`(12) Patent Application Publication (10) Pub. No.: US 2010/0197519 A1
`
` Li et al. (43) Pub. Date: Aug. 5, 2010
`
`
`US 20100197519A1
`
`(54) KETOL-ACID REDUCTOISOMERASE USING
`NADH
`
`(75)
`
`Inventors:
`
`_
`.
`gouglen 11:13 Lavxeincev111e, hgéUS);
`er-.ng lao,
`1 mmgton,
`(US), Marl{ J. Nelson, Newark, 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 25/112213, 4417 LAN-
`CASTER PIKE
`WILMINGTON, DE 19805 (US)
`
`(73) Assignee:
`
`E. I. DU PONT DE NEMOURS
`AND COMPANY, Wilmington, DE
`(US)
`
`(21) APP1~ N0-3
`
`12/637905
`
`(22)
`
`Filed;
`
`Dec. 15, 2009
`
`Related US. 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.
`
`51
`
`(
`
`)
`
`Int. Cl.
`
`Publication Classification
`
`(200601)
`C403 30/04
`(2006.01)
`CIZN 9/02
`(2006.01)
`C0711 21/04
`888288
`gig $32
`‘
`(200601)
`CUP 7”"
`(52) US. Cl. ............ 506/9; 435/189; 536/23.2; 435/243;
`435/25; 435/160
`
`ABSTRACT
`(57)
`Methods for the evolution of NADPH specific ketol-acid
`reductoisomerase enzymes to acquire NADH specificity are
`provided. Specific mutant ketol-acid reductoisomerase
`enzymes isolated from Pseudomonas that have undergone
`co-factor switching to utilize NADH are described.
`
`BUTAMAX 1022
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 1 0f 24
`
`US 2010/0197519 A1
`
`Glucose
`
`D
`
`l
`$
`
`[1
`
`Tim“ WM
`
`U
`
`U
`
`Pyruvote
`
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`flUH
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`
`NADPH + H
`
`"b"
`
`NADP
`
`ketolocid reductoisomerose (KARI)
`
`UH
`
`>6?“
`ocetohydroxy acid
`DH 0
`
` _ . h
`
`2,3-Dlhydroxy Isovolercte
`gellydrotose
`C
`\
`H20
`
`Continued
`’
`on
`Fig. 18
`
`FIG.
`
`1A
`
`
`
`Patent Application Publication
`
`A
`
`2
`
`US 2010/0197519 A1
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`IN..mma:653585+IN..mm+umo+1+:223.._...Su..—
`26:86
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`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 3 0f 24
`
`US 2010/0197519 A1
`
`Sequence ID
`
`17
`
`16
`
`18
`
`(44)
`
`(44)
`
`VGLRKGSATVAKA
`
`VGLRSGSATVAKA
`
`(162)
`
`IGLRKGSNTFAEA
`
`FIG.
`
`2A
`
`Sequence ID
`
`9
`
`10
`
`11
`
`l 7
`
`15
`
`12
`
`13
`
`14
`
`(44) VGLRKNGASWENAK
`
`(44) VGLRKNGASWNNAK
`
`(44) VGLRKNGASWENAK
`
`(4 4 ) VGLRKGSATVAKAE
`
`(44) VGLRKNGASWNKAV
`
`(44)
`
`IGVRKDGASWKAAI
`
`(44) VGLEREGKSWELAK
`
`(44)
`
`IGLRRGGKSWELAT
`
`Consensus
`
`VGLRKNGASWE AK
`
`FIG. 28
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 4 of 24
`
`US 2010/0197519 A1
`
`“aw
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`
`30¢; i
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`FIG. 3
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`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 5 0f 24
`
`US 2010/0197519 A1
`
`0
`@8313
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`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 6 of 24
`
`US 2010/0197519 A1
`
`83$
`
`
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`cozaeamcoo5.0200
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`0
`
`mumuw
`1.23.
`
`mm
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`
`R7//////////%
`m§§
`
`FIG.
`
`5A
`
`8me
`
`
`
`829.3805328
`
`01.23.45.
`nwnwnmnwmu
`
`D25T
`
`Wt
`
`Neg
`
`@
`
`\
`
`FIG.
`
`5B
`
`NADH
`
`NADPH
`
`NADH
`
`NADPH
`
`
`
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 7 0f 24
`
`US 2010/0197519 A1
`
`te V%§
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`E/
`a\\\\
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`.—«'
`
`zo 7
`
`7381
`
`91A9
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`an
`7
`
`7%NADH NNADPH
`
`FIG.6
`
`7/
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`O
`
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`‘9
`
`0a
`<9
`
`W.
`9
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`\\\\\\\\\\\\\\§
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` 7836103904
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`‘7/////////////////////
`24F968F10 /
`
`\\\\\\\
`
`
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 8 0f 24
`
`US 2010/0197519 Al
`
`o
`.00
`
`oc
`
`o
`
`N
`
`A C
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`0 v
`
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`"5 O‘—
`20 ""'""
`0-
`LI.
`
`Ed
`
`.)
`|——
`
`c:
`(\l
`
`cannon-3c:
`gmnm
`
`(Z) KuAnov
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 9 0f 24
`
`US 2010/0197519 A1
`
`Sequence ID
`
`47
`48
`18
`16
`17
`
`47
`48
`18
`16
`17
`
`47
`48
`18
`16
`17
`
`47
`4B
`18
`16
`17
`
`51
`(18) —---KKVAIIG SQGHAH
`
`(17) ———~KTVAVIG ‘SQGHAQ
`(51) FKGIKQIGVIG
`SQAPAQ
`(17)
`-———KKVAIIG
`SQGHAH
`(17) ————KKVAIIG
`SQGHAQ
`
`101
`
`LRD ------ NGFD‘
`
`GLRKGaKSWDKA
`
`GVRPG—KSFEVA
`LRD ****** SGVE
`LKDSLTEAKSDVV‘K GLRKGSNSFAEA
`LKD ------ SGVD T GLRSGSATVAKA
`YLKD ------ SGVD T GLRKGSATVAKA
`
`
`
`————— VYTVAEAAK ADVVMILL DELQPEVYEAEIAPNLQA
`
`----- VMSVSEAVR AQVVQMLL DEQQAHVYKAGVEENLRE
`ISEENGTLGDMWEflI GSDLVLLL SDSAQADNYEKVFSHMK—
`~~~~~ VADVKTA
`DVVMI
`DEFQGRLYKEEIEPNLKK
`————— VTDVAAAVA ADLVMI
`DEFQSQLYKNEIEPNIKK
`
`151
`(102) SLVFAHGFNVHFDQVK---'PENVDVFLVAPKGPGHLVRRTFSEG —————
`
`(101) MLLFSHGFNIHFGQIN-~—_P YVDVAMVAPKSPGHLVRRVFQEG -----
`(150)
`ILGLSHGFLLGHLQSLGQD PINISVIAVCPKGMGPSVRRLYVQGKEVNG
`(102) TLAFAHGFSIHYNQVV7A~'R DLDVIMIAPKAPGHTVRSEFVKG —————
`
`
`(102) TLAFSHGFAIHYNQVV-‘—""DLDVIMIAPKAPGHTVRSEFVKG —————
`
`
`201
`(144) GAVPALFAVYQD TGVATEKALSYAD IGATRAGVLETTFKEE"ETDLFG
`(143) NGVPALVAVHQD TGTALHVALAYAK
`GCTRAGVIETTFQEE”ETDLFG
`(200) AGINSSFAVHQD DGRATDVALGWSI
`G——SPFTFATTLEQEYKSDIFG
`(144) GGIPDLIAIYQD SGNAKNVALSYAC
`GGGRTGIIETTFKDEflETDLFG
`
`(144) GGIPDLIAIYQD SGNAKNVALSY
`GGGRTGIIETTFKDETETDLFG
`
`
`
`
`
`100
`
`150
`
`200
`
`250
`
`FIG. 8
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 10 of 24
`
`US 2010/0197519 A1
`
`............................................................ha
`
`............................................................mv
`
`............................................................vv
`
`............................................................0H
`
`
`
`-I----1|-q..................................................mm
`
`............................................................mm
`
`............................................................Hg
`
`.............................................................mm
`
`............................................................mH
`
`
`
`[n-1----------------ll-llllll---l:---:-----;-i..............ow
`
`............................................................NV
`
`............................................................pm
`
`............................................................wv
`
`............................................................qm
`
`............................................................mm
`
`............................................................mm
`
`............................................................mfl
`
`
`
`-1-1‘|11-1-11-------1|1------------------4..................om
`
`............................................................«H
`
`
`
`....................................---IIIIIII----1l---1----,mm
`
`............................................................Hm
`
`............................................................nv
`
`............................................................wv
`
`wmmq>mmmmammu¢mHBmmqumoqzqmmmxmmoxq¢Mm¢<<memmmmmgmmee<¢emmzmfl
`
`<m.0:
`
`Q_wo:mzumm
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 11 0f 24
`
`US 2010/0197519 A1
`
`12c
`110
`100
`90
`80
`7o
`11111 .M11||
`—————————————————————————————
`FYDKDCDJS ————— I IQG ——————————
`————————————————————————————— KVFYDKDCDJS ————— 1-QG ——————————
`————————————————————————————— KVFYDKDCDJS ————— I QG ——————————
`————————————————————————————— RVFY‘DKDCDJS ————— IIQG ——————————
`———————————————————————————— QVYYDKDADAS ————— IIQG ——————————
`———————————————————————————— QWYDKDCDLS ————— IIQG ——————————
`———————————————————————————— KWYDKDCD35 ————— 1-05 ——————————
`————————————————————————————— NVYYDKDCDLS ————— IVQG ——————————
`—————————— 1. —————————————————— KVE‘YDKDADHS ————— L_KG ——————————
`————————————————————————————— KVYYDKDADJS ————— LIKQ—~j ———————
`————————————————————————————— KVFYDKDCDJS ----- 1 Q6 ——————————
`————————————————————————————— QVYYDKDCDLS ————— IIQG ——————————
`—————————— J —————————————————— KVFYDKDCDAS ————— I_QG ——————————
`—————————— 1 —————————————————— KVYYDSDAD3G ————— L_KS ——————————
`————————————————————————— A-—VSIYYDKDCDIN ————— L_KS ——————————
`————————————————————————————— RVYYDRDADVN ————— L KS ——————————
`————————————————————————— KCTSKIYTDNDAN _.D ~~~~~ LIKG ——————————
`—————————————————————————TD—ATIYYDDDAEST ————— vdDD ——————————
`—————————— J ——————————————A—F-KIYTDREASLE ————— EKG ——————————
`
`
`
`
`
`—————————— 1 ——————————————A——IELLYDADADAS ~~~~~ L_QG ——————————
`———————————————————————— V———KVYYNGDIKEN ————— V-AG ——————————
`—————————————————————————A———KVYYEKDVTVN ————— VJKE ——————————
`————————————————————————————— KTYYEKDANVE ————— L3KG ——————————
`ANGGGSALSAQMVSAPSINTPSATTFDFDSSVFKKEKVTusGHDEYIVRGGRNLEPLLPD
`
`Sequence lD
`
`17
`43
`44
`16
`35
`39
`41
`38
`15
`4o
`42
`37
`46
`34
`36
`33
`13
`30
`14
`
`32
`31
`47
`43
`18
`
`
`
`FIG. 98
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 12 0f 24
`
`US 2010/0197519 A1
`
`SequencelD
`
`17
`43
`44
`l6
`35
`39
`41
`38
`l 5
`4o
`42
`37
`46
`34
`
`35
`
`33
`13
`30
`
`14
`47
`
`32
`31
`
`48
`18
`
`180
`170
`:60
`150
`14C
`130
`
`....‘....1....g....‘....1....4....(‘4.‘,.A..;....:..A.J‘...
`
`LAHGLK—~
`_____ KKVAII
`Q
`KDS —————— GV-
`I‘VGQRKGSATV
`..... KKVAI
`Q
`‘OACN KDS —————— GV—
`‘VGLRKGSATVA
`AHGLK--
`————— KKVAII
`Q
`KDS —————— Gv~
`CVGQRKGSATVA
`EAHGLK—-
`—————KKVAI
`Q
`KDS ——————Gv— pvezRSGSATVAflifiAHGLK——
`_____ KKVAV
`Q
`KES —————— GVQ VVVGLREGSSSAA
`KAGLA--
`_____ KKVAI
`Q
`-;ALN KDS —————— GV—E :VGJRKGSGSWAHAENAGLA*-
`.....KKVAI'F
`Q
`KDS——————GV—3:EVGHRAGSASVAHAEAHGLT--
`77777KKVAII
`Q
`QDS ——————NV— gvcdRADscswxgifiEAsLK——
`_____ KNVTI I
`Q
`1 DS ------ GV—KIVI'VGLRKNGASWN
`AGLQ-—
`
`
`Q
`.
`:qKDs ——————GV—DVEVALRPGSASAKKAENAGLT-—
`_____ RKVAI-
`KDS —————— Gv—Dv“VGQRKGSATVAKA«AHGLK——
`Q
`_____ KKVAI
`Q
`KDS —————— GV~DVVVGQRAGSSSI
`EAYGLK—-
`_____ KKVAI-
`
`
`Q
`CACILKDS —————— GviDvrIGARKGSATVA
`_____ KKVAII
`AHGLK—-
`Q
`RDS —————— GVA J
`..... KKIA:
`:AQRPDSASVK
`DAGFK—-
`
`
`Q
`RDS——————GV—ag IGLKEGGQSWA
`
`
`
`RDS ——————GVK
`VAQRPGSASIKqAEAEGLK——
`Q
`RDS —————— GL—NV fGLEREGKSWELAdSDGIT—-
`
`Q
`DDS —————— GV—ER
`GLREDSSSRsfi
`ADGLD—-
`
`
`Q
`RDS——————GL—EV IGLRRGGKSWEiEESEGFR—-
`
`
`EAGFE—-
`Q
`RDS —————— GV—EV IGLREGSKSAE
`
`
`Q
`KES —————— GV‘DV VGVRQGK—SFT AQEDGHK~~
`
`Q
`HRDN——————GF—av VGLRKGK—SWD§2§EDGFS--
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`KANFI--
`
`
`
`
`
`_____KKVAI'
`
`————— KKVAVI
`————— KRIAV
`————— KTVAV
`
`_____ RKVAI
`————— KTVAV
`
`—————KTIAVI
`_____KKVAII
`
`TDGFE~*
`GVRPGK—SFE
`LRDS —————— Gv— v
`Q
`————— KTVAV-
`AFKGIKQIGVIGWGSQAPAQAQNLKDSLTEAKSDV—VVKIGLRKGSNSFAEARAAGFSEE
`*
`«1‘
`it
`+
`9:
`"k
`*
`
`chxx<G/A)
`
`FIG. 9C
`
`
`
`Patent Application Publication
`
`iAug.5,2010 Sheet130f24
`
`US 2010/0197519 A1
`
`210
`
`220
`
`FQSQLYKNEIEPNIKKGATJAFS
`FQSQLYKNEVEPNLKKGATAAFS
`
`240
`230
`[....[....{
`GFAIHYNQVVPR
`
`HG AIHYNQVVPR
`
`HQAVIYHNQIAPNVKPGAAIAFA
`
`FQGRLYKDEIEPNLKKGATLAF
`FQKELYNDVIEPNIKQGATLAF
`QIADVYKNEVHGNIKQGAALAFA
`FQSRLYRDEIEPNIKQGATLAF
`FQSQLYKNEVEPNLKKGATiAFS
`
`
`
`200
`190
`....\....|....|....
`———VTDVAAAVAGAD4VMI
`
`~——VTDVASAVAAADHVMI
`——~VADVATAVAAADLVM:
`———VADVKTAVAAADVVMI
`———VASIEDAAAQADVVMI
`—-—VKEVAEAVAGADVVMI
`-——VKSVKDAVAAADVVMI
`~—~VAEVEEAVKAADIIMI
`———VKEVAEAVKDADVVMI
`k-“VKSVPEAVAGADLVMI
`
`———VTDVASAVAAAD4VMI
`~4—TSDVA5AVASADVVM4
`~—~VTDVATAVAAADLVMI
`FQGQLYKQEIEPNIKKGAT4AFSHG
`———VLTNAEAAKWADILMI
`HQAAIYAEDLKDNLRPGSAIAFAHG
`{G
`———VKSVKEATKEADLIMI
`IQSEIFNEEIKPELKAGKTJAFA
`———VLTPAEAAAWADVVML
`LQADLYKSELAANLKPGAALVF
`—'—PLHTKDAVKDADIIIFJ
`QRTLWLESVQPYMKKGADJVF
`
`VQPAVYE-QIEDVLQPGDTLQFA,
`———VATPRGAAEQADLVSWJ
`———VYEIGEAVRKADVILVL
`EQPKVWQEQIAPNAKEGVVVDFA
`———VKTTAEAAAWADVIM,
`SQAEIFTNDIEPNiNAGDALLFG
`
`———VFSVKEAAAQAEII
`QQQKVYEAEIKDELTAGKSLVFA
`LQPEVYEAEIAPNqQAGNSJVFA
`———VYTVAEAAKQADVVMIL
`--—VMSVSEAVRTAQVVQPL PD
`QQAHVYKAGVEENAREGQMLLFS
`NGTLGDMWETISGSDLVLLLISDSAQADNYE‘KVPSHMKPNSILGLS
`4e
`&
`
`
`
`FQAQLYKSEIEPNLKSGATJAFEEG
`
`
`
`
`
`SIHYNQIVPR
`SIHYNQVVPR
`
`
`
`AIiYNQVVPR
`NIHFGLIEPR
`NI{YGQIVAP
`
`IHFKLIEAR
`NIHYKLIDPP
`
`NIiYGQIEPS
`NViFGLIKPP
`NIiFDLIKPA
`NVHFHQIVPP
`
`iGFNViFDQVKPP
`G NIiFGQINPP
`
`iGFLLGHLQSLGQ
`
`
`
`
`
`
`L4:
`t“
`
`L L L
`
`“‘L
`
`UCUUUUDUUUU'
`
`H
`L
`LIQWHHHWHH‘HFWHWWWMDUUUUUU
`
`
`u
`
`FF‘L'L'
`F‘L't—‘L—‘L
`
` (“'L1‘}rU'DUDUU
`
`SequencelD
`
`17
`43
`44
`16
`35
`39
`41
`38
`15
`4O
`42
`37
`46
`34
`36
`33
`13
`3O
`14
`32
`31
`47
`48
`18
`
`FIG.
`
`9D
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 14 of 24
`
`US 2010/0197519 A1
`
`SequencelD
`
`17
`43
`44
`16
`35
`39
`41
`3B
`15
`4O
`42
`37
`46
`34
`36
`33
`13
`3O
`14
`32
`31
`47
`48
`18
`
`.\i
`._
`A-~*DL
`A*——DL
`A—v—DL
`A———DL
`A-v—DL
`A7~~DL
`A-——DLEV
`S—i—DLB
`A———DLE
`A———DLD
`A———DL
`K---DL
`A———DL
`K———DI
`K———GIDV
`A———DL
`
`{903%
`
`E———D
`K———N1Uv
`D——-DII
`A———DVDV
`A——«NVEV
`
`S~—-YVE
`
`
`MIAP
`
`MIAP
`MVAP
`MVAP
`
`MVAP
`
`MVAP
`
`MVAP s
`
`
`
`
`
`
`
`j: TV SEFVKG—————GGPDLIggiQ
`9
`- EjvaTEFVKG—————GGIPDLZ
`y
`PGE J SEFVKG—————GG_PDL; 11g
`MIAP
`#6 H SSEYVRG—————GGVPCLdith
`IAP
`MIAP AQGHggfiHEFSIG—————GG”PCL3AIHQ
`A2
`P F
`REYYKAG—————GGVPAL 2&3Q
`AP
`lREEYLAG —————RGVPALMAKYQ
`RRQFVDG ----- KGVPCLIA n
`:E
`JV RTYEQG—————AGVPALEAIY
`LVAP
`4V RTFSEG—————GAVPALSQK Q
`,.
`A
`v RVFQEG—————NGVPAL
`flgg
`DFPKNISVIAVCPKGMGPSVRRLYVQGKEVNGAGINSSFAVHQDVDGRATDVAJGWSIAL
`~x
`*
`
`270
`250
`280
`260
`
`
`.J...\y/I
`
`GHT WSEFVKG—————GG PDLI“IjQ
`-GETV TEFVKG —————GF_PDLL
`Q
`‘G TVSSEFVKG—————GGIPDLI
`IyQ
`
`GETV SEFVKG—————GGIPDLI
`IYQ
`STYVEG 77777 GGVPSL PIHQ
`PG
`
`FGfiflgaSEFVKG—————GG PDL PIEQ
`SEFVRG ————— GG PDLIA YQ
`HG
`
`flSEFAKG ————— GGIPDLLAIYQ
`EGH
`PGHTVRGTYAQG ————— GGVPHLI
`HQ
`
`
`
`
`
`
`PG
`
`GH
`
`I TEFTKG ————— GG_PDL_AIFQ
`
`
`
`RGEYLKG ————— GGVPCL
`
`
`
`
`
`
`
`
`
`
`
`Q
`
`
`
`
`
`JVRRNYEND ————— EGTPGLLA YQ
`
`FIG. 9E
`
`
`
`AtnetaP
`
`0..
`
`US 2010/0197519 A1
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`“a.0:
`
`.mUflm>ntmm0§>qm>e0<0MHmmvEH.HHOHmU0PHm7,W7i__.h2%0mmomm9m
`
`
`
`
`
`vErHH93000vwmnmdjmv5-8HHOHmO0NVAwdm>nrom0§>qm><w«EduwmmeWMHmwmm>1hmmw§>qm>euw><O15-thvE[Hwemwwow5ummeflfiwfiimiuw><SEEEa-HoemouRWeUdm>nHmEU§>Am>Ew>d.ggmhmnmL-U“HoemwwmmmUmenvmmwmqu<quwLiamQWMNEmmEw>ommowvm2.,HHUmmo0mm0UWm>QDmmU<OqumuuoEm.5059oE.MomQ>numwwr§>qu>U«amQfimnmmmD£9290ommwmm>nUMb¢<mzqm2HUH>bQTMHMvErrL56$;0maSomm>nemmowOHQMmquo>>manQumumwarm:Ami/«mag“MDSofia/mmwmmHmzmfiamofiflmzqw5m2953Eng«anym‘Q[m_-mMPZLA>w<m<0am0wdmei>mww>x>qmmrwd.m:d>w<meonvmUmmrw1ummw§>qmmqu‘om91Hmm0§>q<H>0BmmFmHflOmar.4/.ngomv4odo>qemww§>q<H>uUdmQHmHmvamp
`
`
`”HOB/n50maumUmm>qtmmw§>qm>mUmmMQHJHmvainHoemo0mm.wozu>§fo<oflmfio30nomew”EmuH.5580mmMUmm>JHMbw§>qm>u05mL1omwmvinb
`
`
`
`
`
`
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`
`
`Dm.Uam>1tmmw§>qm>90an“DrugmmMagi“HUB/moo3nDrm>nhmfiw§>qm>90&mUEADHMBJLvEmHHUHmoumv
`mQAnQEJHAx:“Hommw6mmmUmm>qemmw§>gm>o0dmenQHMHwv:EHHUHmU0av
`
`Pn:85:36
` Hum>ezmwfiqommzommewmmmqom>Hom>¢oquwmmwmHammmmmumqtrfinmhuuhmmwma
`ommiibfiflmfiwiwombnmEB-6430o2Odmenummw§>qm>d~0Lga
`
`
`
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`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 16 0f 24
`
`US 2010/0197519 A1
`
`SequencelD
`
`l7
`43
`44
`16
`
`41
`
`35
`39
`38
`
`15
`40
`42
`37
`
`46
`36
`13
`14
`
`34
`
`33
`
`30
`
`32
`31
`47
`48
`18
`
`420
`410
`400
`390
`370
`380
`
`1\\I‘r‘\|>\|
`K-LIVDLMYEEG ANMNYSISNNAEYGEYVTGPEVINAESRQAMRNALKRIQEGEYAKMF
`
`K~LIVDLMYE G ANMNYSISNNAEYGEYVTGPEVINAESRQAMRNALKR.Q G YAKMF
`K—LIVDZMYEGG ANMNYS;SNNAEYGEYVTGPEVINEESRKAMRNALKR'QDGEYAKMF
`K~LIVD4MYEGG ANMNYS SNNAEYGEYVTGPEVINAESRAAMRNALKR Q GEYAMMF
`K-LIVDALYQ G ANMRYSESNTAEYGDFTRGPRV-NEESREAMREILAE_QgGEFAREF
`
`K-LIVD1MYE§G ANMNYS SNNAEYGEYVTGPEVINDQSRAAMRNALKR Q G YAKMF
`K—LIVDAMYEGG ADMNYS SNNAEYGEYVTGPEV;NEQSREAMRNALKRIQgG YAKMF
`
`
`
`
`
`
`
`
`
`
`
`K—LIVDLMYEGG ANMN S-SNNAEYGEYVTGPEVINEESRKAMRNALKR-Q§GEYAKMF
`K—LIVDLIYQ G ADMRYSVSNTAEYGDYITGPKI TKETKEAMKGVHKDIQNGEFAKDF
`K—MLVDLVYE G SGMLNA SDTAKYGGMTVGKFV DESVRKRMKEALQR.K36KFAEEW
`K—LIMDLIWQiG YGMLBGVSDTAKYGGLTVGPRV'DENVKRKMKEAAMRVKSGEFAKEW
`
`
`
`
`
`
`
`
`
`K—LIVDLMFE G ANMNYSISNNAEYGEYVTGPEV NEQSRQAMRNALKR'Q G YAKMF
`
`K—LIVDLIYEGG GNMNYSJSNNAEYGEYVTGPRVVTAETKQAMKQCLHD;Q G YAKSF
`K—LIVDLMYEGG ANMNYS'SNNAEYGEYVTGVKVTNEQSRAAMKECLAN-QNGAYAKRF
`KGLIVDlMYEGG ANMNYS’SNNAEYGEYVTGPEV‘NAESRQAMRNALKRIQCGEYAKMF
`K—LIVDJMYEGG ANMNYS SNNAEYGEYVTGPEV-NEQSREAMRNALKR_Q GEYAKMF
`
`K~LIVDLIYEPG ANMRYSESNTAEYGDIVSGPRVINEESKKAMKAILDDIQ G FVSKF
`
`K-LIVD-IYE G ANMRYSISNTAEYGDYVTGSRI TEATKAEMKRVZAD_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 SDTAQWGDFVSGPRVVTEDTKKAMGTVJAEIQ GTFARGW
`K—LIVDLMYE G TNMR S SDTAEFGDYVTGSRIVTDETKKEMKRVGTEIQ G FAKKW
`TGVISKTISTKGMLALYNSLSEEGKK-DFQAAYSASYYPSMDILYECYEDVASGSEIRSV
`
`FIG. 90
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 17 0f 24
`
`US 2010/0197519 A1
`
`480
`470
`460
`450
`440
`430
`....‘
`..A.|.4..|‘...:4...\....1....1i.._a‘...
`._..\....\....
`ISEGATGYP——3MTAKRRNNAA4615-I G QflibMMPWIGANKIVDKAKN —————————
`ITEGATGYP——SMTAKRRNNAE7GIE-VIG K-RSMMPWIAANKIVDKDKN —————————
`
`SequencelD
`
`17
`43
`44
`16
`35
`39
`41
`38
`15
`40
`42
`37
`46
`34
`36
`33
`13
`3O
`14
`32
`31
`47
`48
`18
`
`
`
`ISEGATNYP—-SMTAKRRNNAAHGIE-I G jSfififiPW_SANKIVDKTKN —————————
`ITEGAANYP~#SMTAYRRNNAAHPIE-Q G KL
`PWLAANKIVDKSKN ---------
`VLENQAGCP——TLTARRRLAAE{EIEvVVG R_RGMMPWINANKLVDKDKN —————————
`IAEGAHNYP—-5MTAYRRNNAA4PIE—Q G KURSMMPWIASNKIVDKSKN —————————
`ITEGAANYP-—SMTAYRRNNAAiQIEvVVGEK-RTMMPWLAANKIVDKTKN —————————
`1 SEGATNYP—»SMTARRRNNAE {Q1 E— I TGIAK‘RGMMPWIGGNKI I DKDKN —————————
`LLENKAGAP—-TLISRRRLTADHQIE-QVGRK-RAMMPW-AKNKLVDQSKN ~~~~~~ f“-
`:LEGQANYP~-EMTAWRRNNAAHQIE—VVGRKHRSMMPWIAANKLVDHSKN ---------
`ISEGATGYP—-SM”AKRRNNAA{GIE-IIGEKiRfiMMPWIAANKIVDKDKN —————————
`
`ISEGALNYP-—SM"ARRRQNAA-[EIE—TWGTK_RSMMPW SANKIVDKDKN —————————
`ISEGATNYp——SMTAKRRNNAA{GIE—IIG QZRSMMPWISANKIVDKTKN —————————
`VLDNRAGQP—-ELKAARKRMAA-[PIE—Q G R-R<MMPWIASNKLVDKARN —————————
`ILERRANFA—iRMHAERKLWDSLIE—K G E_RAMMPWISAKKLVDKDKN —————————
`MLECKAGQP?—SFKATRRIQXEHVIE-V‘G KLRGMMPWISKNKLVDKARN —————————
`
`
`
`
`
`VEEYGRGMP—ATVVNGLSNVQNSLEE~KDG QfiEELVQK———————GKPKS—————————
`
`IANVENGNT~-ELEGLRASYNNHPIEgETG KJRnimswvxvnARAETA———————————
`IVENQVNRP—-RFNAINASENEHQIEiij K_R:MMPFVKQGKKKEAVVSVAQN~~~~~
`IAEHKAGRP-—NFHATNEKENEHEIE-V G KHR MMPFV-QPRVKVGMK ——————————
`ILENQAGRP“Ta/NAMKKAEQNHQLE—K G ELREMMSWIDAPKELVKK ———————————
`VLAGRRFYEKEGLPAFPMGKIDQTRMWKVGEKVESVRP-—AGD—-LGPLYPFTAGVYVAL
`
`
`
`
`
`
`
`
`
`
`JFAW ——————— GDD ———————————
`ISENQAGRPv—SYKQLRAAEKNHDIEiAVG DL
`VEEYNRGAP~-TLRKLMEEARTHPIEinG a RKJLF___GP _________________
`
`FIG.
`
`9H
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 18 of 24
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`US 2010/0197519 A1
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`............................................................PM
`
`............................................................mq
`
`............................................................WV
`
`............................................................ofi
`
`...........................................................mm
`
`
`
`1--111-|1-1--------1........................................mm
`
`............................................................fiv
`
`............................................................mm
`
`
`
`.........................4,1--(11--‘1-I,-----I----|-I-------ma
`
`............................................................ow
`
`............................................................Nv
`
`...........................................................rm
`
`............................................................ow
`
`............................................................vm
`
`............................................................mm
`
`............................................................mm
`
`............................................................ma
`
`.............................................................om
`
`............................................................VH
`
`
`
`lllllllllllllllll.lIIllllllll'llllllllIlllullvllllilllll|llllllmm
`
`............................................................Hm
`
`............................................................bv
`
`............................................................mw
`
`mmmzmmqummgymuzo>zhm>wm<mzhm24mo><mH>mmzHHmmwmmummqumHomzzma
`
`5.0:
`
`Q_wocw:cmm
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 19 of 24
`
`US 2010/0197519 A1
`
`
`
`IIIIIIIIIIIIII...........IIIIIIIIIaw
`
`IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIImm
`
`
`
`IIlIIL.llllllllllllllllllllllllllllllllllllllllllllllllllllllmm
`
`............................................................mm
`
`
`
`llllllllllllllllllllllllllllllllllllllllllllllllllllllllllll3......IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIma
`
`............................................................Nw
`
`............................................................pm
`
`............................................................wv
`
`............................................................vm
`
`............................................................mm
`
`............................................................mm
`
`............................................................ma
`
`............................................................om
`
`
`
`
`
`...............IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIVa
`
`
`
`IIIIIIIIIIIIIIII...................IIIIIIIII.mm
`
`
`
`............................................................Hm
`
`............................................................NV
`
`IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII0H
`
`
`
`
`
`
`
`IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIpa
`
`IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIImv
`
`IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII«v
`
`Q_wo:m:cmw
`
`
`
`
`
`mmmED¢Q¢E>mHO>mmmHO¢U>DH<MI>mDmJMZmHAQOZHmflUZQ><>J<OOm1HHMO;mH............................................................wv
`
`
`
`
`
`am.2...
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 20 of 24
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`US 2010/0197519 A1
`
`PH
`
`MW
`
`3“
`
`ma
`
`mm
`
`mm
`
`:V
`
`mm
`
`ma
`
`0v
`
`NV
`
`NM
`
`ow
`
`qm
`
`@m
`
`mm
`
`ma
`
`om
`
`«a
`
`mm
`
`Hm
`
`CV
`
`mq
`
`ma
`
`n:85:35
`
`gm.0:
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 21 0f 24
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`US 2010/0197519 A1
`
`*->quafskVYYDkDadlsGhdeylikGKkVAvI
`
`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 thvaEAvanDvai lPDeane
` kAea+e+ V +va Ava+AD+Vmi +PDefQ++
`
`-EAHEL VTDVAAAVAGADLVMI TPDEFQSQ 87
`
`ereeIeankpGatLaFAHGFNIHf-anvPrakaDiDViMVAPKgPGH
`
`+Y++eIepn+k+GatLaF+HGF+IH+ ulvPra
`
`D+DViM+APK+PGH
`
`Sequence [D 17
`
`88 LYKNEIEPNIKKGATLAFSHGFAIHY
`
`PRA-—-DLDVIMIAPKAPGH 134
`
`tVRreYVngGVPaLiAVyQ
`
`tVR+e+vagG+P+LiA+yQ
`
`Sequence [D 17
`
`135 TVRSEFVKGGGIPDLIAIYQ
`
`GnAk++ALsYA
`
`GnAkdlALsYA‘ {GggRAGVIETTFk
`
`GNAKNVALSY--
`GGGRTGIIETTFK 184
`
`
`GggR+G+IETTFk
`
`eETETDLFGEQaVLCGGVteLVkaGFETLVEaGYaPEmAYFECLHElKLI
`
`+ETETDLFGEQaVLCGG++eLVkaGFETLVEaGYaPEmAYFECLHElKLI
`
`Sequence ID 17
`
`185 DETETDLFGEQAVLCGGTVELVKAGFETLVEAGYAPEMAYFECLHELKLI 234
`
`FIG.
`
`10A
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 22 0f 24
`
`US 2010/0197519 A1
`
`VDLmYEgGIanMrySiSdTAeYGdyvtGprVIdeeskeaMkevadIQSG
`
`VDLmYEgGIanM+ySiS++AeYG+ythp+VI++es++aM+++Lk+IQ+G
`
`Sequence ID 17
`
`235 VDLMYEGGIANMNYSISNNAEYGEYVTGPEVINAESRQAMRNALKRIQDG 234
`
`eFAkewilEnanyPket1talrrneaquIEWkVGekLRsmmpWIaanK
`
`e+Ak++i+E+++GyP ++ta rrn+a+H IE +Ge+LRsmmpWI anK
`
`SequenCe ID 1?
`
`285 EYAKMFISEGATGYP—-SMTAKRRNNAAHGIE—IIGEQLRSMMPWIGANK 331
`
`lvdkdkn<-*
`
`+vdk+kn
`
`Sequence ID 17
`
`332 IVDKAKN
`
`333
`
`FIG.
`
`1OB
`
`
`
`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)
`
`
`
`mammmm
`
`
`mn-nnnn
`
`
`"nu-Inn.-
`
`
`"Hanan-l-
`
`
`"mun-ill
`
`
`
`the cofaotor specificity domain
`
`the cofactor binding affinity domain
`
`FIG.
`
`11
`
`
`
`Patent Application Publication
`
`Aug. 5, 2010 Sheet 24 of 24
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`US 2010/0197519 A1
`
`IIZI?
`
`
`
`
`
`
`
`EmEon36:6me55389:EmEoubEEm96:5BEESm5
`
`Exno>2a§2:9:5:33:33:
`
`
`
`
`EmEoumEEEEva36:6me
`
`
`
`56,9809:EmcozmSE5:5EmEonBEEm96:5.20389:E20:95:.35250
`
`IE.m2iiEliml
`
`
`
`gillall!
`
`
`
`
`
`lililBy,EHIIHIM-IEEHIIIII!EHallIII!EE!!!
`
`:25Ex
`
`
`
`
`
`
`
`
`
`
`
`
`US 2010/0197519 A1
`
`Aug. 5, 2010
`
`KETOL-ACID REDUCTOISOMERASE USING
`NADH
`
`[0001] This application is a continuation-in-part of US.
`Ser. No. 12/337,736, filed Dec. 18, 2008 and claims the ben-
`efit of the US. 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 efficiency 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;
`Nishiyama, 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 c