(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2010/0197519 A1
`Li et al.
`(43) Pub. Date:
`Aug. 5, 2010
`
`US 20l00l975l9Al
`
`(54) KETOL-ACID REDUCTOISOMERASE USING
`NADH
`
`(75)
`
`Inventors:
`
`goufien Ifj,’ Lavegincéville’ I\gE(US);
`er‘ ng mo’
`1 mmgton’
`(US); Mank J. Nelson, Nevvark, DE
`(US); Daniel P. Okeefe, Ridley
`park, pA (US)
`
`Correspondence Address:
`E I DU PONT DE NEMOURS AND COMPANY
`LEGAL PATENT RECORDS CENTER
`BARLEY MILL PLAZA 25/1122B, 4417 LAN-
`CASTER PIKE
`WILMINGTON, DE 19805 (US)
`
`(73) Assignee:
`
`E. I. DU PONT DE NEMOURS
`AND COMPANY, Wilmington, DE
`(US)
`
`(21) APP1~ N05
`
`12/637305
`
`(22)
`
`Filed;
`
`Dec, 15, 2009
`
`Related U.S. Application Data
`(63) Continuation-in-part of application No. 12/337,736,
`filed on Dec. 18, 2008.
`(60) Provisional application No. 61/015,346, filed on Dec.
`20, 2007, provisional application No. 61/109,297,
`filed on Oct. 29, 2008.
`_
`_
`_
`_
`Publication Classification
`
`(51)
`
`Int. Cl.
`(200901)
`C403 30/04
`(2006.01)
`CIZN 9/02
`(2006.01)
`C07H 21/04
`gggggg
`5%
`‘
`(200601)
`C121’ 7/16
`(52) U.S. Cl. .......... .. 506/9; 435/189; 536/23.2; 435/243;
`435/25; 435/160
`
`ABSTRACT
`(57)
`Methods for the evolution of NADPH specific ketol-acid
`reductoisomerase enzymes to acquire NADH specificity are
`provided. Specific mutant ketol-acid reductoisomerase
`enzymes isolated from Pseudomonas that have undergone
`co-factor switching to utilize NADH are described.
`
`BUTAMAX 1022
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 1 of 24
`
`US 2010/0197519 A1
`
`Glucose
`
`D
`
`I
`$
`
`[1
`
`WJKW \”)J\[]H
`
`U
`
`U
`
`Pyruvate
`
`Iloll
`
`CD2
`
`ocetolote synthose
`
`DH
`
`\“>%/UH
`
`U
`
`u
`
`S-Acetolactote
`
`NADPH + H*
`
`"b"
`
`NADp+
`
`ketolocid reductoisomerose (KARI)
`
`UH
`
`><%/DH
`DH 0
`
`2,3-Dihydroxy isovolercte
`
`,,
`
`,.
`
`ocetdoehhyyddrrooxeglsceicid
`°
`\
`H20
`
`Continued
`Fig. 1B
`
`on
`
`D
`
`FIG.
`
`1A
`
`

`
`Patent Application Publication
`
`2
`
`US 2010/0197519 A1
`
` 2_o;8_oIN.-mNM:6535:95+:N.-mN+_W:0:+I+:3:..:N8Em_o>om_3mv_-mmPE%_o::Bo2soa..QN:m./._p.5.5
`In3:..W...a2888.£98umzucosu.EooAQ
`aI4E0.5emo_b.o€oo%
`
`
`
`.mmmocmmofifmub<z<oo-mI<oo-mI
`
`
`
`
`+:N.-mNmmoc_Eomco:
`
`
`mmoc_Eomco:DNI.\JImNmN
`P23}QN5$o$m§..:2.Ioc__o>.-
`
`mc_Eo_b3om__IIa/j\c/_\
`ommEo_:2N8
`
`N124N12mc__o>
`
`mmo_.¢o€oumuGmc__o>
`
`49_._§§8282$.29:B885
`
`
`
`In_.§__%823%m_u>_._mEo
`
`mmo:moo_u>;mu
`
`m.5:
`
`m_ocSBom_\f\..._..
`<8_:338_$335<oo33.5
`
`<oo_b.:Bom_<ou-m/=\/\Q
`
`
`<8-m
`
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 3 of 24
`
`US 2010/0197519 A1
`
`Sequence ID
`
`17
`
`1 6
`
`18
`
`<4 4)
`
`( 4 4)
`
`VGLRKGSATVAKA
`
`VGLRSGSATVAKA
`
`(162)
`
`IGLRKGSNTFAEA
`
`FIG.
`
`2A
`
`Sequence ID
`
`9
`
`10
`
`ll
`
`l 7
`
`1 5
`
`12
`
`13
`
`14
`
`(44) VGLRKNGASWENAK
`
`(44) VGLRKNGASWNNAK
`
`(44) VGLRKNGASWENAK
`
`(4 4 ) VGLRKGSATVAKAE
`
`(4 4 ) VGLRKNGASWNKAV
`
`(44)
`
`IGVRKDGASWKAAI
`
`(44) VGLEREGKSWELAK
`
`(44)
`
`IGLRRGGKSWELAT
`
`Consensus
`
`VGLRKNGASWE AK
`
`FIG.
`
`2B
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 4 of 24
`
`US 2010/0197519 A1
`
`
`
`\...m\..mm
`
`.,.;H
`
`
`
`ww\\\n.\.....3«\\\.\.(.«_
`
`FIG. 3
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 5 of 24
`
`US 2010/0197519 A1
`
`Negohve \\\‘
`
`1k
`
`\
`
`I
`
`%Wt
`
`substrateE]NADHw/osubstrateENADPHw/osubstrate
`flNADHwsubstrate§NADPHw/
`
`A4
`
`FIG.
`
`EZI s
`
`\\\\\\\\\\\\\\\‘«
`
`EQ I .
`
`\\\\\\\\\\\\\\\\\\\\‘a
`
`94
`
`: I
`
`E %
`
`///////////////////////////////’///////////.//////4‘
`
`.\\\\\\\\\\\\\\\\\\\\\‘3 '
`
` :
`
`0.00
`
`LO
`
`I
`0
`
`o
`“'5
`C?
`
`-0.15
`
`(3
`
`C?’
`
`L0
`.
`C.’
`
`G
`.
`C.’
`
`L0
`
`<.=
`
`o
`“Z
`9
`
`-0.45
`
`-0.50
`
`(mg) uogdwnsuoo Joq:>o;o;)
`
`C4012
`
`cscs
`
`C3811
`
`C3A10
`
`C3A7
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 6 of 24
`
`US 2010/0197519 A1
`
`NADH
`
`NADPH
`
`NADH
`
`NADPH
`
`FIG.
`
`5A
`
`m n
`
`O 1
`
`n_u00__
`.23.
`
`nu
`
`4.
`
`83$
`
`
`
`:o:aE:mcoo53200
`
`T
`
`§...,s
`
`
`
`cozacsmcoo_2u2oo
`
`01.23.45.
`n_un_un.un.un_u
`
`j
`
`\
`
`FIG.
`
`5B
`
`
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 7 of 24
`
`US 2010/0197519 A1
`
`3:’
`8»
`
`2gr
`
`\
`
`C3Bl1
`
`91A9
`
`5“%§§
`
`3904V?V83610
`
`24F968F1O07/my
`
`7
`
`7
`
`NADH
`
`NADPH
`
`
`
`FIG.6
`
`-0.1
`
`‘-‘f
`C?
`
`“"?
`o
`
`<'
`(DI
`
`“'3
`o
`
`(9179) uondunnsuoo Jo1:>o;o3
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 8 of 24
`
`US 2010/0197519 A1
`
`3
`(13
`
`CD
`CD
`
`a-5
`
`I‘
`
`L)
`
`O -
`
`.4
`
`E’
`C
`B
`(D
`5
`O‘— |-laid
`«+3
`
`L|_
`
`EC
`
`D
`I-
`
`C)
`("V
`
`C3I.f')®L.l")CJ
`2|“-LDC\!
`
`(z) Kwmov
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 9 of 24
`
`US 2010/0197519 A1
`
`100
`
`150
`
`200
`
`250
`
`51
`(18) ——-—KKVAIIG
`
`SQGHAH
`
`LRD ---- --NGFD‘
`
`GLRKG~KSWDKA
`
`(17) ——-~KTVAVIG ‘SQGHAQ
`(51) FKGIKQIGVIG
`SQAPAQ
`(17)
`-———KKVAIIG
`SQGHAH
`(17)
`-———KKVAIIG
`SQGHAQ
`
`GVRPG—KSFEVA
`LRD '''' -~SGVE
`LKDSLTEAKSDVV‘K GLRKGSNSFAEA
`LKD ---- --SGVD T GLRSGSATVAKA
`YLKD ---- —-SGVD T GLRKGSATVAKA
`
`101
`
`
`
`-~---VYTVAEAAK ADVVMILL DELQPEVYEAEIAPNLQA
`-----VMSVSEAVR AQVVQMLL DEQQAHVYKAGVEENLRE
`lSEENGTLGDMWEflI GSDLVLLL SDSAQADNYEKVFSHMK—
`~~---VADVKTA
`DVVM1
`DEFQGRLYKEEIEPNLKK
`—--——VTDVAAAVA ADLVMI
`DEFQSQLYKNEIEPNIKK
`
`151
`(102) SLVFAHGFNVHFDQVK---'PWNVDVFLVAPKGPGHLVRRTFSEG—--—-
`
`(101) MLLFSHGFNIHFGQIN-~—-P YVDVAMVAPKSPGHLVRRVFQEG-----
`(150)
`ILGLSHGFLLGHLQSLGQD PINISVIAVCPKGMGPSVRRLYVQGKEVNG
`(102) TLAFAHGFSIHYNQVV—-~'R DLDVIMIAPKAPGHTVRSEFVKG—————
`(102) TLAFSHGFAIHYNQVV—-—"FDLDVIMIAPKAPGHTVRS3FVKG—-———
`
`
`
`201
`(144) GAVPALFAVYQD TGVATEKALSYAD IGATRAGVLETTFKEE"ETDLFG
`(143) NGVPALVAVHQD TGTALHVALAYAK
`GCTRAGVIETTFQEE”ETDLFG
`(200) AGINSSFAVHQD DGRATDVALGWSI
`G——SPFTFATTLEQEYKSDIFG
`(144) GGIPDLIAIYQD SGNAKNVALSYAC
`GGGRTG11ETTFKDE”ETDLFG
`(144) GGIPDLIAIYQD SGNAKNVALSY
`GGGRTGIIETTFKDETETDLFG
`
`FIG. 8
`
`Sequence ID
`
`47
`
`48
`18
`16
`17
`
`47
`48
`18
`16
`17
`
`47
`48
`18
`16
`17
`
`47
`48
`18
`16
`17
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 10 of 24
`
`US 2010/0197519 A1
`
`
`
`xxmfillllllllllllllllllllllllllllllllllllllllllllllllllllllllllI:S............................................-:.I-:-::»uauuu«3..........................................................
`
`
`
`
`
`
`
`uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuIn3uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu-«pm
`
`
`
`
`
`uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu«:qm
`
`
`
`............................................................mm
`
`
`
`..........................................................I-mm
`
`
`
`llllllllllllllllllllllllllllllllllllllllllllllllllllllllllI:ma
`
`
`
`:n.||:u»:..:IIIIIII..:romuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuua»3IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
`
`
`
`
`
`..................................!-»:::-:-:--::}uuuanlnu;mm
`
`wmmq>m§vSmmu§HemmmwqmoqzqmmmmmmovE5B¢.<mmBmmmmmqmmHH<m,S?.z3::::::::::::::::::::::::::::::::::::::::::::::::::::::::::u;3uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu
`
`
`
`
`up3uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu1;am
`
`2..5:
`
`
`
`..........................................................u«2
`
`
`uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuno3..........................................................
`
`
`--3..........................................................:1Q.
`
`
`
`uuuuuuuu:q-I---:---------u-uunaxuuuuluui-ufuuuuuuunuuunu-mm
`
`
`
`..........................................................1-mm
`
`
`
`nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnIn.3
`
`
`
`...........................................................1-mm
`
`n__mocmzumm
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 11 of 24
`
`US 2010/0197519 A1
`
`Sequence|D
`
`17
`43
`44
`16
`35
`39
`41
`38
`15
`40
`42
`37
`46
`34
`36
`33
`13
`30
`14
`32
`31
`47
`43
`18
`
`120
`110
`100
`90
`so
`70
`....1....‘....1....1....1....1_...
`....1....p....|...4
`....
`——————————————————————————— ——
`EYDKDcD1s—«———11QG ———————— --
`——————————————————————————— --KvFYDKDcD_s——n-~1-QG ———————— —-
`——————————————————————————— ——KVFYDKDCD-S~~~——I QG ———————— --
`——————————————————————————— <—RVFYDKDCD3S—-—~~IIQG ———————— --
`—————————————————————————— —-QVYYDKDAD3S-—---IIQG ———————— --
`—————————————————————————— ~-QVYYDKDCDLS-----IIQG ———————— --
`—————————————————————————— ——KVYYDKDCD3S-----I-QS ———————— --
`——————————————————————————— ——NVYYDKDCDLS—~---IVQG ———————— —«
`———————— ——4. ———————————————— ~—KVFYDKDAD.S~~——-L_KG ———————— --
`——————————————————————————— --KVYYDKDAD3S——--—LIKQ——j ————— --
`——————————————————————————— --KVFYDKDCD4S—~—--I QG ———————— —~
`——————————————————————————— ——QV\_"{DE<DCDLS~~---IIQG ———————— --
`———————— --J ---------------- —-KVFYDKDCDAS~~---I_QG ———————— --
`———————— —-1 ———————————————— ——KVYYDSDAD3G~~———L_KS ———————— --
`——————————————————————— ——A-—VSIYYDKDCDIN-~-——L_KS ———————— --
`——————————————————————————— -—RVYYDRDADVN—-——~L KS ———————— --
`——————————————————————— ——KCTSKIYTDNDAN4D~—~~ALIKG ———————— --
`——————————————————————— —4TD—ATIYYDDDAEST-----VJDD ———————— —~
`———————— ——J ———————————— ——Ar~—KIYTDREASLE--—--P3KG ———————— --
`—————————————————————— ——A——1ELLyDADAD3s~-~~—L_Qc ———————— --
`———————— ——] ———————————— ——v———KvYYNGD1KEN—————v-Ac ———————— --
`——————————————————————— ——A———Kvyy1-:KDv1:vN—————v3£<E ———————— ~~
`——————————————————————————— -—KTYYEKDANVE——-—-L3KG ———————— --
`ANGGGSALSAQMVSAPSINTPSATTFDFDSSVFKKEKVTHSGHDEYIVRGGRNLFPLLPD
`
`FIG. 9B
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 12 of 24
`
`US 2010/0197519 A1
`
`Sequence ID
`
`130
`
`140
`
`150
`
`160
`
`170
`
`1.80
`
`17
`43
`44
`16
`35
`39
`41
`38
`15
`40
`42
`37
`46
`34
`
`36
`
`33
`13
`30
`
`14
`47
`
`32
`31
`
`48
`18
`
`———--KKVAII
`—~v——KKVAI
`————~KKVAII
`—----KKVAI
`——-——KKVAV
`—————KK'vA[
`---_-KKvA1_F
`—————KKVAII
`——-——KNvTII
`-————RKVAI'
`--—--KKVAI
`----—KKVAI'
`----*KKVAII
`‘~---KKIAI
`
`~A-——KK\/'AI'
`
`~~—--KKVAVI
`—————KRIAV
`~---—KTVAV
`
`—————RKVAI
`—————KTVAV
`
`~————KT1Av:
`-————KK\/AII
`
`Q
`Q
`Q
`Q
`Q
`Q
`Q
`Q
`Q
`Q
`Q
`Q
`Q
`Q
`
`Q
`
`Q
`Q
`
`Q
`Q
`
`Q
`Q
`
`
`
`EAGFE—-
`RDS ———— ——sv—Ev IGLREGSKSAE
`KES ———— ——GV4DV VGVRQGK—SFT AQEDGHK~~
`
`RDS——————GL—EV IGLRRGGKSWE;EESEGFR—-
`IIRDN---—--GE‘-EN VGLRKGK—SWD EDGFS--
`
`TDGFE—~
`GVRPGK—SFE
`LRDS ———— —-Gv— V
`Q
`—-———KTVAV_
`AFKGIKQIGVIGWGSQAPAQAQNLKDSLTEAKSDV—V'VKIGLRKGSNSFAEARAAGFSEE
`1'
`<1‘
`it
`'}
`9: *
`iz
`
`GXGXX (G/A)
`
`FIG. 9C
`
`/‘VG JRKGSATVY
`KDS - - — — ~—GV-
`LAHGLK‘“
`-oacm KDS ———— ~-Gv— ‘VGLRKGSATVA
`AHGLK--
`KDS ———— ~—Gv~
`CVGQRKGSATVA
`EAHGLK——
`KDS ———— ——Gv—
`;vG:RsGsATvAfl§EAHGLK——
`KES ———— ——Gv~ VVVGLREGSSSAA
`KAGLA——
`-J A. J KDS ---- ~-G\f—E ZVG JRE<GSGSWAI<1A.3NAGLA«—
`-1-F: KDS———— ——GV—fl:EVG-RAGSASVAHAEAHGLT--
`QDS ———— ——NV—
`;vc-RADscswK::fi§AsLK——
`IDS ———— ——GV—NVIVGLRKNGASWN
`A£LQ——
`- rqxns ———— ——GV—DV VALRPGSASAKKAENAGLT-—
`KDS ---- --G\/“UV "VG_.R£<GSATVAK}‘«*AHGLK--
`KDS - - - - --GV~DVV\/G_.RAGSSSI
`’EAYGLK—-
`‘Cl LKDS - - - - --GV—E'V'l"IG_.RKGSATVA
`AHGLK--
`RDS — - — - --G‘/A I
`IA_lRPDSASVE<
`DAGF'K—-
`
`RDS- -—--~GV-§:
`
`IGLKEGGQSWA
`
`KANE'I--
`
`VA._iRPGSASIKI‘1AEAEIGLK--
`RDS - - - - -—G\/"K
`RDS ———— ——GL—NV fGLEREGKSWE1AdSDGIT--
`DDS ———— —~GV—ER GLREDsssRsfl ADGLD--
`
`
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 13 of 24
`
`US 2010/0197519 A1
`
`200
`190
`....y....|._..|....
`-——vTDvAAAvAGADivMI
`———vTDvAsAvAAAD-vM:
`——~vADvATAvAAADLvM:
`———VADVKTAVAAADVVMI
`———VASIEDAAAQADVVMI
`—-—VKEVAEAVAGADVVMI
`---VKSVKDAVAAADVVMI
`~——vAEvEEAvKAADIIMI
`———VKEVAEAVKDADVVMI
`~—~VKSVPEAVAGADLVMI
`———VTDVASAVAAAD4VMI
`—A—TSDVASAVASADVVM%
`~—~VTDVATAVAAADLVMI
`FQGQLYKQEIEPNIKKGAT4AFSdG
`———vLTNAEAAKwADILMI
`HQAAIYAEDLKDNLRPGSAIAFAHG
`iG
`———VKSVKEATKEADLIMI
`IQSEIFNEEIKPELKAGKTJAFA
`—-—v1TPAEAAAwADvvML
`LQADLYKSELAANLKPGAALVF
`-v—PLHTKDAvKDADI1IFi
`QRTLWLESVQPYMKKGADJVF
`
`VQPAVYE-QIEDVLQPGDTLQFA.
`———VATPRGAAEQADLVSWJ
`———VYEIGEAVRKADVILVL
`EQPKVWQEQIAPNAKEGVVVDFA
`———VKTTAEAAAWADVIM,
`SQAEIFTNDIEPN4NAGDALLFG
`———VFSVKEAAAQAEII
`QQQKVYEAEIKDELTAGKSLVFA
`LQPEVYEAEIAPN4QAGNSJVFA
`———VYTVAEAAKQADVVMIL
`--—VMSVSEAVRTAQVVQPL pm
`QQAHVYKAGVEEN-REGQMLLFS
`NGTLGDMWETISGSDLVLLLISDSAQADNYEAKVFSHMKPNSILGLS
`it
`$<
`
`
`
`AI{YNQVVPR
`NIHFGLIEPR
`NI{YGQIVAP
`IHFKLIEAR
`NIHYKLIDPP
`NI{YGQIEPS
`NV{FGLIKPP
`NI{FDLIKPA
`NVHFHQIVPP
`4GFNViFDQVKPP
`G NIiFGQINPP
`4GFLLGHLQSLGQ
`
`210
`
`220
`
`FQSQLYKNEIEPNIKKGATJAFS
`FQSQLYKNEVEPNLKKGATAAFS
`
`240
`230
`[....[_...1
`GFAIHYNQVVPR
`
`HG AIHYNQVVPR
`
`HQAVIYHNQIAPNVKPGAAIAFA
`
`FQAQLYKSEIEPNLKSGATJAFSEG
`
`FQGRLYKDEIEPNLKKGATLAF
`FQKELYNDVIEPNIKQGATLAF
`QIADVYKNEVHGNIKQGAALAFA
`FQSRLYRDEIEPNIKQGATLAF
`FQSQLYKNEVEPNLKKGAT4AFS
`
`SIHYNQIVPR
`SIHYNQVVPR
`
`TLSL
`
`LA
`t“
`
`L L t
`
`"L“L'L'L“‘L
`'L“"t“L—"
`
`UOCJUCJUDUUUUi
`
`H2:'zLr*:S‘:L3s-4»4L«‘»—;‘En4»J‘»4::sqTa»-:1
`rvw:r:1m_:v.1_:3$m:JUUUUUUU
`
` ("LLITE’U'DUDUU
`
`FIG.
`
`9D
`
`Sequence|D
`
`17
`43
`44
`16
`35
`39
`41
`38
`15
`40
`42
`37
`46
`34
`36
`33
`13
`30
`14
`32
`31
`47
`48
`18
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 14 of 24
`
`US 2010/0197519 A1
`
`..
`.1.
`A———DL
`A—-—oL
`A—-—DL
`A———DL
`A-~—DL
`A—~—DL
`
`K---DL
`
`A———DL
`
`E———D
`
`Sequence|D
`
`17
`43
`44
`16
`35
`39
`41
`38
`15
`40
`42
`37
`46
`34
`36
`33
`13
`30
`14
`32
`31
`47
`48
`18
`
`280
`270
`260
`...;....;....:.... ....
`....
`GHT flSEFVKG—~——AGG PDLI*IjQ
`-GETV TEFVKG————-GG_PDLL
`Q
`~c TVgSEFVKG———-GGIPDLI
`Iyq
`GETV SEFVKG—~———GGIPDLI
`IYQ
`PG
`sTYvaG—————esvpsL PIHQ
`PGfi§$aSEFVKG—-———GG PDL PIEQ
`
`
`
`SEFVRG—~——-GG PDLIA YQ
`as
`flSEFAKG—----GGIPDLLAIYQ
`ass
`PGHTVRGTYAQG-----GGVPHLI
`HQ
`
`
`
`' TEFTKG-—--—GG.PDL.AIFQ
`
`Q
`
`
`
`
`
`250
`4...
`
`\
`
`MIAP
`
`A-——DLEV MIAP
`S———DLB MVAP
`A—--DLE MVAP
`
`jg TV SEFVKG—————GGPDLIE%YQ
`9
`A——-DLD
`-
`fi7v§TEFvKG—~—-GGIPDL:
`y
`A———DL
`PGE J SEFVKG--—--GG_PDLL 119
`MIAP
`A———DL
`fie “ 3sEYvRc~-\\\ycvpcLd:hdQ
`IAP
`K———DI
`.§GH§§fiHEFSIG——-——GG”PCLgAIHQ
`K———GIDV MIAP
`P F
`REYYKAG————-GGVPAL i§flQ
`K———D3a§
`A2
`/REEYLAG—~———RGVPALMAE¥Q
`K———NIDV
`Am
`RRQFVDG--——-KGVPCLIA D
`D——-DII
`JV RTYEQG—————AGvPAL#AIY
`A———DVDV
`ii
`.v RTFSEG-———-GAVPAL;:fi Q
`A——«Nv:v LVAP
`v RVFQEG-——~-NGVPAL
`flgfi
`S~——YVE
`A
`9:
`az
`DFPKNI SV IAVC PKGMG P SVRRLYVQG KEVNGAGIN S S FAVHQDVDG RAT DVAVJGW S IAL
`
`MVAP
`
`PG
`
`MVAP
`
`cu
`
`RGEYLKG—————GGVPCL
`
`MVAP s
`
`3VRRNYEND~—~——EGTPGL1A YQ
`
`FIG. 9E
`
`

`
`tnmaP
`
`US 2010/0197519 A1
`
`
`
`
`
`H556u2.mu$>1.:Eo§>1E>eo<0anewmvE-DE05oSW2%ommommo..mM.D_mocmzcmw
`.momm>flEo§>qm><omMWWSSEvii..B,Eoomm.mo?fi>w3S<oSmEo>3mmmumEmuSofia0mmbo.E>n.eEw§>qm>uo-3.LwemfimEmuA02003pmo.%>u:mrS§>fi,_>ewan.ornommVEHE
`HHQHMUo3nowm>n..fl._r$§>aE>eoammoEE.n:_fvm:
`
`
`
`
`
`
`EL556uowLwemwoRWeo¢m>1..amEu§>1E>ew>d.wmnm-mn_mV5-5Hoemo0SAwmm>n:Eu§>qm><o->mouBwoTmHmVEHH..5,onmnmnmLNLoiswmmMumenbmzufifimiwo->mmgmmi5:Eommowvm2,wmm>1,..Eo§>qm>Sw»>d.O%goWuEV::5,56mS5ummeH.m:G§>qm><uw><ubemnmMommaomm0wmmiummumofimmquo593$oSSomm>u.EmBoH1Emquo>>manmmmumEL.1wzm>u>Eov§Sm2qo5mg953Em:..>o/ReoEmo,S>u.H§9§>qme>u3mmfimnmEp>.>o$§oomhomm>num:2§zqmzHoH>pBEEVCCL EH6,059oSfufifi1uEo§>q¢mGo>4m.o:m_-mE;£654oHmMw<mew>mEw>m>1EmEm3m:.>Si1.:n;.. Humizvfimqommzommfiémfiumiwm><o1EHummBHomv§.mom7FHEeIEmo3.omH1Hmm@¢.M>1H<H>oHmQ5305:Sufi;o3Mo,B>1EEo§>q§>uomm%Em,E
`
`o$>n.ErG§Sm>Hw3,wombnmEH-Ho§oo3o¢E1.nEo§>qm>muLEmoznoefigxiMommawmm%wm.m>uEmEw§>qm>oDd.MnmamwmviEHoemo0av
`
`
`
`
`
`
`
`
`
`
`
`
`H>o<m<wmm
`
`
`
`
`
`mm.0:
`
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 16 of 24
`
`US 2010/0197519 A1
`
`Sequence|D
`
`370
`
`380
`
`390
`
`400
`
`410
`
`420
`
`K—LIVDLMYE§G ANMYSISNNAEYGEYVTGPEVINAESRQAMRNALKRIQEGEYAKMF
`K—LIVDLMYE G ANMYSISNNAEYGEYVTGPEVINAESRQAMRNALKR_Q G YAKMF
`K—LIVDZMYEGG ANMNYS;SNNAEYGEYVTGPEVINEESRKAMRNALKR'QDGEYAKMF
`K~LIVD3MYEGG ANMNYS SNNAEYGEYVTGPEVINAESRAAMRNALKR Q GEYANMF
`
`K—LIVDLMFE G ANMYSISNNAEYGEYVTGPEV NEQSRQAMRNALKR'Q G YAKMF
`
`K-LIVDGLYQ G ANMRYSESNTAEYGDFTRGPRV_NEESREAMREILAE_Q%GEFAREF
`K-LIVD3MYE§G ANMNYS SNNAEYGEYVTGPEV1NDQSRAAMRNALKR Q G YAKMF
`K—LIVD3MYEGG ADMNYS SNNAEYGEYVTGPEV;NEQSREAMRNALKRIQQG YAKMF
`
`
`
`K—LIVDLIYEGG GNMYSJSNNAEYGEYVTGPRVVTAETKQAMKQCLHDLQ G YAKSF
`K—LIVDLMYEGG ANMNYS'SNNAEYGEYVTGVKVTNEQSRAAMKECLAN-QNGAYAKRF
`KGLIVDLMYEGG ANMNYS”SNNAEYGEYVTGPEV'NAESRQAMRNALKRIQCGEYAKMF
`K—LIVD.MYEGG ANMNYS SNNAEYGEYVTGPEV_NEQSREAMRNALKR_Q GEYAKMF
`
`K—LIVDLMYEGG ANMN S-SNNAEYGEYVTGPEVINEESRKAMRNALKR_QSGEYAKMF
`K—LIVDLIYQ G ADMRYSVSNTAEYGDYITGPKI TKETKEAMKGV.KDIQNGEFAKDF
`K—MLVDLVYE G SGMLNA SDTAKYGGMTVGKFV DESVRKRMKEALQR.K3GKFAEEW
`K—L1MDLIWQ_G YGMLBGVSDTAKYGGLTVGPRV’DENVKRKMKEAAMRVKSGEFAKEW
`
`K~LIVDLIYEPG ANMRYSESNTAEYGDIVSGPRVINEESKKAMKAILDDIQ G FVSKF
`
`K-LIVD_IYE G ANMRYSISNTAEYGDYVTGSRI TEATKAEMKRV1AD_Q G FVRDW
`
`K-LIVDLMYE G SEMWDS SDTAEYGGLTRGDRIVDDHAREKMEEV-EEVQ G FAREW
`
`K-LIVDLMFE G SNMNYS SDTAEFGGYLSGPRVIDADTKSRMKDILTDTQDG FTKRL
`K—LIVDLMYE G AGMRYS SDTAQWGDFVSGPRVVDAKVKESMKEVLKDIQ GTFAKEW
`K—LIVDLMYE G ENMRYS SDTAQWGDFVSGPRVVTEDTKKAMGTV.AEIQ GTFARGW
`K—L1VDLMYE G TNMR S SDTAEFGDYVTGSRIVTDETKKEMKRVGTEIQ G FAKKW
`TGVISKTISTKGMLALYNSLSEEGKK-DFQAAYSASYYPSMDILYECYEDVASGSEIRSV
`
`FIG. 90
`
`17
`43
`44
`16
`
`41
`
`35
`39
`38
`
`15
`40
`42
`37
`
`46
`36
`13
`14
`
`34
`
`33
`
`30
`
`32
`31
`47
`48
`18
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 17 of 24
`
`US 2010/0197519 A1
`
`430
`470
`460
`450
`440
`430
`....1
`....|....u.__.:....\....1._..:..._a....
`....\....1....
`ISEGATGYP—-SMTAKRRNNAA4GIE-I G QHEBMPWIGANKIVDKAKN ——————— --
`ITEGATGYP——SMTAKRRNNAE1GIE—VIG K_RSMMPWIAANKIVDKDKN ——————— ——
`
`ISEGATNYP—-SMTAKRRNNAAHGIE-I G Q1R MPW_SANKIVDKTKN ——————— --
`ITEGAANYP~~SMTAYRRNNAAHPIE-Q G KLR§MPWjAANKIVDKSKN ——————— ——
`VLENQAGCP——TLTARRRLAAE{EIE—VVG R_RGMPWINANKLVDKDKN ——————— --
`IAEGAHNYP-~SMTAYRRNNAA4FIE-Q G KURSMMPWIASNKIVDKSKN ——————— ~-
`ITEGAANYP——SMTAYRRNNAA{QIE~VVGEK_RTMMPWLAANKIVDKTKN ——————— --
`ISEGATNYP—*SMTARRRNNAE{QIE—ITGQK_RGMPWIGGNKIIDKDKN ——————— --
`LLENKAcAP——TLISRRRLTADHQIE-QuGmK-RAMPw-AKNKLvDQsKN ~~~~ ~4¢«—
`ILEGQANYP~-EMTAWRRNNAAHQIE—VVGRKflRSMMPWIAANKLVDHSKN ------- --
`ISEGATGYP--SM”AKRRNNAA{GIE-IIGEK_R§MMPWIAANKIVDKDKN ——————— --
`ISEGALNYP-—SM”ARRRQNAA4EIE-TWGVK_RSMMPW SANKIVDKDKN ——————— --
`ISEGATNYP——SMTAKRRNNAA{GIE-IIG QZRSMMPWISANKIVDKTKN ——————— --
`VLDNRAGQP—-ELKAARKRMAA{PIE—Q G R_R<MPWIASNKLVDKARN ——————— --
`ILERRANFA——RMHAERKLMNDSLIE—K G E_RAMpw:sAKKLvDKDKN ——————— --
`MLECKAGQP~-SFKATRRIQXEHVIE-V‘G KLRGMPWISKNKLVDKARN ——————— --
`
`VEEYGRGMP—ATVVNGLSNVQNSLEE~KDG Qfia LVQK————— --GKPKS ——————— ——
`ISENQAGRP——SYKQLRAAEKNHDIE—AVG DL
`JFAW ————— ——GDD ————————— --
`vEEYNRsAP——TLRKLMEEARTHpIE—Kdq E RK3LF—--GP ——————————————— ——
`IANVENGNT~-ELEGLRASYNNHPIE—ETG K RDJMSWVKVDARAETA ————————— --
`IVENQVNRP—-RFNAINASENEHQIE—VjG K_R7MPFVKQGKKKEAVVSVAQN~-—~—
`IAEHKAGRP-—NFHATNEKENEHEIE—V G KHR MPFV—QPRVKVGMK ———————— --
`ILENQAGRP——TYNAMKKAEQNHQLE~K G ELREMSWIDAPKELVKK ————————— --
`VLAGRRFYEKEGLPAFPMGKI DQTRMWKVGEKVESVREV —Z\GD— - LG PLYPFTAGVYVAL
`
`
`
`Sequence ID
`
`17
`43
`44
`16
`35
`39
`41
`38
`15
`40
`42
`37
`46
`34
`36
`33
`13
`30
`14
`32
`31
`47
`48
`18
`
`FIG.
`
`9H
`
`

`
`Patent Application Publication
`
`.Aug.5,2010 Sheet18of24
`
`US 2010/0197519 A1
`
`
`
`..........................................................--FM
`
`..........................................................--mq
`
`
`
`..........................................................--vv
`
`..........................................................»-ofi
`
`
`
`..........................................................--mm
`
`
`
`..................-1------------x;--------------{------,----mm
`
`
`
`..........................................................--fiv
`
`
`
`..........................................................-xmm
`
`
`
`:::::::::::::::::::::::IJlllllillllillllIilllllllllllllllliima
`
`..........................................................--ow
`
`............................................................N¢
`
`..........................................................1-pm
`
`..........................................................--ow
`
`..........................................................-;vm
`
`
`
`..........................................................1-mm
`
`
`
`..........................................................-1mm
`
`
`
`..........................................................--ma
`
`
`
`............................................................--om
`
`..........................................................1-Va
`
`
`
`...............--:--1----1-------:----I---------------------mm
`
`
`
`..........................................................--am
`
`
`
`............................................................hv
`
`
`
`..........................................................-1aw
`
`mm<zmmmwqmmHemuzo>zum>wm¢mzmmzqmo><mH>mmzHHmm»mmuxmmqHmHomzma
`
`3.0:
`
`Q_wo:w:cmm
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 19 of 24
`
`US 2010/0197519 A1
`
`
`
`
`
`............[J---------------r-L---E----r-=I-ma
`
`IIIIIIIIIIIlIIFIIIPII|III|II|IIIIIIIIIiIIlIIIIIIIIIIIIITIIII
`
`IIIIIPII|P.III.I[.II.||r-...I|||IIIII.||I.r|II.I|I||I|.III|ur.III!.!.||l|||I
`
`---------;--;-.........----1------Hq
`
`III|III|IIIlIIIl|IIIIIII|IIIIIIII7IIIIIIIIIIIIIIIIIIIiIIII||
`
`IIIIIIIII||Ill|lI||IIIl.I||.I|.|.||l|l|l.l.l|||.l.||
`
`............................................................ow
`
`..........................................................--mv
`
`..........................................................--pm
`
`..........................................................--wv
`
`..........................................................--vm
`
`..........................................................--mm
`
`
`
`..........................................................-1mm
`
`IIIIII|||I||||I|||IIIIII|II|II||||||I|||I|IIl|II||I||||||Ill
`
`I|I|iIIIII‘I’III‘i|I[|IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII|ll:
`
`uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuunu2II|IIIIIII|IIIIIIIIIIIIIIIIIlIIIIIIJIIIII|I|1III1IJIIIJ|IIll
`
`Il1]III|lJIIIJII|iI|III1IIIII|IIIIIIIIIIIIIIIIIIIIIIII|'I|'l
`
`o_wo:m:cmw
`
`
`
`
`
`IIIIIIlIIIIIIIIIIIIIII.IIIIII!|.I||I.lIu|l|.lI||.l.I|l|
`
`
`
`IIIIIII|I|I|II|J.|ll.|.|.l||.l|l|||ll|||||l.l|IIll.
`
`IIIIIIII’IIIIUIIIII7IIIII|III|IIII|llI1IIIIII1IIIIIIIIIIIIII
`
`..........................................................--nv
`
`mmm>.ma¢o§L>mE>mmmu8<u>oH<mm>nEm«EzmSeam;m<wzm><>q<oom«E»nE3..........................................................--mv
`
`3.o_.._
`
`

`
`tnm3P
`
`M.
`
`t
`
`US 2010/0197519 A1
`
`M.n:mocmzamm
`
`.m2I}MH.,1.Pmm
`
`MMMS-5-
`n31..
`
`.m2II
`
`nmmll
`
`we3llASI!2II
`
`5,2l}
`
`
`
` umI}mEII23ll
`
`Smmi..
`.mmmll03iiaomllm2ll
`
`mq---:5IIMHmIIII
`
`
`Va.0:
`
`mmmama
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 21 of 24
`
`US 2010/0197519 A1
`
`*->qMfafskVYYDkDadlsGhdeylikGKkVAvI
`
`SQG
`
`M
`
`kV+YDkD+dls
`
`+i+GKkVA+I
`
`SQGHA+
`
`LrD
`
`L+D
`
`Sequence [D 17
`
`1
`
`~M----KVFYDKDCDLS-———-IIQGKKVAII
`
`HAQ
`
`SQG
`
`LKD 37
`
`SGVdVvVG
`
`SGVdV+VG
`
`Sequence ID 17
`
`38 SGVDVTVG
`
` -kAeaaef VktvaEAvaqADvVmi
` kAea+e+ V +va Ava+AD+Vmi +PDefQ++
`
`-EAHEL VTDVAAAVAGADLVMI TFDEFQSQ 87
`
`lPDefQae
`
`vYeeeIepnLkpGatLaFAHGFNIHf-anvPrafPkDiDViMVAPKgPGH
`
`+Y++eIepn+k+GatLaF+HGF+IH+ -|vPra
`
`D+DViM+APK+PGH
`
`Sequence [D 1?
`
`88 LYKNEIEPNIKKGATLAFSHGFAIHY
`
`PRA---DLDVIMIAPKAPGH 134
`
`tVRreYvkGgGVPaLiAVyQ
`
`GnAkdlALsYA-
`
`iGggRAGvIETTFk
`
`GnAk++ALsYA
`
`GggR+G+IETTFk
`
`
`
`GNAKNVALSY--
`
`GGGRTGIIETTFK 184
`
`Sequence [D 17
`
`135 TVRSEFVKGGGIPDLIAIYQ
`
`tVR+e+vkGgG+P+LiA+yQ
`
`eETETDLFGEQaVLCGGvteLVkaGFETLVEaGYaPEmAYFECLHElKLI
`
`+ETETDLFGEQaVLCGG++eLVkaGFETLVEaGYaPEmAYFECLHElKLI
`
`SGQUGHCG ID 17
`
`185 DETETDLFGEQAVLCGGTVELVKAGFETLVEAGYAPEMAYFECLHELKLI 234
`
`FIG.
`
`10A
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 22 of 24
`
`US 2010/0197519 A1
`
`VDLmYEgGIanMrySiSdTAeYGdyvtGprVIdeeskeaMkevLkdIQSG
`
`VDLmYEgGIanM+ySiS++AeYG+yvtGp+VI++es++aM+++Lk+IQ+G
`
`SGQUGDCB ID 17
`
`235 VDLMYEGGIANMYSISNNAEYGEYVTGPEVINAESRQAMRNALKRIQDG 234
`
`eFAkewi1EnqaGyPket1talrrneaeHqIEWkVGekLRsmmpWIaanK
`
`e+Ak++i+E+++GyP ++ta rrn+a+H IE +Ge+LRsmmpWI anK
`
`Sequence [D 1?
`
`235 EYA.KMFISEGATGYP—-SMTAKRRNNAAHGIEFIIGEQLRSMMPWIGANK 331
`
`lvdkdkn<-*
`
`+vdk+kn
`
`Sequence ID 17
`
`332 IVDKAKN
`
`333
`
`FIG. 10B
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 23 of 24
`
`US 2010/0197519 A1
`
`
`170
` 156
`
`Saturation Mutagenesis targeting positions 47, 50, 52
`& 53 (the cofactor specificity determining domain)
`
`
`
`EEEEEIIEIIEIIEI
`IIIIEIEII
`
`IIIIIEIIEIII
`
`
`!I3IIIIIIII-
`
`
`IIIIIIEIIIII
`
`
`
`
`
`the cofaotor specificity domain
`
`the cofactor binding affinity domain
`
`FIG.
`
`11
`
`

`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 24 of 24
`
`US 2010/0197519 A1
`
`E
`
`IE.9:iiii?
`
`
`
`Eli-ill!
`
`s:8552.Em:5:,mega).
`
`
`
`_oUmv8ome.Emco:mSE5:5:_mEon3_:_tmmsvcfi.Bom.6ome.:_m:o_fiSEm:_:_nEoo
`
`
`
`
`:_mEoum:E_E_9wub_o=_omqm
`
`
`
`
`
`
`
`ENEOUb_oEowamBfifloo9:
`
`||Z.%
`
`
`
`ENEOD>..=:EmmC_U:_fl_9om.5om£
`
`
`
`
`
`
`
`
`
`
`
`
`
`IulliEmaElilililEEliEEEli".III.‘EEl‘!
`
`
`
`
`

`
`US 2010/0197519 A1
`
`Aug. 5, 2010
`
`KETOL-ACID REDUCTOISOMERASE USING
`NADH
`
`[0001] This application is a continuation-in-part of U.S.
`Ser. No. 12/337,736, filed Dec. 18, 2008 and claims the ben-
`efit of the U.S. Provisional Applications, 61/015,346, filed
`Dec. 20, 2007, and 61/109,297, filed Oct. 29, 2008.
`
`FIELD OF THE INVENTION
`
`[0002] The invention relates to protein evolution. Specifi-
`cally, ketol-acid reductoisomerase enzymes have been
`evolved to use the cofactor NADH instead of NADPH.
`
`BACKGROUND OF THE INVENTION
`
`[0003] Ketol-acid reductoisomerase enzymes are ubiqui-
`tous in nature and are involved in the production ofvaline and
`isoleucine, pathways that may affect the biological synthesis
`of isobutanol.
`Isobutanol
`is specifically produced from
`catabolism of L-valine as a by-product of yeast fermentation.
`It is a component of “fusel oil” that forms as a result of
`incomplete metabolism of amino acids by yeasts. After the
`amine group of L-valine is harvested as a nitrogen source, the
`resulting ot-keto acid is decarboxylated and reduced to isobu-
`tanol by enzymes ofthe Ehrlich pathway (Dickinson, et al., J.
`Biol. Chem., 273: 25752-25756, 1998).
`[0004] Addition of exogenous L-valine to the fermentation
`increases the yield ofisobutanol, as described by Dickinson et
`al., supra, wherein it is reported that a yield of isobutanol of 3
`g/L is obtained by providing L-valine at a concentration of 20
`g/L in the fermentation. In addition, production of n-pro-
`panol, isobutanol and isoamylalcohol has been shown by
`calcium alginate immobilized cells of Zymomonas mobilis
`(Oaxaca, et al., Acta Biotechnol., 11: 523-532, 1991).
`[0005] An increase in the yield of C3-C5 alcohols from
`carbohydrates was shown when amino acids leucine, isoleu-
`cine, and/or valine were added to the growth medium as the
`nitrogen source (WO 2005040392).
`[0006] While methods described above indicate the poten-
`tial of isobutanol production via biological means these meth-
`ods are cost prohibitive for industrial scale isobutanol pro-
`duction. The biosynthesis of isobutanol directly from sugars
`would be economically viable and would represent an
`advance in the art. However, to date the only ketol-acid reduc-
`toisomerase (KARI) enzymes known are those that bind
`NADPH in its native form, reducing the energy efiiciency of
`the pathway. A KARI that would bind NADH would be ben-
`eficial and enhance the productivity of the isobutanol biosyn-
`thetic pathway by capitalizing on the NADH produced by the
`existing glycolytic and other metabolic pathways in most
`commonly used microbial cells. The discovery of a KARI
`enzyme that can use NADH as a cofactor as opposed to
`NADPH would be an advance in the art.
`
`[0007] The evolution of enzymes having specificity for the
`NADH cofactor as opposed to NADPH is known for some
`enzymes and is commonly referred to as “cofactor switch-
`ing”. See for example Eppink, et al. (J. Mol. Biol., 292: 87-96,
`1999), describing the switching of the cofactor specificity of
`strictly NADPH-dependent p-Hydroxybenzoate hydroxylase
`(PHBH) from Pseudomonas fluorescens by site-directed
`mutagenesis; and Nakanishi, et al., (J. Biol. Chem., 272:
`2218-2222, 1997), describing the use of site-directed
`mutagenesis on a mouse lung carbonyl reductase in which
`
`Thr-38 was replaced by Asp (T38D) resulting in an enzyme
`having a 200-fold increase in the KMvalues for NADP(H) and
`a corresponding decrease of more than 7-fold in those for
`NAD(H). Co-factor switching has been applied to a variety of
`enzymes including monooxygenases, (Kamerbeek, et al.,
`Eur. J, Biochem., 271: 2107-2116, 2004); dehydrogenases;
`Nishiyarna, et al., J. Biol. Chem., 268: 4656-4660, 1993;
`Ferredoxin-NADP reductase, Martinez-Julvez, et al., Bio-
`phys. Chem., 115: 219-224, 2005); and oxidoreductases
`(US2004/0248250).
`[0008] Rane et al., (Arch. Biochem. Biophys., 338: 83-89,
`1997) discuss cofactor switching of a ketol acid reductoi-
`somerase isolated from E. coli by targeting four residues in
`the enzyme for mutagenesis, (R68, K69, K75, and R76,);
`however the effectiveness of this method is in doubt.
`
`[0009] Although the above cited methods suggest that it is
`generally possible to switch the cofactor specificity between
`NADH and NADPH, the methods are enzyme specific and the
`outcomes unpredictable. The development of a ketol-acid
`reductoisomerase having a high specificity for NADH with
`decreased specificity for NADPH would greatly enhance this
`enzyme’s effectiveness in the isobutanol biosynthetic path-
`way and hence increase isobutanol production. However, no
`such KARI enzyme has been reported.
`
`SUMMARY OF THE INVENTION
`
`[0010] Applicants have solved the stated problem by iden-
`tifying a number of mutant ketol-acid reductoisomerase
`enzymes that either have a preference for specificity for
`NADH as opposed to NADPH or use NADH exclusively in
`their reaction. The method involves mutagenesis of certain
`specific residues in the KARI enzyme to produce the co-
`factor switching.
`[0011] Accordingly the invention provides A mutant ketol-
`acid reductoisomerase enzyme comprising the amino acid
`sequence as set forth in SEQ ID NO: 29; a nucleic acid
`molecule encoding a mutant ketol-acid reductoisomerase
`enzyme having the amino acid sequence as set forth in SEQ
`ID NO: 1 9; a mutant ketol-acid reductoisomerase enzyme as
`set for in SEQ ID NO:19; a mutant ketol-acid reductoi-
`somerase enzyme having the amino acid sequence selected
`from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28,
`67, 68, 70, 75, 79, 80, 81 and 82; and a mutant ketol-acid
`reductoisomerase enzyme as set forth in SEQ ID NO:17
`comprising at least one mutation at a residue selected from the
`group consisting of 24, 33, 47, 50, 52, 53, 61, 80, 115, 156,
`165, and 170.
`[0012]
`In another embodiment the invention provides a
`method for the evolution of an NADPH binding ketol-acid
`reductoisomerase enzyme to an NADH using form compris-
`mg:
`a) providing a ketol-acid reductoisomerase
`[0013]
`enzyme which uses NADPH having a specific native
`amino acid sequence;
`[0014]
`b) identifying the cofactor switching residues in
`the enzyme of (a) based on the amino acid sequence of
`the Pseudomonas fluorescens ketol-acid reductoi-
`somerase enzyme as set for the in SEQ ID NO: 17
`wherein the cofactor switching residues are at positions
`selected from the group consisting of: 24, 33, 47, 50, 52,
`53, 61, 80, 115, 156, 165, and 170; and
`[0015]
`c) creating mutations in at least one of the cofac-
`tor switching residues of (b) to create a mutant enzyme
`wherein said mutant enzyme binds NADH.
`
`

`
`US 2010/0197519 A1
`
`Aug. 5, 2010
`
`In another embodiment the invention provides a
`[0016]
`method for the production of isobutanol comprising:
`[0017]
`a) providing a recombinant microbial host cell
`comprising the following genetic constructs:
`[0018]
`i) at least one genetic construct encoding an
`acetolactate synthase enzyme for the conversion of
`pyruvate to acetolactate;
`[0019]
`ii) at least one genetic construct encoding a
`ketol-acid reductoisomerase enzyme of either of
`Claims 1 or 6;
`[0020]
`iii) at least one genetic construct encoding an
`acetohydroxy acid dehydratase for the conversion of
`2,3-dihydroxyisovalerate to ot-ketoisovalerate, (path-
`way step c);
`[0021]
`iv) at least one genetic construct encoding a
`branched-chain keto acid decarboxylase, of the con-
`version of ot-ketoisovalerate to isobutyraldehyde,
`(pathway step d);
`[0022]
`v) at least one genetic construct encoding a
`branched-chain alcohol dehydrogenase for the con-
`version of isobutyraldehyde to isobutanol (pathway
`step e); and
`[0023]
`b) growing the host cell of (a) under conditions
`where iso-butanol is produced.
`[0024]
`In another embodiment the invention provides a
`method for the evolution and identification of an NADPH
`
`binding ketol-acid reductoisomerase enzyme to an NADH
`using form comprising:
`[0025]
`a) providing a ketol-acid reductoisomerase
`enzyme which uses NADPH having a specific native
`amino acid sequence;
`[0026]
`b) identifying the amino acid residues in the
`native amino acid sequence whose side chains are in
`close proximity to the adeno syl 2'-pho sphate ofNADPH
`as mutagenesis targets;
`[0027]
`c) creating a library of mutant ketol-acid reduc-
`toisomerase enzymes from the class I ketol-acid reduc-
`toisomerase enzyme of step (a), having at least one
`mutation in at least one ofthe mutagenesis target sites of
`step (b); and
`[0028]
`d) screening the library of mutant ketol-acid
`reductoisomerase enzymes of step (c) to identify NADH
`binding mutant of ketol-acid reductoisomerase enzyme.
`[0029] Alternatively the invention provides a method for
`evolution of an NADPH specific ketol-acid reductoisomerase
`enzyme to an NADH using form comprising:
`[0030]
`a) providing a mutant enzyme having an amino
`acid sequence selected from the group consisting of
`SEQ ID NOs: 28, 67, 68, 69, 70, and 84;
`[0031]
`b) constructing a site-saturation library targeting
`amino acid positions 47, 50, 52 and 53 of the mutant
`enzyme of (a); and
`[0032]
`c) screening the site-saturation library of (b) to
`identify mutants which accept NADH instead of
`NADPH as cofactor.
`
`Similarly the invention provides a method for evo-
`[0033]
`lution of an NADPH specific ketol-acid reductoisomerase
`enzyme to an NADH using form comprising:
`[0034]
`a) providing a DNA fragment encoding a mutant
`enzyme having an amino acid sequence selected