - -VLDD---------- gil 18313972 ref NP 560639. A- - -KYTDREASE- - - - - PLKG---------- gil 19552493 ref NP 600495. -------------A- - ELLYDADADS- - - - - LQG---------- gil 1607.9881 ref|NP 390707. -------------W---KWYYNGDKEN- - - - -VLAG---------- gil 42780593 ref INP 977840. -------------A- - -KWYYEKdVTVN- - - - -VLKE---------- gil 42781005 ref INP 978252. War w w - - - - - - - r ur m KTYYEKDANVE- - - - - LLKG---------- gil 266346 spi Q01292. ILV5 SPIOL ANGGGSALSAQM SAPSINTPSATTFDFDSSVFKKEKVTLSGHDEYIVRGGRNLFPLLPD Patent Application Publication Jun. 25, 2009 Sheet 13 of 15 US 2009/0163376 A1 gil 70732562 ref YP 26.2325.1/1 gil 28868203 ref INP 790822. 1/1 gil 26991362 ref INP 746787.1/1 gil 15599888 |ref NP 253382. 1 1/1 gil 114319705 ref YP 741388.1 / gi83648555 ref YP 436990.1/l gil 67159493 ref ZP 0.042001.1.1 gil 71065099 ref YP 263826.1/1 gil 1754.6794 refNP 520196.1/1 gil 74318.007 ref YP315747, 1/1 gil 66044103 ref YP 233944.1/1 gil 120553816|ref|YP 958167.1/ gi 146306044 ref|YP 001186509. gil 56552037 ref IYP 162876, 1/1 gi5724.0359 refzP_003.683.08.1 gi 6225553 spo32414 (ILvc RHOM ------------------------------------------------------------ gil 15897495 ref INP 342100.1/1 gil 76801743 ref YP 326751.1/1 gi 18313972 refNP 560639.1/1 gil 19552493 ref NP 600495. 1/1 gil 1607.9881 Iref NP-390707. 1/1 gi 42780593 ref INP 977840.1/1 gi 42781005 ref INP 978252.1/1 gil 266346 sp|Q01292. ILV5 SPIOL MMAQIEILRKKGHSYSEIINESVIEAVDSLNPFMHARGVSFMVDNCSTTARLGSRKWAPR gi70732562 ref YP 262325.1/1 gi 28868203 ref INP 790822.1/1 gi 269,91362 ref INP 746787. 1/1 gil 15599888 ref INP 253382.1/1 gil 114319705 ref YP 741388.1 / gil 83648555 ref (YP 436990.1/1 gil 67159493 refizP 0.042001l. 1 gil 71065099 ref YP 263826.1/1 gil 1754.6794 Iref INP 520196.1/1 gil 74318.007 ref YP315747. 1/1 gil 660.44103 ref YP 233944, 1/1 gil 120553816 ref YP 9581 67.1 / gil 146306044 ref YP 001186509. gi56552037 ref YP 162876.1/1 gi57240359 refizP_003683.08.1 gil 6225553 spo32414 ILvo RHOM gil 15897.495 ref NP 342100.1/1 gil 76801743 ref|YP 326751.1/1 gil 1831.3972 ref INP 560639.1/1 gil 19552493 refNP 600495. 1/1 gil 1607.9881 ref INP 390707. 1/1 gi 42780593 ref INP 977840.1/1 gil 42781005 refNP 978252.1/1 gil 266346 splg01292 |ILv.5 SPIOL FDYILSQQALVAVDNGAPINQDLISNFLSDPVHEAIGVCAQLRPSVDISVTADADFVRPE Patent Application Publication Jun. 25, 2009 Sheet 14 of 15 US 2009/O163376 A1 gil 70732562 ref|YP 26.2325. 1/1 ---- gi 28868203 refNP 790822. 1 1/1 ---- gi 26991362 ref|NP 746787. 1/1 ---- gil 15599888 |ref INP 253382. 1/1 ---- gill 14319705 ref YP 741388.1 / ---- gi 8364,8555 ref|YP 436990.1/1 ---- gil 67159493 ref ZP 0.042001.1.1 - - - - gi 71065099 ref YP 263826.1/1 ---- gi 1754.6794 ref INP 520196.1/1 ---- gi 74318.007 ref YP315747. 1/1 ---- gil 660.44103 ref YP 233944.1 / 1. ---- gil 12055381.6 ref YP 9581.67.1 / ---- gi 146306044 (ref YP 001186509. ---- gi 56552037 ref (YP 162876. 1 1/1 ---- gi 57240359 ref ZP 003.683.08.1 | ---- gi 6225553 spo32414 ILVC RHOM ---- gil 15897.495 ref INP 342100. 1/1 ---- gi 76801743 ref YP326751. 1 1/1 ---- gil 18313972 ref INP 560639. 1/1 ---- gil 19552493 ref INP 600495.