(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2009/0163376 A1
`Li et al.
`(43) Pub. Date: Jun. 25, 2009
`
`
`US 20090163376A1
`
`(54) KETOL-ACID REDUCTOISOMERASE USING
`NADH
`
`Related US. Application Data
`
`(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.
`(2006.01)
`C40B 30/04
`(2006.01)
`C12N 9/90
`(2006.01)
`C12N 15/11
`(2006.01)
`C12N 5/06
`(2006.01)
`0121) 7/16
`(52) use. ............ 506/9; 435/233; 536/232; 435/325;
`435/160
`
`(57)
`
`ABSTRACT
`
`Methods for the evolution of NADPH binding ketol-acid
`reductoisomerase enzymes to acquire NADH binding func-
`tionality are provided. Specific mutant ketol-acid reductoi-
`somerase enzymes isolated from Pseudomonas that have
`undergone co-factor switching to bind NADH are described.
`
`(75)
`
`Inventors:
`
`Yougen Li, Lawrenceville, NJ (US);
`Der-Ing Liao, Wilmington, DE
`(US); Mark 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 25/1122B, 4417 LAN-
`CASTER PIKE
`
`WILMINGTON, DE 19805 (US)
`
`(73) Assignee:
`
`E.I. DU PONT DE NEMOURS
`AND COMPANY
`
`(21) Appl. No.:
`
`12/337,736
`
`(22)
`
`Filed:
`
`Dec. 18, 2008
`
`Glucose
`
`O
`l
`0
`$0.. ya...
`
`0
`
`Pyruvate O
`
`C 02 meta/are syn those
`
`OH
`
`5&1
`
`0
`0
`S-Acetolactate
`II
`VI
`
`NADPH + H+
`NADP’
`
`b
`szo/aadreducfaisamerase {KARI}
`
`OH 0
`2 3-Dihydroxy isovalerale
`
`'dd h d I 5
`1 h a’
`ace 0 y raxy an
`E y M a e
`"c"
`
`0
`-Ketoisovalerale
`:1
`’
`II
`I
`2 e , 2 H
`
`1
`
`H20
`
`f‘
`HS—CoA
`branched chain ke faac/d
`dehydragenase
`
`Hkll
`10
`
`I /' b 1‘ /6' A
`MW)“ 152,35!” 0
`butyryl CoA
`
`o
`isobulyryl CoA
`
`Isobutanol
`
`BUTAMAX 1015
`
`°
`
`valine
`
`N
`
`“2
`
`va/ine
`decarbaXy/asz
`"ln
`lsubutylamlne
`0T .
`.
`N":
`4
`”,0
`omega
`rmnsammas'e
`
`NH _
`_3'
`2 e . 2 H
`
`’
`
`D00
`val/'ne
`transalmnase
`"h" dehydrayenase or
`
`NHa;
`'
`2 e‘ 2 H‘
`
`0
`
`,
`
`H
`
`0
`
`OH branched draw [(2 to acid
`
`decarbaxy/ase
`
`)
`"d"
`Isobutyraldehyde
`
`C02
`
`+
`
`branchedcham
`0/60/10/
`dehydrogenase
`
`" NADH + H
`
`"
`
`2 e', 2 H”
`
`9
`
`HS-CoA
`aldehyde ehydragenase
`
`an acy/ar/Z?
`S-CuA
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 1 0f 15
`
`US 2009/0163376 A1
`
`Figure 1
`
`Glucose
`I
`
`o
`\[KlLOH
`
`'
`+
`
`' o
`WHkOH
`
`0
`
`Pyruvate O
`
`Ilall
`
`(302%acera/afesynrhase
`
`OH
`OH
`
`O
`O
`S-Acetolactate
`
`°
`
`va/ine
`decarbaxy/as‘e
`
`,
`
`4.
`NADPH H
`NADP+
`
`'lb"
`
`.
`
`k2 fol ac/dreducia/s‘amerase (KARI)
`
`_
`
`valine
`
`NH
`
`1
`
`Hill
`°H i.
`cc:
`
`isobuiylamine
`
`NH?
`4
`”w
`omega
`rransammas'e
`
`NH .
`3‘
`2 e . 2 H
`
`‘
`
`0
`
`H
`
`NHa;
`2 6.. 2 H‘
`
`A we
`valine
`transammas‘z
`"h" dzhydragenase or
`
`.
`
`O
`
`OH branched aha/n ks to acid
`
`deca’baWb“
`
`"d"
`Isobutyraldehyde
`
`
`0
`a-Ketoisovaleraie
`
`OH
`
`OH
`
`‘
`OH O
`2.3-Dihydroxyisovalera1e
`
`'dd h drafase
`f )1 o’r x
`ace 0 y a y ac,
`e y
`"C“
`<
`H20
`
`)
`002
`
`2 e' 2 H’
`'
`" NADH + H
`9
`'
`NAD ‘
`HS—C A
`
`0
`an acy/afin
`aldehyde aihydrogenase
`
`2 e’. 2 H‘
`
`"f"
`HS—CoA
`branched chain ke faac/d
`dehydrogenase
`
`
`
`.0
`u kl l
`/\/'k /s-cm
`Isabur
`/CoA
`mufas'fry
`buiyryl CoA
`
`S-CDA
`
`o
`isobutyryl CoA
`
`+
`
`"e
`branchedchain
`alcohol
`dehydragenasz
`
`OH
`
`Y
`
`Isobutanol
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 2 0f 15
`
`US 2009/0163376 A1
`
`Figure 2’
`
`a P
`
`FS-KARI
`
`(44) VGLRKGSATVAKA
`
`PAC-KARI
`
`(44) VGLRSGSATVAKA
`
`Spinach_KARI
`
`(162)1GLRKGSNSFAEA
`
`b
`
`MMC5
`MMSZ
`MVSB
`
`59
`72
`(44).VGLRKNGASWENAK
`(44) VGLRKNGASWNNAK
`(44) VGLRKN ASWENAK
`
`PFS—ilVC
`
`(44) VGLRKGSATVAKAE
`
`KARI—81
`
`(44) VGLRKNGASWNKAV
`
`IGVRKDQAsuKAAI
`ile (59)
`KARI—D1
`(48) VGLEREGKS%ELAK
`KARI—D2
`(45)
`IGLRRGGKsiELAT
`Consensus
`(59) VGLRKN AS‘E AK
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 3 0f 15
`
`US 2009/0163376 A1
`
`Figure 3
`
`
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 4 0f 15
`
`US 2009/0163376 A1
`
`’ Figure 4
`
`63A10
`N5
`
`C3B11
`
`csca
`
`
`
`iii:llIllllIIInun
`
`Wfifi‘fimQfi'PaWWWRWA
`
`Wt
`C4012
`III.
`a .
`mu
`....
`55““ mun
`r-u
`4» u
`anIIII
`mnflmgfifi
`III-
`
`[0
`
`Negative
`
`
`
`
`
`‘95..
`
`iiR
`
`E NADHw/ substrate
`NADPH w/ substrate
`
`E] NADH w/o substrate
`NADPH w/o substrate
`
`0.00
`
`-0‘.05
`
`-0.10
`
`-0.15
`
`-0.20
`
`—0.25
`
`-0.30
`
`—0.35
`
`—0.40
`
`—0.45
`
`-0.50
`
`
`
`
`
`Cofactorconsumption(A340)
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 5 0f 15
`
`US 2009/0163376 A1
`
`Figure 5
`
`R47F’
`
`$500
`
`
`
`v
`0°
`3:
`0-.
`#9
`00
`o.
`z:
`o.
`O9
`06
`M
`”
`>’ 9
`*
`
`
`
`..
`§
`4 -0.1
`‘g
`o
`a
`s
`g 02
`U
`L
`2
`3 43.3
`'8
`o
`
`-0.4
`
`T520
`A
`3
`g 43.1
`c
`c,
`:7
`a. 0.2
`s
`2
`0 —0.3
`u
`‘6
`‘6
`w —0.4
`‘3
`
`
`
`m NADH m NADPH
`
`
`‘5 NADH
`E ”ADP“
`
`-0.5
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 6 0f 15
`
`US 2009/0163376 A1
`
`Figure 6
`
`24F9
`
`68F10
`
`83G10
`
`39G4
`
`91A9
`
`C3B11
`
`Wt
`
`Negative
`
`
`
`
`-O.1 —
`
`Q0‘0‘OOQOOQOOQ.§OO§QQQQOQOOOO
` ‘v.
`
`"OOOQOQOéOOOQOWOO‘OQ6%.'OOOQOOOOO§000§O§QOO
`
`
`
`vQ‘OQ‘OQOOOOQOQfiififi-OO
`
`
`
`
`
`
`Cofactorconsumption(A340) -o.4
`
`'0 -3 ‘
`
`
`
`4‘
`
`::::::
`
`
`
`E NADH [21 NADPH
`
`'
`0.5
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 7 0f 15
`
`US 2009/0163376 A1
`
`Figure 7
`
`
`
`Activity(%)
`
`m\l6‘O01O
`
`N001
`
`20
`
`40
`
`' 60
`
`8O
`
`Tem perature °C
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 8 of 15
`
`US 2009/0163376 A1
`
`Figure 8
`
`51
`100
`----KKVAIIGYGSQGHAHAQNLRD------NGFDVMVGLRKG-KSWDKA
`----KTVAVIGYGSQGHAQAQNLRD------ SGVEVMVGVRPG- KSFEVA
`FKGIKQIGVIGWGSQAPAQAQNLKDSLTEAKSDVVVKIGLRKGSNSFAEA
`--——KKVAIIGYGSQGHAHACNLKD ------ SGVDVTVGLRSGSATVAKA
`----KKVAIIGYGSQGHAQA§NLKD —————— SGVDVEVGLRKGSATVAKA
`
`101
`150
`KEDGES -----VYTVAEAAKQADVVMILLPDELQPEVYEAEIAPNLQAGN
`KTDGfiE-----VMSVSEAVRTAQVVQMLfiPDEQQAHVYKAGVEENLREGQ
`RAAGFSEENGTLGDMWETISGSDLVLLLLSDSAQADNYEKVFSHMK PNS
`EAHGLK-----VADVKTAVflAADVVMILLPDEFQGRLYKEEIEPNLKKGA
`EAHGfiK-----VTDVAAAVAGADLVMILQPDEFQSQLYKNEIEPNIKKGfl
`
`151
`
`200
`
`SLVFAHGFNVHFDQVK-—-PPANVDVFLVAPKGPGHLVRRTFSEG -----
`MLLFSHGFNIHFGQIN---PRSYVDVAMVAPKSPGHLVRRVFQEG-----
`ILGLSHGFLLGHLQSLGQDFPKNISVIAVCPKGMGPSVRRLYVQGKEVNG
`TLAFAHGFSIHYNQVV---PRADLDVIMIAPKAPGHTVRSEFVKG -----
`PEI
`TLAFSHGFAIHYNQVV---PRADLDVIMIAPKAPGHTVRSEFVKG -----
`
`250
`201
`GAVPALFAVYQDATGVATEKALSYADGIGATRAGVLETTFKEETETDLFG
`NGVPALVAVHQDATGTALHVALAYAKGVGCTRAGVIETTFQEETETDLFG
`AGINSSFAVHQDVDGRATDVALGWSIALG--SPFTFATTLEQEYKSDIFG
`GGIPDLIAIYQDASGNAKNVALSYACGVGGGRTGIIETTFKDETETDLFG
`GGIPDLIAIYQDASGNAKNVALSYAAGVGGGRTGIIETTFKDETETDLFG
`
`) ) ) ) )
`
`) ) ) ) )
`
`(18
`(17
`(51
`(l7
`(17
`
`(57
`(56
`(101
`(57
`(57
`
`ival B cereus
`iva2 B cereus
`
`Spinach KARI
`PAO KARI
`PFS KARI
`
`ival B cereus
`iva2 B cereus
`
`Spinach KARI
`PAO KARI
`PF5 KARI
`
`ival B cereus
`ivaZ B cereus
`
`Spinach KARI
`PAO KARI
`PF5 KARI
`
`ival B cereus
`iva2 B cereus
`
`Spinach KARI
`PAO KARI
`PF5 KARI
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 9 0f 15
`
`US 2009/0163376 A1
`
`gil70732562|ref|YP_262325 1|/1 ------------------------------------------------------------
`gil28868203|ref|NP_790822 1|/1 ------------------------------------------------------------
`gil26991362|ref|NP_746787.1|/1 ------------------------------------------------------------
`gil15599888lreleP_253382.1]/1 ------------------------------------------------------------
`gil114319705|xef|YP_741388.1|/
`------------------------------------------------------------
`gil83648555|ref|YE_436990.1|/1 ------------------------------------------------------------
`gil67159493Iref|ZP_00420011.1|
`------------------------------------------------------------
`gil71065099|ref|YP_263826.1|/1 ------------------------------------------------------------
`giI17546794lzeleP_520196.1|/1 ------------------------------------------------------------
`gil74318007|xef|YP_315747.1|/1 ------------------------------------------------------------
`gil66044103|re£|YP_233944.1|/1 ------------------------------------------------------------
`gil120553816]:e£|YP_958167.ll/
`------------------------------------------------------------
`gil146306044|xefIYP_001186509.
`—————————————————————————————————————— ----------------------
`gil56552037lrefIYP_162876.1|/1
`————————————————————————————————————————————————————————————
`gi|57240359lzef|ZP_00368308.1|
`------------------------------------------------------------
`gi|6225553|sp|032414IIch_aaon ------------------------------------------------------------
`gil15897495|releP_342100.11/1 ------------------------------------------------------------
`gil76801743|ref|YP_326751.11/1 —-—----—-——-—————-—; ————————————————————————————————————————
`gil18313972|xef|NP_560639.11/1
`------------------------------------------------------------
`gil19552493|ref|NP_600495.1|/1
`------------------------------------------------------------
`giI16079881|releP_390707.1|/1 ------------------------------------------------------------
`gil42780593|releP_977840.1|/1 ------------------------------------------------------------
`gil42781005|xef|NP_978252.1l/1
`------------------------------------------------------------
`91126634“ SpIQ01292 IILV5_SPIOL MAATMTTFSLSSSSSTSAMSKALKQSPKPSALNLGFLGSSSTIKACRSLKMRV‘LPSG
`80
`90
`100
`110
`120
`.l....l....I....|....I....I....I....I....I....I
`
`
`
`-~errnxncnns ————— rigs----------
`E-------
`gi|70732562IrefIYP_262325.ll/1
`I‘— —————————————————KVFYDKDCDLS _____ 1.19:; __________
`91 I28863203 I ref INP_790822 . 1| /1
`-----------I‘l——————————————————KVFYDKDCDLS _____ IIQG__________
`gi I25991362 I ref INP__746787 . 1| /1
`giI15599888|releP_253382.1|/1 -----------I‘v------------------RVFYDKDCDLS—————IifQG..........
`giI114319705IrefIYP_741388.1|/
`———————————I‘v- -----------------QWYDKDADLS _____ IIQG__________
`gi183643555lrefIYP__436990.1I ll
`-----------I‘— -----------------QV‘IYDKDCDLS _____ IIQG __________
`giI67159493IrefIZP_00420011. 1|
`———————————I‘I------------------KWYDKDCDLS_____IfQS __________
`gil71065099IrefIYP_263826.1| /1
`-----------I‘v- —————————————————NVYYDKDCDLS ————— IVQG__________
`9iI17546794lrefINP_520196.1|/1
`-----------I‘I——————————————————KVFYDKDADLS—————121:1(3__________
`gil74318007|refIYP_315747.1|/1
`-----------I‘r— —————————————————KVYYDKDADLS_____L’I‘KQ__________
`gil66044103|re£IYP_233944.1I/1
`-----------I‘l— -----------------KVFYDKDCDLS ————— 1.599__________
`gi l 120553816| ref I YP_958167 .1 | /
`-----------1‘1——————————————————QW‘IDKDCDLS..... 1:ng__________
`gill46306044lreleP_001186509.
`———————————til——————————————————KVFYDKDCDLS ————— IEIQG__________
`gil56552037IreleP_162876.1|/1 ———————————I‘-------------------KVYYDSDADLG————— 1%}(5 __________
`gil57240359lref|ZP_00368308 1|
`———————————~‘I——————————————A--VSIYYDKDCDIN—————1.sz __________
`gi 1 6225553 I sp l032414 IILVC_RHOM ———————————\‘I——————————————————RVYYDRDADVN—————1.1:}(3__________
`gi115897495lreleP__342100. 1I/1
`-----------1‘.——————————————KCTSKIYTDNDANLD—————1.1ny..........
`gi176801743lrefIYP_326751 ll/l
`-----------1‘.——————————————TD—ATIYYDDDAEST—————vying__________
`gi|18313972lreflNP_560639.1|/1
`———————————w——————————————A——-KIYTDREASLE..... page__________
`gi I19552493 I ref INP_6004 95. 1 | /1
`———————————I‘v——————————————A--IELLYDADADLS ————— LEQG__________
`91 | 16079881 I ref INP_390707 . 1 | /1
`———————————I‘I- —————————————V---KVYYNGDIKEN—————mg__________
`gi I42780593Ire£INP_977840. 1|/1 ———————————1‘:--------------A-—-KVYYEKDVTVN—————VLKE __________
`gi |42781005lrefINP_978252.1I/1
`-----------Iv——————————————————K'ryyamm-----LLKG__________
`gi I266346 I Sp I QO 12 92 I ILV5__SPIOL ANGGGSALSAQ; SAPSINTPSATTFDFDSSVFKKEKVTLSGHDEYIVRGGRNLFPLLPD
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 10 of 15
`
`US 2009/0163376 A1
`
`giI70732562|ref|YP_262325.1l/1
`9iI28863203|zef|NP_790822.1l/1
`gi|26991362|ref|NP_746787.1|/1
`gil15599888|zef|NP_253382.1|/1
`gi|114319705|tef|YP_741388.1I/
`gil83648555|ref|YP_436990.1I/1
`gil67159493|ref|ZP_00420011.1|
`gi|71065099|releP 263826.1I/1
`gil17546794|ref|NP:520196.1l/l
`gil74318007|re£IYP_315747.11/1
`gil66044103lref|YP_233944.1I/l
`gil120553816|zef|YP_958167.1I/
`gil1463060441releP_001186509.
`gil56552037|zef|YP_162876.1|/1
`gil57240359|ref|ZP_00368308.1l
`gil6225553lspl032414IILVC_REOM
`gil15897495|zeleP_342100. 1|/1
`gil76801743|ref|YP_326751 1|/1
`gil18313972|re£|NP_560639.11/1
`gil19552493IrefINP_600495.1I/1
`giI16079881|ref|NP_390707.11/1
`gi|42780593Izef|NP_977840.1l/1
`giI42781005Iref|NP_978252.11/1
`gi|266346|splQOlZ92|ILV5_SPIOL
`
`M
`
`_a:i:
`'
`GLRAGSSSI-
`IGERKGSAxV *
`
`GXGXX(G/A)
`
`giI70732562IreleP_262325.1|/1
`gi|28868203|releP_790822.ll/l
`giI26991362|releP_746787.1|/1
`gil15599388I:e£lNP_253382.1l/1
`gi|114319705|re£|YP_741388.13/
`giI83648555|ref|YP_436990.1I/l
`gil67159493|ref|ZP_00420011.1|
`giI71065099|re£|YP4263826.11/1
`gi|17546794|ref|NP_520196.1l/1
`gil74318007|ze£|YP_315747.ll/l
`gil66044103|releP_233944.1l/1
`gi|120553816lre£|YP 958167.1l/
`gil146306044|releP:001186509.
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`gil15897495lref|NP_342100.1I/1
`gil76801743lrefIYP‘326751.1I/1
`gi|18313972|ref|NP_560639.1|/1
`gi|19552493|reflNP_600495.1|/1
`gil16079881|releP_390707.1|/1
`gil42780593IreleP_977840.1I/1
`gil42781005|ref|NP_978252.1|/1
`gi|266346lspl901292|ILV5_SPIOL
`
`240
`200
`190
`....1....I....i....I....I....l....|....I....l....l....l
`DVAAAVAG5DLVMI
`
`
`AIHYNQVVPR
`—--VIDVASAVAA5DLVMI
`——-VADVATAVAAADLVMI
`s5HYNQVVPa
`
`
`
`s5aynqvvpn
`FQGRLYKEEIEPNLKKGATLAF'
`—-—VADVKTAVAA5LVVMI
`HQAVIYHNQIAPNVKPGAAIAF-
`5HFGQquA
`s5EYNQvaR
`
`s5nynqvvva
`
`
`
`
`
`
`EQAAIYAEDLKDNLRPGSAIAF
`
`IQSEIFNEEEKPELKAGKTflAF‘
`
`---PLHTKDAVXD5DIII
`---VATPRGAAEQADLVS
`—--VYEIGEAVRK§DYIL
`—-—VXTTAEAAAW%DV;
`---VFSVKEAAAQ&E%Q
`---VYTVAEAAKQ%D¥¥MI
`—--VMSVSEAVRT§QVVQ
`NGTLGDMWETISGSDLVL
`
`{HYGQIEPS
`-FGLIKPP
`QPKVWQEQ5APNLKEGVVVDF
`NIHFDLIKPA
`SQAEIFTND};PN}NAGDALLF
`FEQIVPP
`QQQKVYEAERKDEPTAGKSPVF
`FDQVKPP
`LQPEVYEAEIAPNLQAGNSLVF
`IHFGQINPP
`QQAHVYKAGVEENLREGQMLLFSHGI
`ISHSAQRDNYE- KVFSHMKPNSILGLSHGILLGHLQSLGQ
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 11 0f 15
`
`US 2009/0163376 A1
`
`
`
`
`gil70732562Iref|YP_262325.1I/1 A———0LDTIMr-PK--Gn
`giI28868203Ire£INP_790322 . 1 I /1 A---DLD IMI-PK‘ 'GI:
`“
`E'VKG----- GGIPDLI
`'
`‘
`-
`gil26991362 I releP_746787. 1I/1 A--—DLD IMI'PK‘ 'Glt
`'SEEWKG-----GGIPDLIHI
`‘
`LSYg‘aSGV
`
`giI15599888Iref|NP_253382 . 1I/1 A-—-DLD IMI‘PK'
`'G‘:
`A
`‘
`'
`LSYACGV
`'SEE’VKG-----GGIPDL
`gi I 114319705 IreleP_741388 .1 I / A---DLD IMXP
`'G"
`' STMYVEG----- GGVPSLI IATG"AI“I'LSY§1$AN
`gil83648555I refIYP__436990. 1I/1 B---DLD IM-PK'
`'GI-
`'SEEW'KG-----GGIPDLI
`‘
`"
`LSYASGV
`giI67159493IrefIZP_00420011.1I
`A—--DLD IMI'PK‘ 'Gh
`EEVRG-----GGIPDLI
`‘
`LSYlAHCGV
`giI71065099IreleP__263826.1I/1 ' S---DLD IMV'PK'
`'Gli
`'SEflAKG-----GGIPDLI'I‘ QIASGOAV
`LSY’A‘AGV
`giI17546794|ref|NP_520196.1I/1 A---DLD IMV'PK‘ 'G"
`TXAQG-----GGVPHLI
`‘
`‘
`LSYATAN
`
`giI74316007Iref|YP_315747.1I/1 A---DLD IMI'PK'
`'G"
`'SEFEVKG-----GGIPDLI I CIASG
`gil66044103|re£|YP_233944.1I/1 A--—DLD IMf‘PK"GI
`Egvxc-----GGIPDLIHVX I
`
`gi|120553816|refIYP_958167.1l/ K---DLD IMV-PK"GI
`A
`gil146306044IrefIYP_001186SO9. A---DLD IM:m ‘PK‘ 'G"
`'SEFVKG-----GGIPDLIRI; O V
`
`gi|56552037lxefIYP_162876.1|/1
`-snyvns—————GGVPCLVHVDQ
`x———010¥FM§-p c-cv
`
`gi|57240359|re£IZP_00368308 1: K---GID IM -
`gi|6225553|sp|032414|Ich_RHOM A---DLD¥Hii-P‘ -e.
`gi|15897495IrefINP_342100.1l/1 K---DSD YMV-
`a}.
`E—--DVN¥TMZ
`gi|76801743IrefIYP_326751.1I/l
`
`gi|18313972Ire£INP_560639.1I/1
`x-——nzn I
`gi|19552493|ref|NP_600495.1|/1 D-—~DII e W p
`
`gi|16079881lreleP_390707.1I/1 A---DVD L¥-P
`.
`
`.
`If?“
`‘
`'
`—————
`'PTFSEG—————GAVPAL
`gi|42780593|ref|NP_977840.1I/1 A—--NVDHFL 1p
`'6»
`
`g;I42781005|ref|NP_978252.1I/1 S--—W GI-
`"VfiQEG
`NGVPAL
`H
`
`GPS -v gVQGxEVNGAcINss * ”1*
`gi|266346lspI901292IILV5_SPIOL nrpxn
`
`
`
`pP
`
`t 3
`
`gi|70732562lref|YP_262325.1I/1
`gi|28868203|ref|NP_790822.1l/1
`gi|26991362I:ef|NP_746787.1|/1
`giI15599888IrefINP_253382.1II1
`gil114319705IrefIYP_741383.1I/
`gil83648555Iref|YP_436990.1I/1
`gil67159493|ref|ZP_00420011.1I
`gil71065099|reflYP_263826.1I/1
`gi|1754679d|ref|NP_520196.1|/1
`gil74318007|ref|YP_315747.1|/1
`gil66044103|reE|YP_233944.1l/1
`giI120553816lrefIYP_958167.1|/
`gi|146306044IrefIYP_001186509.
`giI56552037|ref|YP_162876.1|l1
`gil57240359|ref|ZP_00368308.1I
`gil6225553l5p|032414IILVC_RHOM
`giI15897495IreleP_342100.1I/1
`gi|76801743lreleP_326751.1|/1
`gil18313972|ref|NP_560639.1|/1
`gil19552493Izef|NP_600495.1|/1
`gil16079881|ref|NP_390707.1I/1
`giI42780593IrefINP_9'/7840.1I/1
`giI42781005|ref|NP_978252.11/1
`gi I2 66345 I 8p I 9012 92 I ILV5_SPIOL
`
`60
`....:....
`l
`.
`.l
`GRTGIIE
`Facfiann
`GRTGIIE
`FECLEEL
`GRTGIIE
`FacyaaL
`GRTGIIE
`PECFHEL
`GRAGVIE
`FEC‘IéEET
`GRTGII
`FECLHEL
`GRTGII
`rEcgaEL
`GRSGIIE
`FECIL‘HEL
`annex:
`FECLHEL
`GRTGII
`rchazn
`carer:
`rncyaan
`GRTGIIE
`rachnzn
`GRTGIIE
`FECLEEL
`cascvra
`,rzcgnnm
`GRTGIIE
`FEC‘L‘HFM
`GRSGIIE
`FECLEEV
`TRAGVIP
`FETINEL
`TRAGVVE
`4FECLNEL
`TRAGVIE
`FEVLNEA
`ARAGVIP
`:FEVLEEL
`ARAGVL
`SW FECLEEL
`TRAGV'LE
`%:"FEC‘LIHEL
`
`TRAGVIE
`"H‘2rzcganL
`SPF--TF
`SEDW‘wKNTEECI
`
`
`
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 12 of 15
`
`US 2009/0163376 A1
`
`gil70732562ireleP_262325.1|/1
`gi|28868203IreleP_790822.1|/1
`gi|26991362lreleP_746787.1|/1
`gil15599888Iref|NP_253382.1|/1
`gil114319705|re£|YEL741388.1|/
`giI83648555tref|YP_436990.1I/1
`gil67159493tref|ZP_00420011.1|
`9iI71065099lref|YP_263826.1l/1
`gil17546794|releP_520196.1l/1
`gi|74318007|releP_315747.1|/1
`gi|66044103|releP_233944.1|/1
`gi1120553816lref|YP_958167.1|/
`gi|146306044Ire£|¥P_001186509.
`giI56552037|xef|¥9_162876.1|/1
`gi|57240359|releP_00368308.1|
`gi|6225553|sp|032414|ILVC_RHOM
`gil15897495|ref|NP_342100.1|/1
`gil76801743|releP_326751.1|/1
`gil18313972|ref|NP_560639.1|/1
`gil19552493lre£|NP_600495.1|/1
`gil16079881|re£|NP_390707.1[/1
`gi|42780593|tef|NP_977a4o.1I/1
`gi|42781005|ref|NP_978252 11/1
`911266346|spl901292|ILv5_SPIOL
`
`
`
`
`
`AEYGEYVTGPEVINAESRQAMRNALKRIQD
`
`REYGEYVTGPEVINEESRKAMRNALKRIQD
`
`AEYGEYVTGPEVINAESRAAMRNALKRIQD
`
`
`AEYGDFTRGPRVINEESREAMREILAEIQE
`
`AEYGEYVTGPEVINDQSRAAMRNALKRIQD
`
`AEYGEYVTGPEVINEQSRQAMRNALKRIQD
`
`
`
`
`
`
`SMMIWIAANKIVDKDKN---------
`
`
`
`AEYGDYITGPKIITKETKEAEKGVLKDIQ SFAKDF
`IVDL§IQ
`K
`AMYGDYVTGSRIITEATKAEMKRVLADIQS
`FVRDW
`K-gEVDLIYE
`ISGMLKA 5wTAKYGGMTVGKFVIDESVRKRMKEALQRIKS
`AEEW
`K-MIVDLxYE
`SEMWDS SmTAEYGGLTRGDRIVDDHAREKMEEVLEEMQ
`FAREW
`K--EEVDLMYE
`K-EEMDLIWQ IYGMLN SmTAKYGGLTVGPRVIDENVKRKMKEAAMRVKS
`FAKEW
`x—EEVDLMFE
`ISNMNYS SHTAEFGGYLSGPRVIDADTKSRMKDILTDIQD FTKRL
`K--E$anMYE
`GMRYSISmTAQWGDFVSGPRVVDAKVKESMKEVLKDIQ
`FAKEW
`K-EEVDLMYE
`ENMRISII SmTA§WGDFVSGPRVVTEDTKKAMGTVLAEIQD
`FARGW
`K—EEVDLMYE
`TNMRESISHTAEFGDYVTGSRIVTDETKKEMKRVLTEI
`FAKKW
`TGgISKTIsr
`_SIEGKK—DFQAAYSASYYPSMDILYECYEDVAS SEIRSV
`430
`440
`450
`460
`470
`480
`.I....|....l....l....1....l....I....l....l....|....l....
`1I/1
`gil70732562lreleP_262325.
`
`ISEGATGYP--SMTAKRRNNAABGIE IIGIfiEESMMPWIGANKIVDKAKN—————————
`ITEGATGYP--SMTAKRRNNAEBGIE-VIG|
`1|/1
`gil28868203|ze£|NP_790822.
`gi|26991362lreleP_746787.1I/1
`ISEGATNYP--SMTAKRRNNAAHGIE IIGIQ: SMMPWISANKIVDKTKN---------
`
`ITEGAANIP--SMTAYRRNNAAHPIE—QIGIKgaggziganNKIvnxsxu---------
`gi|15599888|ref|NP_253382 ll/1
`
`gil114319705|re£|YP_741388. 11/
`VLENQAGCP--TLTARRRLAAEEEIE-
`uni
`INANKLVDKDKN---------
`IAEGAHNYP--SMTAYRRNNAAHPIE—Q GI
`SMMPWIASNKIVDKSKN_________
`gil83648555Iref|¥Pu436990. 1|/1
`ITEGAANYP——SMTAYRRNNAAHQIE-
`IAAMKIVDKTKN.........
`gil67159493IreleP_00420011.1I
`ISEGATNYP--SMTARRRNNAEEQIE I G
`IGGNKIIDKDKN—————————
`gil71065099|ref|YP_263826.1|/1
`
`gil17546794Iref|NP_520196.11/1
`
`IAKNKLVDQSKN_________
`LLENKAGAP--TLISRRRLTADHQIE-Q G
`-. -
`gil74318007|ref|YP_315747.1I/1' :
`ILEGQANYP--EMTAWRRNNAAHQIE-
`"S¥MPWIAANKLVDHSKN_________
`ISBGATGYP--SMTAKRRNNAAHGIE IIG:
`SMMPWIAANKIVDKDKN—————————
`gil66044103|ze£|YP_233944.1|/1
`ISEGALNYP--SMTARRRQNAAHEIE—
`SMMIWISAMKIVDKDKN—————————
`gil120553816|re£|!P_958167.1l/
`ISEGATNYP--SMTAKRRNNAAHGIE IIG:
`SMMPWISANKIVDKTKN---------
`gil146306044|ref|YP_001186509.
`VLDNRAGQP--ELKAARKRMAABPIE-Q
`IASNKLVDKARN_________
`gil56552037tref|YP_162876.1|/1
`ILERRANFA-—RMHAERKLMNDSLIE~K G:
`ISAKKLVDKDKN---------
`gil57240359tref|ZP_00368308.1|
`MLECKAGQP--SFKATRRIQXEHVIE-
`ISKNKLVDKARN.........
`gil6225553lspl032414IILVC_RHOM
`gil15897495|ref|NP_342100.1I/1
`XEEYGRGMP--TVVNGLSNVQNSLEE KIG’Q: qux-------prxs—————————
`gil76801743|releP_326751.1I/1
`ESENQAGRP--SYKQLRAAEKNHDIE-A
`:an-------sun___________
`
`gil18313972lref|NP_560639.1I/1
`LF-——GP _________________
`zEEYNRGAP--TLRKLMEEARTHPIE—
`gi|19552493|ref|NP_600495.ll/l
`
`IAMVENGNI——ELEGLRASINNHPIE-E
`aMswvxvnARAETA-----------
`gil16079881lref|NP_390707.1I/1
`PFVKQGKKKEAVVSVAQN-----
`IVENQVNRP—-RFNAINASENEHQIE-
`
`m IIFV-QPRVKVGMK__________
`IAEHKAGRP--NFHAINEKENEHEIE-
`gil42780593|ref|NP_977840.1l/1
`gil42781005lref|NP_973252 1|/1
`ILENQAGRP—-T2NAMKKAEQNHQLE—
`WIDAPKELVKK———————————
`
`SEEP--AGD--LGPLYPFIAGVYVAL
`gi|266346lsp|901292|ILv5_SPIOL
`
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 13 0f 15
`
`US 2009/0163376 A1
`
`
`
`gil70732562|ref|YP_262325.1|/1
`gil28868203|ref|NP_790822.1|/1
`gi|26991362|re£|NP_746787.1l/1
`giI15599888IreleP_253382.1l/1
`giI114319705|ref|YP_741388.1I/
`gil836485553reflYP_436990.1|/1
`giI67159493|releP_00420011.1l
`gil71065099|refl¥9_263826.1I/1
`giI17546794|releP_520196.1I/1
`gi|74318007IreleP_315747.1|/1
`giI66044103|releP_233944.1l/1
`giI120553816|ref|YP_958167.ll/
`gil146306044|ref|¥P_001186509.
`gi|56552037lteleP_162876.1l/1
`gi|57240359|zef|ZP_00368308.1|
`gil6225553|ap|032414IILVC_RHOM ------------------------------------------------------------
`giI15897495|re£|NP_342100.1|/1
`gil76801743lrefl!P_326751.1|/1
`gi|18313972|zefINP_560639.1I/1
`giI19552493|ref|NP_600495.1|/1
`gi|16079881IrefINP_390707.1|/1
`gil42780593|zeleP_977840.1I/1
`gil42781005lzef|NP_978252.1I/1
`giI266346Isp|Q01292lILV5_SPIOL MMAQIEILRKKGESYSEIINESVIEAVDSLNPFMBARGVSFMVDNCSTTARLGSRKWAPR
`
`
`
`
`
`gil70732562|re£|YP_262325.1|/l
`gil28868203|zef|NP_790822.1I/1
`gi|26991362|re£lNP_746787.1|/1
`giI15599888|tef|NP_253382.1I/1
`gi|114319705IreleP_741388.1I/
`gil83648555|ref|YP_436990.1|/1
`gil67159493|ref|ZP_00420011.1i
`gil71065099|ref|YP_263826.1|/1
`gil17546794|zeleP_520196.1|/1
`gi|74318007|releP_315747.1|/1
`gil66044103lte£IYP_233944.1I/1
`giI120553816|zeleP_958167.1I/
`gil146306044|releP_001186509.
`9i|56552037|re£|YP_162876.1|/1
`gil57240359|ref|ZP_00368308.1|
`gil6225553lsp|032414IILVC_RBOM ----------------------------------7 —————————————————————————
`giI15897495IreleP_342100.13/1
`giI76801743ireleP_326751.ll/l
`gil18313972lre£|NP_560639.1|/1
`gil19552493|ze£|NP_600495.1|/1
`gil16079881|ref|NP_390707.1|/1
`gil42780593lzefINP_977840.1I/1
`gil42781005IrefINP_978252.1|/1
`gil265346lSplQOlZQZIILV5_SPIOL FDYILSQQALVAVDNGAPINQDLISNFLSDPVBEAIGVCAQLRPSVDISVTADADFVRPE
`
`
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 14 0f 15
`
`US 2009/0163376 A1
`
`gil70732562|ref|YB_262325.1l/1 —«-—
`gi|28868203lref|NB_790822.1l/1
`-——-
`gil26991362IreleR_7467B7.1|/1
`-———
`gil15599888|ref|NB_253382.1I/1 —---
`gi|114319705|releB_741388.1l/
`-—--
`gi|83648555|ref|¥2_436990.1|/1 ----
`gil67159493|ref|ZP_OO420011.1|
`--—-
`gi171065099Iref|YP_263826.1|/1
`----
`giI17546794lreleP_520196.1|/1 ----
`gi|74318007lreleP_315747.ll/1 ————
`gi|66044103|ref|YP_233944.11/1
`----
`gil120553816lref|YB_958167.ll/
`————
`gii146306044|releB_001186509. —--—
`gi|56552037lreleB_162876.1|/1
`--—-
`gil57240359|ref|ZE_00368308.1|
`————
`gi|6225553|sp|032414|Iqu_RHOM ----
`gi|15897495lreflNP_342100.1|/1
`-—-—
`gi|76801743|ref|YP_326751.1|/1 ——--
`gi|18313972lreleP_560639.1|/1 ————
`'gil19552493lref|NP_600495.1|/1 ——--
`giI16079881lreleP_390707.1l/1 -—--
`giI42780593lraleP_977840.1l/l
`----
`giI42781005lref|NB_973252.11/1
`--—-
`gi|266346|splQ01292|ILv5_SPIOL LRQA
`
`

`

`Patent Application Publication
`
`Jun. 25, 2009 Sheet 15 0f 15
`
`US 2009/0163376 A1
`
`Figure 10
`
`gi|70732562|refl¥P_262325.1|: domain 1 of 1,
`1e—27O
`
`from 1 to 338: score 926.8,
`
`[*1 H
`
`*—>quafskVYYDkDad1sGhdeylikGKkVAvI
`
`SQG
`
`M
`
`kV+YDkD+dls
`
`+i+GKkVA+I
`
`SQGHA+
`
`rD
`
`L+D
`
`gi|7073256
`
`1
`
`-M---—KVFYDKDCDLS ————— IIQGKKVAII
`
`SQGHAQ
`
`LKD 37
`
`SGVdVvYG
`SGVdV+VG
`
`gi|7073256
`
`38 SGVDVTVG
`
`
`
`kAeaaEf thvaEAvanDvai lPDeane
`kAea+e+ V +va Ava+AD+Vmi +PDefQ++
`
`
`
`‘
`‘ AHe
`PDEFQSQ 87
`
`IVAAAVAGADLVMI
`
`ereeIeankpGatLaFAHGFNIHf-qlvPrakaDiDViMVAPKgPGH
`
`+Y++eIepn+k+GatLaF+HGF+IH+ q vPra
`
`D+DViM+APK+PGH
`
`gil7073256
`
`88 LYKNEIEPNIKKGATLAFSHGFAIH
`
`
`
`RA——-DLDVIMIAPKAPGH 134
`
`tVRreYvagGVPaLiAVyQ
`
`GnAkd1ALsY
`
`'GggRAGvIETTFk
`
`tVR+e+vagG+P+LiA+yQ
`
`GnAk++ALsYA
`
`GggR+G+IETTFk
`
`gi|7073256
`
`135 TVRSEFVKGGGIPDLIAIYQ
`
`GNAKNVALSY
`
`GGGRTGIIETTEK 184
`
`eETETDLFGEQaVLCGtheLVkaGFETLVEaGYaPEmAYFECLHElKLI
`
`+ETETDLFGEQaVLCGG++eLVkaGFETLVEaGYaPEmAYFECLHElKLI
`
`gi|7073256
`
`185 DETETDLFGEQAVLCGGTVELVKAGFETLVEAGYAPEMAYFECLHELKLI 234
`
`VDLmYEgGIanMrySiSdTAeYGdythprVIdeeskeaMkevadIQsG
`
`VDLmYEgGIanM+ySiS++AeYG+ythp+VI++es++aM+++Lk+IQ+G
`
`gil7073256
`
`235 VDLMYEGGIANMNYSISNNAEYGEYVTGPEVINAESRQAMRNALKRIQDG 284
`
`eFAkewilEnanyPketltalrrneaquIEWkVGekLRsmmpWIaanK
`
`e+Ak++i+E+++GyP ++ta rrn+a+H IE +Ge+LRsmmpWI anK
`
`gil7073256
`
`285 EYAKMFISEGATGYP--SMTAKRRNNAAHGIE-IIGEQLRSMMPWIGANK 331
`
`1vdkdkn<—*
`
`+vdk+kn
`
`gi|7073256
`
`332 IVDKAKN
`
`338
`
`

`

`US 2009/0163376 A1
`
`Jun. 25, 2009
`
`KETOL-ACID REDUCTOISOMERASE USING
`NADH
`
`[0001] This application claims the benefit of US. Provi-
`sional 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 of the 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., Acla 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., (1999), 292,
`87-96, 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., (1997),
`272, 2218-2222, describing the use of site-directed mutagen-
`esis 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 Km values for NADP(H) and a cor-
`responding 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., (2004), 271, 2107-2116); dehydrogenases;
`Nishiyama, et al., J. Biol. Chem., (1993), 268, 4656-4660;
`Ferredoxin-NADP reductase, Martinez-Julvez, et al., Bio-
`phys. Chem., (2005), 115, 219-224); and oxidoreductases
`(US2004/0248250).
`[0008] Rane et al., (Arch. Biochem. Biophys., (1997), 338,
`83-89) 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 as
`opposed to NADPH would greatly enhance its effectiveness
`in the isobutanol biosynthetic pathway, however, no such
`KARI enzyme has been reported.
`[0010] Applicants have solved the stated problem by iden-
`tifying a number of mutant ketol-acid reductoisomerase
`enzymes that have a preference for binding NADH as
`opposed to NADPH.
`
`SUMMARY OF THE INVENTION
`
`[0011] The invention relates to a method for the evolution
`of ketol-acid reductoisomerase (KARI) enzymes from bind-
`ing the cofactor NADPH to binding NADH. The method
`involves mutagenesis of certain specific residues in them
`KARI enzyme to produce the co-factor switching.
`[0012] Accordingly the invention provides a mutant ketol-
`acid reductoisomerase enzyme comprising the amino acid
`sequence as set forth in SEQ ID NO: 29.
`[0013] Alternatively the invention provides a mutant ketol-
`acid reductoisomerase enzyme having the amino acid
`sequence selected from the group consisting of SEQ ID NO:
`19, 24, 25, 26, 27, 28, 67, 68, 69, and 70.
`[0014]
`In a preferred embodiment a mutant ketol-acid
`reductoisomerase enzyme is provided 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.
`[0015]
`In a specific embodiment the invention provides a
`mutant ketol-acid reductoisomerase enzyme as set forth in
`SEQ ID NO:17 wherein:
`[0016]
`a) the residue at position 47 has an amino acid
`substation selected from the group consisting ofA, C, D,
`F, G, I, L, N, P, andY;
`[0017]
`b) the residue at position 50 has an amino acid
`substitution selected from the group consisting of A, C,
`D, E, F, G, M, N, V, W;
`[0018]
`c) the residue at position 52 has an amino acid
`substitution selected from the group consisting of A, C,
`D, G, H, N, S;
`[0019]
`d) the residue at position 53 has an amino acid
`substitution selected from the group consisting ofA, H,
`I, W;
`e) the residue at position 156 has an amino acid
`[0020]
`substitution ofV;
`[0021]
`f) the residue at position 165 has an amino acid
`substitution of M;
`
`

`

`US 2009/0163376 A1
`
`Jun. 25, 2009
`
`g) the residue at position 61 has an amino acid
`[0022]
`substitution of F;
`[0023]
`h) the residue at position 170 has an amino acid
`substitution ofA;
`[0024]
`i) the residue at position 24 has an amino acid
`substitution of F;
`[0025]
`j) the residue at position 33 has an amino acid
`substitution of L;
`[0026]
`k) the residue at position 80 has an amino acid
`substitution of 1; and
`[0027]
`l) the residue at position 115 has an amino acid
`substitution of L.
`
`In another embodiment the invention provides a
`[0028]
`method for the evolution of a NADPH binding ketol-acid
`reductoisomerase enzyme to an NADH using form compris-
`mg:
`a) providing a ketol-acid reductoisomerase
`[0029]
`enzyme which uses NADPH having a specific native
`amino acid sequence;
`[0030]
`b) identifying the cofactor switching residues in
`the enzyme ofa) based on the amino acid sequence ofthe
`Pseudomonas fluorescens ketol-acid reductoisomerase
`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;
`[0031]
`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.
`[0032]
`In an alternate embodiment the invention provides a
`method for the production of isobutanol comprising:
`[0033]
`a) providing a recombinant microbial host cell
`comprising the following genetic constructs:
`[0034]
`i) at least one genetic construct encoding an
`acetolactate synthase enzyme for the conversion of
`pyruvate to acetolactate;
`[0035]
`ii) at least one genetic construct encoding a
`mutant ketol-acid reductoisomerase enzyme of the
`invention;
`[0036]
`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);
`[0037]
`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);
`[0038]
`v) at leas