Us 20100221802

US 20100221802A1
( 19 ) United Sta tes

( 12) Pa tent Applica tion Publica tion ( 10) Pub. N o. : US 2010/0221802 A1
GRADY et a l. ( 4 3 ) Pub. Da te: Sep. 2, 2010
( 5 4 ) METHOD FOR PRODUCIN G BUTAN OL ( 21) Appl. N o. : 12/7 5 9 , 283
USIN G TWO-PHASE EXTRACTIVE ~
22 F1 d: A . 13 2010
FERMEN TATION ( ) 1 e Pr
Rela ted US. Applica tion Da ta
( 7 5 ) Inv entors MICHAEL CHARLES GRADY ( 6 3 ) Continua tion-in-pa rt of a pplica tion N o. 12/4 7 8, 3 89 ,
' OAKLYN N J ( Us ) ?led on Jun. 4 , 2009 .
MEHMEbALIJA Z IAHIC ( 6 0) Prov is iona l a pplica tion N o. 6 1/05 8, 5 6 7 , ?led on Jun.
WILMIN GTON , DE ( US) ; 4 20 8
RAN JAN PATN AIK, N EWARK, Publica tion Cla s s i?ca tion
DE ( Us ) ( 5 1) Int. Cl.
C12P 7 /16 ( 2006 . 01)
Corres pondence Addres s : C07 C 29 /86 ( 2006 01)
E I U-S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LEGAL PATEN T RECORDS CEN TER ( 5 7 ) ABSTRACT
BARLEY MILL PLAZ A 25 /1122B, 4 4 17 LAN - _
C ASTER PIKE A method of ma kmg buta nol f rom a t lea s t one f ermenta ble
WILMIN GTON , DE 19 805 ( Us ) ca rbon s ource tha t ov ercomes the is s ues of tox icity res ulting
in a n increa s e in the ef f ectiv e titer, the ef f ectiv e ra te, a nd the
ef f ectiv e y ield of buta nol production by f ermenta tion utiliZ
( 7 3 ) As s ignee; BUTAMAX( TM) ADVAN CED ing a recombina nt microbia l hos t Wherein the buta nol is
BIOFUELS LLC, ex tra cted into s peci?c orga nic ex tra cta nts during f ermenta
WILMIN GTON , DE ( U S)
tion
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US 2010/0221802 A1
METHOD FOR PRODUCIN G BUTAN OL
USIN G TWO-PHASE EXTRACTIVE
FERMEN TATION
[ 0001] This a pplica tion is a continua tion-in-pa rt of Us .
a pplica tion Ser. N o. 12/4 7 8, 3 89 , ?led Jun. 4 , 2009 , herein
incorpora ted by ref erence, a nd Which cla ims the bene?t of
priority f rom Us . Prov is iona l Applica tion N o. 6 1/05 8, 5 6 7 ,
?led Jun. 4 , 2008, herein incorpora ted by ref erence.
FIELD OF THE IN VEN TION
[ 0002] The inv ention rela tes to the ?eld of biof uels . More
s peci?ca lly , the inv ention rela tes to a method f or producing
buta nol through microbia l f ermenta tion, in Which the buta nol
product is remov ed by ex tra ction into a Wa ter immis cible
orga nic ex tra cta nt during the f ermenta tion.
BACKGROUN D OF THE IN VEN TION
[ 0003 ] Buta nol is a n importa nt indus tria l chemica l, With a
v a riety of a pplica tions , s uch a s us e a s a f uel a dditiv e, a s a
f eeds tock chemica l in the pla s tics indus try , a nd a s a f ood
gra de ex tra cta nt in the f ood a nd ?a v or indus try . Ea ch y ea r 10
to 12 billion pounds of buta nol a re produced by petrochemi
ca l mea ns a nd the need f or this chemica l Will likely increa s e.
[ 0004 ] Sev era l chemica l s y nthetic methods a re knoWn;
hoWev er, thes e methods of producing buta nol us e s ta rting
ma teria ls deriv ed f rom petrochemica ls a nd a re genera lly
ex pens iv e a nd a re not env ironmenta lly f riendly . Sev era l
methods of producing buta nol by f ermenta tion a re a ls o
knoWn, f or ex a mple the ABE proces s Which is the f ermentiv e
proces s producing a mix ture of a cetone, 1-buta nol a nd etha
nol. Acetone-buta nol-etha nol ( ABE) f ermenta tion by
Clos lridium a celobuly licum is one of the oldes t knoWn indus
tria l f ermenta tions ; a s is a ls o the pa thWa y s a nd genes res pon
s ible f or the production of thes e s olv ents . Production of 1 -bu
ta nol by the ABE proces s is limited by the tox ic ef f ect of the
1-buta nol on Clos lridium a celobuly licum. In s itu ex tra ctiv e
f ermenta tion methods us ing s peci?c orga nic ex tra cta nts
Which a re nontox ic to the ba cterium ha v e been reported to
enha nce the production of 1-buta nol by f ermenta tion us ing
Clos lridium a celobuly licum ( Rof ?er et a l. , Biotechnol.
Bioeng. 3 1: 13 5 -14 3 , 19 88; Rof ?er et a l. , Bioproces s Engi
neering 2: 1-12, 19 87 ; a nd Ev a ns et a l. , Appl. Env iron. Micro
biol. 5 4 : 16 6 2-16 6 7 , 19 88) .
[ 0005 ] In contra s t to the na tiv e Clos lridium a celobuly licum
des cribed a bov e, recombina nt microbia l production hos ts
ex pres s ing 1-buta nol, 2-buta nol, a nd is obuta nol bios y nthetic
pa thWa y s ha v e a ls o been des cribed. Thes e recombina nt hos ts
ha v e the potentia l of producing buta nol in higher y ields com
pa red to the ABE proces s beca us e they do not produce
by products s uch a s a cetone a nd etha nol. HoWev er, the prob
lem With thes e recombina nt hos ts is tha t biologica l produc
tion of buta nol a ppea rs to be limited by buta nol tox icity
thres holds to the hos t microorga nis m us ed in the f ermenta
tion. Ex tra ctiv e f ermenta tion methods ha v e not been a pplied
to the production of buta nols us ing recombina nt microbia l
s tra ins .
[ 0006 ] The pres ent inv ention s a tis ?es the a bov e need a nd
prov ides a method of ma king buta nol f rom a t lea s t one f er
menta ble ca rbon s ource tha t ov ercome the is s ues of tox icity
res ulting in a n increa s e in the ef f ectiv e titer, the ef f ectiv e ra te,
a nd the ef f ectiv e y ield of buta nol production by f ermenta tion
Sep. 2, 2010
utiliZ ing a recombina nt microbia l ho s t Wherein the buta nol is
ex tra cted into s peci?c orga nic ex tra cta nts during f ermenta
tion.
SUMMARY OF THE IN VEN TION
[ 0007 ] The inv ention prov ides a method f or recov ering
buta nol f rom a f ermenta tion medium, the method compris
mg:
[ 0008] a ) prov iding a f ermenta tion medium compris ing
buta nol, Wa ter, a nd a genetica lly modi?ed microorga n
is m tha t produces buta nol f rom a t lea s t one f ermenta ble
ca rbon s ource;
[ 0009 ] b) conta cting the f ermenta tion medium With i) a t
lea s t one ?rs t Wa ter immis cible orga nic ex tra cta nt
s elected f rom the group cons is ting of C12 to C22 f a tty
a lcohols , Cl2 to C22 f a tty a cids , es ters of Cl2 to C22 f a tty
a cids , C l 2 to C22 f a tty a ldehy des , C l 2 to C22 f a tty a mides
a nd mix tures thereof , a nd optiona lly ( ii) a s econd Wa ter
immis cible orga nic ex tra cta nt s elected f rom the group
cons is ting of C12 to C22 f a tty a lcohols , Cl2 to C22 f a tty
a cids , es ters of C12 to C22 f a tty a cids , Cl2 to C22 f a tty
a ldehy des , C 12 to C22 f a tty a mides a nd mix tures thereof
to f orm a tWo-pha s e mix ture compris ing a n a q ueous
pha s e a nd a buta nol-conta ining orga nic pha s e;
[ 0010] c) s epa ra ting the buta nol-conta ining orga nic
pha s e f rom the a q ueous pha s e; a nd
[ 0011] d) recov ering the buta nol f rom the buta nol-con
ta ining orga nic pha s e to produce recov ered buta nol.
[ 0012] The inv ention prov ides a method f or the production
of buta nol compris ing the s teps of :
[ 0013 ] a ) prov iding a genetica lly modi?ed microorga n
ca rbon s ource;
[ 0014 ] b) groWing the microorga nis m in a bipha s ic f er
menta tion medium compris ing a n a q ueous pha s e a nd a
Wa ter immis cible orga nic ex tra cta nt s elected f rom the
group cons is ting of Cl2 to C22 f a tty a lcohols , Cl2 to C22
f a tty a cids , es ters of C12 to C22 f a tty a cids , Cl2 to C22
f a tty a ldehy des , a nd mix tures thereof , Wherein s a id
bipha s ic f ermenta tion medium compris es f rom a bout
3 % to a bout 6 0% by v olume of s a id Wa ter immis cible
orga nic ex tra cta nt, f or a time s u?icient to a lloW ex tra c
tion of the buta nol into the orga nic ex tra cta nt to f orm a
buta nol-conta ining orga nic pha s e;
[ 0015 ] c) s epa ra ting the buta nol-conta ining orga nic
[ 0016 ] d) recov ering the buta nol f rom the buta nol-con
[ 0017 ] An embodiment of the inv ention prov ides a method
f or the production of buta nol compris ing the s teps of :
[ 0018] a ) prov iding a genetica lly modi?ed microorga n
ca rbon s ource;
[ 0019 ] b) groWing the microorga nis m in a f ermenta tion
medium Wherein the microorga nis m produces s a id
buta nol into the f ermenta tion medium to produce a
buta nol-conta ining f ermenta tion medium;
[ 0020] c) conta cting the buta nol-conta ining f ermenta
tion medium With i) a t lea s t one ?rs t Wa ter immis cible
orga nic ex tra cta nt s elected f rom the group cons is ting of
C12 to C22 f a tty a lcohols , Cl2 to C22 f a tty a cids , es ters of
C12 to C22 f a tty a cids , Cl2 to C22 f a tty a ldehy des , Cl2 to
C22 f a tty a mides a nd mix tures thereof , a nd optiona lly
( ii) a s econd Wa ter immis cible orga nic ex tra cta nt
US 2010/0221802 A1
a cids , Cl2 to C22 f a tty a ldehy des , Cl2 to C22 f a tty a mides
a nd mix tures thereof to f orm a tWo-pha s e mix ture com
pris ing a n a q ueous pha s e a nd a buta nol-conta ining
orga nic pha s e;
[ 0021] d) s epa ra ting the buta nol-conta ining orga nic
[ 0022] e) recov ering the buta nol f rom the buta nol-con
ta ining orga nic pha s e.
[ 0023 ] An embodiment of the inv ention prov ides a method
f or the production of buta nol compris ing the s teps of :
[ 0024 ] a ) prov iding a genetica lly modi?ed microorga n
ca rbon s ource;
[ 0025 ] b) groWing the microorga nis m in a f ermenta tion
medium under a erobic conditions f or a time s uf ?cient to
rea ch a pres elected groWth lev el;
[ 0026 ] c) s Witching to microa erobic or a na erobic condi
tions to s timula te buta nol production into the f ermenta
tion medium to f orm a buta nol-conta ining f ermenta tion
medium;
[ 0027 ] d) conta cting the buta nol-conta ining f ermenta
tion medium With i) a t lea s t one ?rs t Wa ter immis cible
orga nic ex tra cta nt s elected f rom the group cons is ting of
C12 to C22 f a tty a lcohols , Cl2 to C22 f a tty a cids , es ters of
C12 to C22 f a tty a cids , Cl2 to C22 f a tty a ldehy des , Cl2 to
C22 f a tty a mides a nd mix tures thereof , a nd optiona lly
( ii) a s econd Wa ter immis cible orga nic ex tra cta nt
a cids , Cl2 to C22 f a tty a ldehy des , Cl2 to C22 f a tty a mides
a nd mix tures thereof to f orm a tWo-pha s e mix ture com
pris ing a n a q ueous pha s e a nd a buta nol-conta ining
orga nic pha s e;
[ 0028] e) s epa ra ting the buta nol-conta ining orga nic
[ 0029 ] f ) recov ering the buta nol f rom the buta nol-con
ta ining orga nic pha s e.
[ 003 0] Another embodiment of the inv ention prov ides a
method f or the production of buta nol compris ing the s teps of :
[ 003 1] a ) prov iding a f ermenta tion medium compris ing
buta nol, Wa ter, a nd a genetica lly modi?ed microorga n
is m tha t produces buta nol f rom a f ermenta tion medium
compris ing a t lea s t one f ermenta ble ca rbon s ource;
[ 003 2] b) conta cting the f ermenta tion medium v ia a co
current or counter-current ex tra cta nt s trea m With i) a t
lea s t one ?rs t Wa ter immis cible orga nic ex tra cta nt
a cids , Cl2 to C22 f a tty a ldehy des , a nd mix tures thereof ,
a nd optiona lly ( ii) a s econd Wa ter immis cible orga nic
ex tra cta nt s elected f rom the group cons is ting of C12 to
C22 f a tty a lcohols , Cl2 to C22 f a tty a cids , es ters of Cl2 to
C22 f a tty a cids , Cl2 to C22 f a tty a ldehy des , Cl2 to C22
f a tty a mides a nd mix tures thereof to f orm a tWo-pha s e
mix ture compris ing a n a q ueous pha s e a nd a buta nol
conta ining orga nic pha s e;
[ 003 3 ] c) s epa ra ting the buta nol-conta ining orga nic
[ 003 4 ] d) recov ering the buta nol f rom the buta nol-con
[ 003 5 ] The pres ent ex tra ctiv e f ermenta tion methods pro
v ide buta nol, including a ll buta nol is omers , Which is knoWn
Sep. 2, 2010
to ha v e a n energy content s imila r to tha t of ga s oline a nd Which
ca n be blended With a ny f os s il f uel. Buta nol is f a v ored a s a
f uel or f uel a dditiv e a s it y ields only CO2 a nd little or no SOX
or N OX When burned in the s ta nda rd interna l combus tion
engine. Additiona lly buta nol is les s corros iv e tha n etha nol,
the mos t pref erred f uel a dditiv e to da te.
[ 003 6 ] In a ddition to its utility a s a biof uel or f uel a dditiv e,
the buta nol produced f rom the pres ent methods ha s the poten
tia l of impa cting hy drogen dis tribution problems in the
emerging f uel cell indus try . Fuel cells toda y a re pla gued by
s a f ety concerns a s s ocia ted With hy drogen tra ns port a nd dis
tribution. Buta nol ca n be ea s ily ref ormed f or its hy drogen
content a nd ca n be dis tributed through ex is ting ga s s ta tions in
the purity req uired f or either f uel cells or v ehicles .
[ 003 7 ] Fina lly , the pres ent methods produce buta nol f rom
pla nt deriv ed ca rbon s ources , a v oiding the nega tiv e env iron
menta l impa ct a s s ocia ted With s ta nda rd petrochemica l pro
ces s es f or buta nol production.
BRIEF DESCRIPTION OF THE FIGURE AN D
SEQ UEN CE DESCRIPTION S
[ 003 8] FIG. 1 is a gra ph s hoWing the concentra tion of
is obuta nol in the f ermenta tion medium ( i. e. , a q ueous pha s e)
during a f ermenta tion us ing oley l a lcohol a s the orga nic
ex tra cta nt With ga s s tripping ( I) a s des cribed in Ex a mple 6 ,
a nd during a f ermenta tion With ga s s tripping a lone ( Q ) , a s
des cribed in Ex a mple 7 . FIG. 1 repres ents da ta genera ted
us ing a recombina nt Es cherichia coli producing is obuta nol.
[ 003 9 ] FIG. 2 is a gra ph s hoWing the concentra tion of
is obuta nol in the f ermenta tion medium ( i. e. , a q ueous pha s e)
during a f ermenta tion us ing oley l a lcohol a s the orga nic
ex tra cta nt With ga s s tripping ( I) a s des cribed in Ex a mple 8,
a nd during a f ermenta tion With ga s s tripping a lone ( Q ) , a s
des cribed in Ex a mple 9 . FIG. 2 repres ents da ta genera ted
us ing a recombina nt Sa ccha romy ces cerev is ia e producing
is obuta nol.
[ 004 0] FIG. 3 s chema tica lly illus tra tes one embodiment of
the methods of the inv ention, in Which the ?rs t Wa ter immis
cible ex tra cta nt a nd the optiona l s econd Wa ter immis cible
ex tra cta nt a re combined in a v es s el prior to conta cting the
f ermenta tion medium With the ex tra cta nt in a f ermenta tion
v es s el.
ex tra cta nt a re a dded s epa ra tely to a f ermenta tion v es s el in
Which the f ermenta tion medium is conta cted With the ex tra c
ta nt.
ex tra cta nt a re a dded s epa ra tely to dif f erent f ermenta tion v es
s els f or conta cting of the f ermenta tion medium With the
ex tra cta nt.
[ 004 3 ] FIG. 6 s chema tica lly illus tra tes one embodiment of
the methods of the inv ention, in Which ex tra ction of the prod
uct occurs doWns trea m of the f ermentor a nd the ?rs t Wa ter
immis cible ex tra cta nt a nd the optiona l s econd Wa ter immis
cible ex tra cta nt a re combined in a v es s el prior to conta cting
the f ermenta tion medium With the ex tra cta nt in a dif f erent
v es s el.
US 2010/0221802 A1
cible ex tra cta nt a re a dded s epa ra tely to a v es s el in Which the
f ermenta tion medium is conta cted With the ex tra cta nt.
cible ex tra cta nt a re a dded s epa ra tely to dif f erent v es s els f or
conta cting of the f ermenta tion medium With the ex tra cta nt.
uct occurs in a t lea s t one ba tch f ermentor v ia co-current How
of a n ex tra cta nt a t or nea r the bottom of a f ermenta tion ma s h
to ?ll the f ermentor With ex tra cta nt Which ?oWs out of the
f ermentor a t a point a t or nea r the top of the f ermentor.
[ 004 7 ] The f ollow ing s eq uences conf orm With 3 7 C. F. R.
1 . 821 1 . 825 ( Req uirements f or Pa tentApplica tions Conta in
ing N ucleotide Seq uences a nd/orAmino Acid Seq uence Dis
clos ures ithe Seq uence Rules ) a nd a re cons is tent With
World Intellectua l Property Orga niz a tion ( WIPO) Sta nda rd
ST. 25 ( 19 9 8) a nd the s eq uence lis ting req uirements of the
EPO a nd PCT ( Rules 5 . 2 a nd 4 9 . 5 ( a bis ) , a nd Section 208 a nd
Annex C of the Adminis tra tiv e Ins tructions ) .
TABLE 1
Summa g of Gene a nd Protein SEQ ID N umbers
SEQ ID N O: SEQ ID N O:
Des cription N ucleic a cid Peptide
Klebs iella pneumonia e budB 1 2
( a cetola cta te s y ntha s e)
E. coli ilv C ( a cetohy drox y a cid 3 4
reductois omera s e)
E. coli ilv D ( a cetohy drox y a cid 5 6
dehy dra ta s e)
La clococcus Z a cris kiv D 7 8
( bra nched-cha in ot-keto a cid
deca rbox y la s e) , codon
optimiz ed
Achromoba cler x y los ox ida ns . 9 10
buta nol dehy drogena s e
( s a dB) gene
Ba cillus s ubriZ is a ls S 3 2 3 3
( a cetola cta te s y ntha s e)
Pf 5 . llv C-Z 4 B8 ( KARI) 3 6 3 7
S. cerev is ia e ILV5 4 0 4 1
( a cetohy drox y a cid
reductois omera s e; KARI)
B. s ubriZ is ketois ov a lera te 4 3 4 4
deca rbox y la s e ( Kiv D) codon
optimiz ed
Hors e liv er a lcohol 4 5 4 6
dehy drogena s e ( HADH)
codon optimiz ed
Streptococcus mula ns ilv D 5 8 5 9
a cetohy drox y a cid
dehy dra ta s e
[ 004 8] SEQ ID N Os : 11-22 a re the nucleotide s eq uences of
the primers us ed to cons truct the recombina nt Es cherichia
coli s tra in des cribed in the Genera l Methods s ection of the
Ex a mples herein beloW.
[ 004 9 ] SEQ ID N O: 23 is the nucleotide s eq uence of the
p?B gene f rom Es cherichia coli s tra in K-12 MG16 5 5 .
[ 005 0] SEQ ID N O: 24 is the nucleotide s eq uence of the
IdhA gene f rom Es cherichia coli s tra in K-12 MG16 5 5 .
a dhE gene f rom Es cherichia coli s tra in K-12 MG16 5 5 .
Sep. 2, 2010
f rdA gene f rom Es cherichia coli s tra in K-12 MG16 5 5 .
f rdB gene f rom Es cherichia coli s tra in K-12 MG16 5 5 .
f rdC gene f rom Es cherichia coli s tra in K-12 MG16 5 5 .
f rdD gene f rom Es cherichia coli s tra in K-12 MG16 5 5 .
[ 005 6 ] SEQ ID N O; 3 0 is the nucleotide s eq uence of
pLH4 7 5 -Z 4 B8.
[ 005 7 ] SEQ ID N O; 3 1 is the nucleotide s eq uence of the
CUP1 promoter.
[ 005 8] SEQ ID N O; 3 4 is the nucleotide s eq uence of the
CYCl termina tor.
ILV5 promoter.
ILV5 termina tor.
FBAl promoter.
[ 006 2] SEQ ID N O; 4 2 is the nucleotide s eq uence of
pLH4 6 8.
[ 006 3 ] SEQ ID N O; 4 7 is the nucleotide s eq uence of pN Y8.
GPD1 promoter.
[ 006 5 ] SEQ ID N Os z 4 9 , 5 0, 5 4 , 5 5 , 6 2-7 1, 7 3 -83 a nd 85 -86
a re the nucleotide s eq uences of primers us ed in the ex a mples .
[ 006 6 ] SEQ ID N O; 5 1 is the nucleotide s eq uence of
pRS4 25 z z GPM-s a dB.
GPM1 promoter.
[ 006 8] SEQ ID N O: 5 3 is the nucleotide s eq uence of the
ADH1 termina tor.
[ 006 9 ] SEQ ID N O: 5 6 is the nucleotide s eq uence of
pRS4 23 FBA ilv D( Strep) .
FBA termina tor.
GPM-s a dB-ADHt s egment.
[ 007 2] SEQ ID N O16 1 is the nucleotide s eq uence of
pUC19 -URA3 r.
[ 007 3 ] SEQ ID N O: 7 2 is the nucleotide s eq uence of the
ilv D-FBAlt s egment.
URA3 r2 templa te DN A.
DETAILED DESCRIPTION
[ 007 5 ] As us ed a bov e a nd throughout the des cription of the
inv ention, the f olloWing terms , unles s otherWis e indica ted,
s ha ll be de?ned a s f olloWs :
[ 007 6 ] The term buta nol a s us ed herein, ref ers to 1-bu
ta nol, 2-buta nol, is obuta nol, or mix tures thereof .
[ 007 7 ] The term a erobic conditions a s us ed herein,
mea ns groWth conditions in the pres ence of ox y gen.
[ 007 8] The term microa erobic conditions a s us ed herein,
mea ns groWth conditions With loW lev els of ox y gen ( i. e. ,
beloW norma l a tmos pheric ox y gen lev els ) .
[ 007 9 ] The term a na erobic conditions a s us ed herein,
mea ns groWth conditions in the a bs ence of ox y gen.
[ 0080] The term f ermenta ble ca rbon s ource a s us ed
herein, ref ers to a ca rbon s ource ca pa ble of being meta boliz ed
by the microorga nis ms dis clos ed herein. Suita ble f erment
a ble ca rbon s ources include, but a re not limited to, monos a c
cha rides , s uch a s glucos e or f ructos e; dis a ccha rides , s uch a s
US 2010/0221802 A1
la ctos e or s ucros e; oligos a ccha rides ; poly s a ccha rides , s uch
a s s ta rch or cellulos e; one ca rbon s ubs tra tes ; a nd mix tures
thereof .
[ 0081] The term ex tra cta nt a s us ed herein ref ers to
orga nic s olv ent us ed to ex tra ct a ny buta nol is omer.
[ 0082] The term bipha s ic f ermenta tion medium a s us ed
herein, ref ers to a tWo-pha s e grow th medium compris ing a
f ermenta tion medium ( i. e. , the a q ueous pha s e) a nd a s uita ble
a mount of a Wa ter immis cible orga nic ex tra cta nt.
[ 0083 ] The term buta nol bios y nthetic pa thWa y a s us ed
herein, ref ers to a n enz y me pa thWa y to produce l-buta nol,
2-buta nol, or is obuta nol.
[ 0084 ] The term l-buta nol bios y nthetic pa thWa y a s us ed
herein, ref ers to a n enZ y me pa thWa y to produce l-buta nol
f rom a cety l-coenZ y me A ( a cety l-CoA) .
[ 0085 ] The term 2-buta nol bios y nthetic pa thWa y a s us ed
herein, ref ers to a n enZ y me pa thWa y to produce 2-buta nol
f rom py ruv a te.
[ 0086 ] The term is obuta nol bios y nthetic pa thWa y a s us ed
herein, ref ers to a n enZ y me pa thWa y to produce is obuta nol
f rom py ruv a te.
[ 0087 ] The term f a tty a cid a s us ed herein, ref ers to a
ca rbox y lic a cid ha v ing a long, a lipha tic cha in ( i. e. , Cll to
C22) , Which is either s a tura ted or uns a tura ted.
[ 0088] The term f a tty a lcohol a s us ed herein, ref ers to a n
a lcohol ha v ing a long, a lipha tic cha in ( i. e. , C 1 1 to C22) , Which
is either s a tura ted or uns a tura ted.
[ 0089 ] The term f a tty a ldehy de a s us ed herein, ref ers to
a n a ldehy de ha v ing a long, a lipha tic cha in ( i. e. , Cll to C22) ,
Which is either s a tura ted or uns a tura ted.
[ 009 0] The term ef f ectiv e titer a s us ed herein, ref ers to
the tota l a mount of buta nol produced by f ermenta tion per liter
of f ermenta tion medium. The tota l a mount of buta nol
includes the a mount of buta nol in the f ermenta tion medium,
a nd the a mount of buta nol recov ered f rom the orga nic ex tra c
ta nt a nd f rom the ga s pha s e, if ga s s tripping is us ed.
[ 009 1] The term minima l media a s us ed herein, ref ers to
groWth media tha t conta in the minimum nutrients pos s ible f or
groWth, genera lly Without the pres ence of a mino a cids . A
minima l medium ty pica lly conta ins a f ermenta ble ca rbon
s ource a nd v a rious s a lts , Which ma y v a ry a mong microorga n
is ms a nd groWing conditions ; thes e s a lts genera lly prov ide
es s entia l elements s uch a s ma gnes ium, nitrogen, phos phorus ,
a nd s ulf ur to a lloW the microorga nis m to s y nthes iZ e proteins
a nd nucleic a cids .
[ 009 2] The term de?ned media a s us ed herein, ref ers to
groWth media tha t ha v e knoWn q ua ntities of a ll ingredients .
e. g. , a de?ned ca rbon s ource a nd nitrogen s ource, a nd tra ce
elements a nd v ita mins req uired by the microorga nis m.
[ 009 3 ] The term OD a s us ed herein, ref ers to optica l
dens ity .
[ 009 4 ] The term OD6 OO a s us ed herein, ref ers to the opti
ca l dens ity a t a Wa v elength of 6 00 nm.
[ 009 5 ] The term id a s us ed herein, ref ers to interna l dia m
eter.
[ 009 6 ] The term Aq a s us ed herein, ref ers to a q ueous
pha s e.
[ 009 7 ] The term Org a s us ed herein, ref ers to orga nic
pha s e.
[ 009 8] The term IPTG a s us ed herein, ref ers to is opropy l
[ 3 -D-thioga la ctopy ra nos ide.
[ 009 9 ] The term v v m a s us ed herein, ref ers to v olume to
v olume per minute.
Sep. 2, 2010
[ 0100] The term ATCC a s us ed herein, ref ers to the
America n Ty pe Culture Collection, Ma na s s a s , Va .
[ 0101] The term v ol mea ns v olume.
[ 0102] The term rpm mea ns rev olutions per minute.
[ 0103 ] The term s ec mea ns s econd( s ) .
[ 0104 ] The term min mea ns minute( s ) .
[ 0105 ] The term h mea ns hour( s ) .
[ 0106 ] The term uL mea ns microliter.
[ 0107 ] The term mL mea ns milliliter( s ) .
[ 0108] The term L mea ns liter( s ) .
[ 0109 ] The term mL/min mea ns milliliters per minute.
[ 0110] The term mmol mea ns millimole( s ) .
[ 0111] The term mM mea ns millimola r.
[ 0112] The term M mea ns mola r.
[ 0113 ] The term um mea ns micrometer.
[ 0114 ] The term g mea ns gra ms .
[ 0115 ] The term pg mea ns microgra m.
[ 0116 ] The term g/g mea ns gra m per gra m.
[ 0117 ] The term g/L mea ns gra ms per liter.
[ 0118] The term ug/mL mea ns microgra m per liter.
[ 0119 ] The term mg/L mea ns micgra m per liter.
[ 0120] The term mmol/min/mg mea ns millimole per
minute per milligra m.
[ 0121] The term g/L/h mea ns gra ms per liter per hour.
[ 0122] The term HPLC mea ns high pres s ure liq uid chro
ma togra phy .
[ 0123 ] The term GC mea ns ga s chroma togra phy .
Genetica lly Modi?ed Microorga nis ms
[ 0124 ] Microbia l hos ts f or buta nol production ma y be
s elected f rom ba cteria , cy a noba cteria , ?la mentous f ungi a nd
y ea s ts . The microbia l hos t us ed s hould be tolera nt to the
buta nol product produced, s o tha t the y ield is not limited by
tox icity of the product to the hos t. The s election of a microbia l
hos t f or buta nol production is des cribed in deta il beloW.
[ 0125 ] Microbes tha t a re meta bolica lly a ctiv e a t high titer
lev els of buta nol a re not Well knoWn in the a rt. Although
buta nol-tolera nt muta nts ha v e been is ola ted f rom s olv ento
genic Clos tridia , little inf orma tion is a v a ila ble concerning the
buta nol tolera nce of other potentia lly us ef ul ba cteria l s tra ins .
Mos t of the s tudies on the compa ris on of a lcohol tolera nce in
ba cteria s ugges t tha t buta nol is more tox ic tha n etha nol ( de
Ca v a lho et a l. , Micros c. Res . Tech. 6 4 : 215 -22 ( 2004 ) a nd
Ka belitZ et a l. , FEMS Microbiol. Len. 220: 223 -227 ( 2003 ) ) .
Toma s et a l. ( J. Ba cteriol. 186 : 2006 -2018 ( 2004 ) ) report tha t
the y ield of l-buta nol during f ermenta tion in Clos lridium
a celobuz y licum ma y be limited by buta nol tox icity . The pri
ma ry ef f ect of l-buta nol on Clos lridium a celobuz y licum is
dis ruption of membra ne f unctions ( Herma nn et a l. , Appl.
Env iron. Microbiol. 5 0: 123 8-124 3 ( 19 85 ) ) .
[ 0126 ] The microbia l hos ts s elected f or the production of
buta nol s hould be tolera nt to buta nol a nd s hould be a ble to
conv ert ca rbohy dra tes to buta nol us ing the introduced bio
s y nthetic pa thWa y a s des cribed beloW. The criteria f or s elec
tion of s uita ble microbia l hos ts include the f olloWing: intrin
s ic tolera nce to buta nol, high ra te of ca rbohy dra te utiliZ a tion,
a v a ila bility of genetic tools f or gene ma nipula tion, a nd the
a bility to genera te s ta ble chromos oma l a ltera tions .
[ 0127 ] Suita ble hos t s tra ins With a tolera nce f or buta nol
ma y be identi?ed by s creening ba s ed on the intrins ic toler
a nce of the s tra in. The intrins ic tolera nce of microbes to
buta nol ma y be mea s ured by determining the concentra tion
of buta nol tha t is res pons ible f or 5 0% inhibition of the groWth
ra te ( IC5 0) When groWn in a minima l medium. The IC5 0
US 2010/0221802 A1
v a lues ma y be determined us ing methods know n in the a rt.
For ex a mple, the microbes of interes t ma y be grow n in the
pres ence of v a rious a mounts of buta nol a nd the grow th ra te
monitored by mea s uring the optica l dens ity a t 6 00 na nom
eters . The doubling time ma y be ca lcula ted f rom the loga rith
mic pa rt of the grow th curv e a nd us ed a s a mea s ure of the
grow th ra te. The concentra tion of buta nol tha t produces 5 0%
inhibition of grow th ma y be determined f rom a gra ph of the
percent inhibition of grow th v ers us the buta nol concentra tion.
Pref era bly , the ho s t s tra in s hould ha v e a n IC5 0 f or buta nol of
grea ter tha n a bout 0. 5 %. More s uita ble is a hos t s tra in w ith a n
IC5 0 f or buta nol tha t is grea ter tha n a bout 1. 5 %. Pa rticula rly
s uita ble is a hos t s tra in w ith a n IC5 0 f or buta nol tha t is grea ter
tha n a bout 2. 5 %.
[ 0128] The microbia l hos t f or buta nol production s hould
a ls o utiliZ e glucos e a nd/ or other ca rbohy dra tes a t a high ra te.
Mos t microbes a re ca pa ble of utiliZ ing ca rbohy dra tes . How
ev er, certa in env ironmenta l microbes ca nnot ef ?ciently us e
ca rbohy dra tes , a nd theref ore w ould not be s uita ble hos ts .
[ 0129 ] The a bility to genetica lly modif y the hos t is es s en
tia l f or the production of a ny recombina nt microorga nis m.
Modes of gene tra ns f er technology tha t ma y be us ed include
by electropora tion, conjuga tion, tra ns duction or na tura l tra ns
f orma tion. A broa d ra nge of hos t conjuga tiv e pla s mids a nd
drug res is ta nce ma rkers a re a v a ila ble. The cloning v ectors
us ed w ith a n orga nis m a re ta ilored to the hos t orga nis m ba s ed
on the na ture of a ntibiotic res is ta nce ma rkers tha t ca n f unc
tion in tha t hos t.
[ 013 0] The microbia l hos t a ls o ma y be ma nipula ted in
order to ina ctiv a te competing pa thw a y s f or ca rbon ?ow by
ina ctiv a ting v a rious genes . This req uires the a v a ila bility of
either tra ns pos ons or chromos oma l integra tion v ectors to
direct ina ctiv a tion. Additiona lly , production hos ts tha t a re
a mena ble to chemica l muta genes is ma y undergo improv e
ments in intrins ic buta nol tolera nce through chemica l
muta genes is a nd muta nt s creening.
[ 013 1] Ba s ed on the criteria des cribed a bov e, s uita ble
microbia l hos ts f or the production of buta nol include, but a re
not limited to, members of the genera , Z y momona s , Es cheri
chia , Sa lmonella , Rh0d0c0ccus , Ps eudomona s , Ba cillus ,
La cloba cillus , Enlerococcus , Pediococcus , Alca ligenes ,
Klebs iella , Pa eniba cillus , Arlhroba cler, Cory neba clerium,
Brev iba clerium, Pichia , Ca ndida , Ha ns enula a nd Sa ccha ro
my ces . Pref erred hos ts include: Es cherichia coli, Alca li genes
eulrophus , Ba cillus lichenif ormis , Pa eniba cillus ma cera ns ,
Rh0d0c0ccus ery lhropol is , Ps eudomona s pulida , La cloba cil
lus pla nla rum, Enlerococcus f a ecium, Enlerococcus ga lli
na rium, Enlerococcus f a eca lis , Pediococcus perllos a ceus ,
Pediococcus a cidila clici, Ba cillus s ublilis a nd Sa ccha romy
ces cerev is ia e.
[ 013 2] Microorga nis ms mentioned a bov e ma y be geneti
ca lly modi?ed to conv ert f ermenta ble ca rbon s ources into
buta nol, s peci?ca lly l-buta nol, 2-buta nol, or is obuta nol,
us ing methods know n in the a rt. Pa rticula rly s uita ble micro
orga nis ms include Es cherichia La cloba cillus , a nd Sa ccha ro
my ces , w here E. coli, L. pla nla rum a nd S. cerev is ia e a re
pa rticula rly pref erred. Additiona lly , the microorga nis m ma y
be a buta nol-tolera nt s tra in of one of the microorga nis ms
lis ted a bov e tha t is is ola ted us ing the method des cribed by
Bra mucci et a l. ( copending a nd commonly ow ned US. pa tent
a pplica tion Ser. N o. 11/7 6 1 , 4 9 7 ; a nd WO 2007 /14 6 3 7 7 ) . An
ex a mple of one s uch s tra in is La cloba cillus pla nla rum s tra in
PN 05 12 ( ATCC: PTA-7 7 27 , biologica l depos it ma de Jul. 12,
2006 f or US. pa tent a pplica tion Ser. N o. 1l/7 6 1, 4 9 7 ) .
Sep. 2, 2010
[ 013 3 ] Thes e microorga nis ms ca n be genetica lly modi?ed
to conta in a l-buta nol bios y nthetic pa thw a y to produce l-bu
ta nol, a s des cribed by Dona lds on et a l. in WO 2007 /04 126 9 ,
incorpora ted herein by ref erence. For ex a mple, the microor
ga nis m ma y be genetica lly modi?ed to ex pres s a l-buta nol
bios y nthetic pa thw a y compris ing the f ollow ing enZ y me-ca ta
ly Z ed s ubs tra te to product conv ers ions :
[ 013 4 ] a ) a cety l-CoA to a cetoa cety l-CoA;
[ 013 5 ] b) a cetoa cety l-CoA to 3 -hy drox y buty ry l-CoA;
[ 013 6 ] c) 3 -hy drox y buty ry l-CoA to crotony l-CoA;
[ 013 7 ] d) crotony l-CoA to buty ry l-CoA;
[ 013 8] e) buty ry l-CoA to buty ra ldehy de; a nd
[ 013 9 ] f ) buty ra ldehy de to l-buta nol.
[ 014 0] The microorga nis ms ma y a ls o be genetica lly modi
?ed to ex pres s a 2-buta nol bios y nthetic pa thw a y to produce
2-buta nol, a s des cribed by Dona lds on et a l. in US. Pa tent
Applica tion Publica tion N os . 2007 /025 9 4 10 a nd 2007 /
029 29 27 , WO 2007 /13 05 18 a nd WO 2007 /13 05 21, a ll of
w hich a re incorpora ted herein by ref erence. For ex a mple, in
one embodiment the microorga nis m ma y be genetica lly
modi?ed to ex pres s a 2-buta nol bios y nthetic pa thw a y com
pris ing the f ollow ing enZ y me-ca ta ly Z ed s ubs tra te to product
conv ers ions :
[ 014 1] a ) py ruv a te to a lpha -a cetola cta te;
[ 014 2] b) a lpha -a cetola cta te to a cetoin;
[ 014 3 ] c) a cetoin to 2, 3 -buta nediol;
[ 014 4 ] d) 2, 3 -buta nediol to 2-buta none; a nd
[ 014 5 ] e) 2-buta none to 2-buta nol.
[ 014 6 ] The microorga nis ms ma y a ls o be genetica lly modi
?ed to ex pres s a n is obuta nol bios y nthetic pa thw a y to produce
is obuta nol, a s des cribed by Dona lds on et a l. in US. Pa tent
Applica tion Publica tion N o. 2007 /009 29 5 7 a nd WO 2007 /
05 06 7 1, both of w hich a re incorpora ted herein by ref erence.
For ex a mple, the microorga nis m ma y be genetica lly modi?ed
to conta in a n is obuta nol bios y nthetic pa thw a y compris ing the
f ollow ing enZ y me-ca ta ly Z ed s ubs tra te to product conv er
s 1ons :
[ 014 7 ] a ) py ruv a te to a cetola cta te;
[ 014 8] b) a cetola cta te to 2, 3 -dihy drox y is ov a lera te;
[ 014 9 ] c) 2, 3 -dihy drox y is ov a lera te to a -ketois ov a lera te;
[ 015 0] d) ot-ketois ov a lera te to is obuty ra ldehy de; a nd
[ 015 1] e) is obuty ra ldehy de to is obuta nol.
[ 015 2] The microorga nis m genetica lly modi?ed to be
ca pa ble of conv erting f ermenta ble ca rbon s ources into
buta nol ma y be a recombina nt Es cherichia coli s tra in tha t
compris es a n is obuta nol bios y nthetic pa thw a y , a s des cribed
a bov e, a nd deletions of the f ollow ing genes to elimina te com
peting pa thw a y s tha t limit is obuta nol production, p?B, giv en
a s SEQ ID N O: 23 , ( encoding f or py ruv a te f orrna te ly a s e) ,
IdhA, giv en a s SEQ ID N O: 24 , ( encoding f or la cta te dehy
drogena s e) , a dhE, giv en a s SEQ ID N O: 25 , ( encoding f or
a lcohol dehy drogena s e) , a nd a t lea s t one gene compris ing the
f rdABCD operon ( encoding f or f uma ra te reducta s e) , s peci?
ca lly , f rdA, giv en a s SEQ ID N O: 26 , f rdB, giv en a s SEQ ID
N O: 27 , f rdC, giv en a s SEQ ID N O: 28, a nd f rdD, giv en a s
SEQ ID N O: 29 ,
[ 015 3 ] The Es cherichia coli s tra in ma y compris e: ( a ) a n
is obuta nol bios y nthetic pa thw a y encoded by the f ollow ing
genes : budB ( giv en a s SEQ ID N O: 1) f rom Klebs iella pneu
monia e encoding a cetola cta te s y ntha s e ( giv en a s SEQ ID
N O: 2) , ilv C ( giv en a s SEQ ID N O: 3 ) f rom E. coli encoding
a cetohy drox y a cid reductois omera s e ( giv en a s SEQ ID
N O: 4 ) , ilv D ( giv en a s SEQ ID N O: 5 ) f rom E. coli encoding
a cetohy drox y a cid dehy dra ta s e ( giv en a s SEQ ID N O: 6 ) ,
US 2010/0221802 A1
kiv D ( giv en a s SEQ ID N 0: 7 ) f rom La clococcus la clis encod
ing the bra nched-cha in keto a cid deca rbox y la s e ( giv en a s
SEQ ID N O: 8) , a nd s a dB ( giv en a s SEQ ID N 0: 9 ) f rom
Achromoba cler x y los ox ida ns encoding a buta nol dehy droge
na s e ( giv en a s SEQ ID N 0: l0) ; a nd ( b) deletions of the
f ollow ing genes : p?B ( SEQ ID N 0: 23 ) , IdhA ( SEQ ID
N 0: 24 ) a dhE ( SEQ ID N 0: 25 ) , a nd f rdB ( SEQ ID N 0: 27 ) .
The enz y mes encoded by the genes of the is obuta nol bios y n
thetic pa thWa y ca ta ly Z e the s ubs tra te to product conv ers ions
f or conv erting py ruv a te to is obuta nol, a s des cribed a bov e.
Speci?ca lly , a cetola cta te s y ntha s e ca ta ly Z es the conv ers ion
of py ruv a te to a cetola cta te, a cetohy drox y a cid reductoi
s omera s e ca ta ly Z es the conv ers ion of a cetola cta te to 2, 3 -di
hy drox y is ov a lera te, a cetohy drox y a cid dehy dra ta s e ca ta
ly Z es the conv ers ion of 2, 3 -dihy drox y is ov a lera te to
ot-ketois ov a lera te, bra nched-cha in keto a cid deca rbox y la s e
ca ta ly Z es the conv ers ion of ot-ketois ov a lera te to is obuty ra l
dehy de, a nd buta nol dehy drogena s e ca ta ly Z es the conv ers ion
of is obuty ra ldehy de to is obuta nol. This recombina nt Es cheri
chia coli s tra in ca n be cons tructed us ing methods knoWn in
the a rt, a s ex empli?ed in the Genera l Methods Section of the
Ex a mples herein beloW.
[ 015 4 ] The Sa ccha romy ces cerev is ia e s tra in ma y com
pris e: a n is obuta nol bios y nthetic pa thWa y encoded by the
f olloWing genes : a ls S coding region f rom Ba cillus s ublilis
( SEQ ID N 0: 3 2) encoding a cetola cta te s y ntha s e ( SEQ ID
N 0: 3 3 ) , ILVS f rom S. cerev is ia e ( SEQ ID N 0: 4 0) encoding
a cetohy drox y a cid reductois omera s e ( KARI; SEQ ID
N 0: 4 l) a nd/or a muta nt KARI s uch a s encoded by Pf 5 . Ilv C
Z 4 B8 ( SEQ ID N 0: 3 6 ; protein SEQ ID N 0: 3 7 ) , ilv D f rom
Streptococcus muta ns ( SEQ ID N 0: 5 8) encoding a cetohy
drox y a cid dehy dra ta s e ( SEQ ID N 0: 5 9 ) , kiv D f rom Ba cillus
s ublilis ( SEQ ID N 0: 4 3 ) encoding the bra nched-cha in keto
a cid deca rbox y la s e ( SEQ ID N 0: 4 4 ) , a nd s a dB f rom Achro
moba cler x y los ox ida ns ( SEQ ID N 0: 9 ) encoding a buta nol
dehy drogena s e ( SEQ ID N 0: l0) . The enZ y mes encoded by
the genes of the is obuta nol bio s y nthetic pa thWa y ca ta ly Z e the
s ubs tra te to product conv ers ions f or conv erting py ruv a te to
is obuta nol, a s des cribed herein.
[ 015 5 ] A pref erred y ea s t s tra in ex pres s ing a n is obuta nol
pa thWa y ha s a cetola cta te s y ntha s e ( ALS) a ctiv ity in the cy to
s ol a nd ha s deletions of the endogenous py ruv a te deca rbox y
la s e ( PDC) genes a s des cribed in commonly oWned a nd co
pending US. Pa tent Applica tion N o. 6 1/05 8, 9 7 0, Which is
herein incorpora ted by ref erence. This combina tion of cy to
s olic ALS a nd reduced PDC ex pres s ion Wa s f ound to grea tly
increa s e ?ux f rom py ruv a te to a cetola cta te, Which then ?oWs
to the pa thWa y f or production of is obuta nol.
[ 015 6 ] This recombina nt Sa ccha romy ces cerev is ia e s tra in
ca n be cons tructed us ing methods knoWn in the a rt, a s ex em
pli?ed in the Genera l Methods s ection of the Ex a mples herein
beloW.
0rga nic Ex tra cta nts
[ 015 7 ] Ex tra cta nts us ef ul in the Methods des cribed herein
a re Wa ter immis cible orga nic s olv ents . Suita ble orga nic
ex tra cta nts s hould meet the criteria f or a n idea l s olv ent f or a
commercia l tWo-pha s e ex tra ctiv e f ermenta tion f or the pro
duction or recov ery of buta nol. Speci?ca lly , the ex tra cta nt
s hould ( i) be nontox ic to the buta nol-producing microorga n
is ms s uch a s , f or ex a mple, genetica lly modi?ed Es cherichia
coli, La cloba cillus pla nla rum, a nd Sa ccha romy ces cerev i
s ia e, ( ii) be s ubs ta ntia lly immis cible With the f ermenta tion
medium, ( iii) ha v e a high pa rtition coe?icient f or the ex tra c
Sep. 2, 2010
tion of buta nol, ( iv ) ha v e a loW pa rtition coe?icient f or the
ex tra ction of nutrients , ( v ) ha v e a loW tendency to f orm emul
s ions With the f ermenta tion medium, a nd ( v i) be loW cos t a nd
nonha Z a rdous . Suita ble orga nic ex tra cta nts f or us e in the
Methods dis clos ed herein a re s elected f rom the group con
s is ting of C12 to C22 f a tty a lcohols , Cl2 to C22 f a tty a cids ,
es ters of Cl2 to C22 f a tty a cids , Cl2 to C22 f a tty a ldehy des , Cl2
to C22 f a tty a mides a nd mix tures thereof . As us ed herein, the
term mix tures thereof encompa s s es both mix tures Within
a nd mix tures betWeen thes e group members , f or ex a mple
mix tures Within Cl2 to C22 f a tty a lcohols , a nd a ls o mix tures
betWeen Cl2 to C22 f a tty a lcohols a nd C12 to C22 f a tty a cids ,
f or ex a mple.
[ 015 8] In s ome ins ta nces ex tra cta nts ha v ing les s tha n
l2-ca rbon cha in lengths ca n be ha rmf ul to the microorga nis m
a nd theref ore ha rmf ul to the proces s of prov iding buta nol v ia
a bios y nthetic pa th. In the ca s e of a n 1 1 -ca rbon ex tra cta nt, the
ef f ect on a microorga nis m ca n be dependent on the condi
tions , but ca n be ha rmf ul. In the ca s e Where a Cl 1 f a tty a lco
hol, C 1 1 f a tty a cid, a n es ter of a C 1 2 f a tty a cid, a C 1 1 a ldehy de,
a nd mix tures thereof ca n be deleterious to the proces s , f or
ex a mple in the ca s e Where a microorga nis m is a dv ers ely
a f f ected by the C l 1 compound under the conditions us ed, s uch
us e is to be a v oided. Suita ble orga nic ex tra cta nts a re f urther
s elected f rom the group cons is ting of oley l a lcohol ( CAS N o.
14 3 -28-2) , beheny l a lcohol ( CAS N o. 6 6 1-19 -8) , cety l a lco
hol ( CAS N o. 3 6 6 5 3 -82-4 ) , la ury l a lcohol, a ls o ref erred to a s
l-dodeca nol ( CAS N o. 112-5 3 -8) , my ris ty l a lcohol ( 112-7 2
1) , s tea ry l a lcohol ( CAS N o. 112-9 2-5 ) , l-undeca nol ( CAS
N o. 112-4 2-5 ) , oleic a cid ( CAS N o. 112-80-1) , la uric a cid
( CAS N o. 14 3 -07 -7 ) , my ris tic a cid ( CAS N o. 5 4 4 -6 3 -8) ,
s tea ric a cid ( CAS N o. 5 7 -11-4 ) , methy l my ris ta te CAS N o.
124 -10-7 ) , methy l olea te ( CAS N o. 112-6 2-9 ) , undeca na l
( CAS N o. 112-4 4 -7 ) , la uric a ldehy de ( CAS N o. 112-5 4 -9 ) ,
2-methy lundeca na l ( CAS N o. l 10-4 1-8) , olea mide ( CAS N o.
3 01-02-0) , linolea mide ( CAS N o. 3 9 9 9 -01-7 ) , pa lmita mide
( CAS N o. 6 29 -5 4 -9 ) a nd s tea ry la mide ( CAS N o. 124 -26 -5 )
a nd mix tures thereof . Thes e orga nic ex tra cta nts a re a v a ila ble
commercia lly f rom v a rious s ources , s uch a s Sigma -Aldrich
( St. Louis , M0) , in v a rious gra des , ma ny of Which ma y be
s uita ble f or us e in ex tra ctiv e f ermenta tion to produce or
recov er buta nol. Technica l gra des conta in a mix ture of com
pounds , including the des ired component a nd higher a nd
loWer f a tty components . For ex a mple, one commercia lly
a v a ila ble technica l gra de oley l a lcohol conta ins a bout 6 5 %
oley l a lcohol a nd a mix ture of higher a nd loWer f a tty a lcohols .
[ 015 9 ] One of rea s ona ble s kill in the a rt ca n a pprecia te tha t
it ma y be a dv a nta geous to us e a mix ture of the orga nic ex tra c
ta nts . For ex a mple, s olv ent mix tures ma y be us ed to increa s e
the pa rtition coef ?cient of the product. Additiona lly , s olv ent
mix tures ma y be us ed to a djus t a nd optimiZ e phy s ica l cha r
a cteris tics of the s olv ent, s uch a s the dens ity , boiling point,
a nd v is cos ity .
Methods f or Producing Buta nol Us ing TWo-Pha s e Ex tra ctiv e
Fermenta tion
[ 016 0] The microorga nis m ma y be cultured in a s uita ble
f ermenta tion medium in a s uita ble f ermentor to produce
buta nol. Any s uita ble f ermentor ma y be us ed including a
s tirred ta nk f ermentor, a n a irlif t f ermentor, a bubble f ermen
tor, or a ny combina tion thereof . Ma teria ls a nd methods f or
the ma intena nce a nd groWth of microbia l cultures a re Well
knoWn to thos e s killed in the a rt of microbiology or f ermen
ta tion s cience ( See f or ex a mple, Ba iley et a l. , Biochemica l
US 2010/0221802 A1
Engineering Funda menta ls , s econd edition, McGra W Hill,
N eWYork, 19 86 ) . Cons idera tion mus t be giv en to a ppropria te
f ermenta tion medium, pH, tempera ture, a nd req uirements f or
a erobic, microa erobic, or a na erobic conditions , depending on
the s peci?c req uirements of the microorga nis m, the f ermen
ta tion, a nd the proces s . The f ermenta tion medium us ed is not
critica l, but it mus t s upport groWth of the microorga nis m us ed
a nd promote the bios y nthetic pa thWa y neces s a ry to produce
the des ired buta nol product. A conv entiona l f ermenta tion
medium ma y be us ed, including, but not limited to, complex
media conta ining orga nic nitrogen s ources s uch a s y ea s t
ex tra ct or peptone a nd a t lea s t one f ermenta ble ca rbon s ource;
minima l media ; a nd de?ned media . Suita ble f ermenta ble ca r
bon s ources include, but a re not limited to, monos a ccha rides ,
s uch a s glucos e or f ructos e; dis a ccha rides , s uch a s la ctos e or
s ucros e; oligos a ccha rides ; poly s a ccha rides , s uch a s s ta rch or
cellulos e; one ca rbon s ubs tra tes ; a nd mix tures thereof . In
a ddition to the a ppropria te ca rbon s ource, the f ermenta tion
medium ma y conta in a s uita ble nitrogen s ource, s uch a s a n
a mmonium s a lt, y ea s t ex tra ct or peptone, minera ls , s a lts ,
cof a ctors , buf f ers a nd other components , knoWn to thos e
s killed in the a rt ( Ba iley et a l. s upra ) . Suita ble conditions f or
the ex tra ctiv e f ermenta tion depend on the pa rticula r microor
ga nis m us ed a nd ma y be rea dily determined by one s killed in
the a rt us ing routine ex perimenta tion.
Methods f or Recov ering Buta nol Us ing TWo-Pha s e Ex tra c
tiv e Fermenta tion
[ 016 1] Buta nol ma y be recov ered f rom a f ermenta tion
medium conta ining buta nol, Wa ter, a t lea s t one f ermenta ble
ca rbon s ource, a nd a microorga nis m tha t ha s been genetica lly
modi?ed ( tha t is , genetica lly engineered) to produce buta nol
v ia a bios y nthetic pa thWa y f rom a t lea s t one ca rbon s ource.
Such genetica lly modi?ed microorga nis ms ca n be s elected
f rom the group cons is ting of Es cherichia coli, La cloba cillus
pla nla rum, a nd Sa ccha romy ces cerev is ia e. The ?rs t s tep in
the proces s is conta cting the f ermenta tion medium With a
Wa ter immis cible orga nic ex tra cta nt, des cribed a bov e, to
f orm a tWo-pha s e mix ture compris ing a n a q ueous pha s e a nd
a buta nol-conta ining orga nic pha s e. Conta cting mea ns the
f ermenta tion medium a nd the orga nic ex tra cta nt a re brought
into phy s ica l conta ct a t a ny time during the f ermenta tion
proces s . In one embodiment, the f ermenta tion medium f ur
ther compris es etha nol, a nd the buta nol-conta ining orga nic
pha s e ca n conta in etha nol.
[ 01 6 2] The orga nic ex tra cta nt ma y conta ct the f ermenta tion
medium a t the s ta rt of the f ermenta tion f orming a bipha s ic
f ermenta tion medium. Alterna tiv ely , the orga nic ex tra cta nt
ma y conta ct the f ermenta tion medium a f ter the microorga n
is m ha s a chiev ed a des ired a mount of groWth, Which ca n be
determined by mea s uring the optica l dens ity of the culture.
[ 016 3 ] Further, the orga nic ex tra cta nt ma y conta ct the f er
menta tion medium a t a time a t Which the buta nol lev el in the
f ermenta tion medium rea ches a pres elected lev el, f or
ex a mple, bef ore the buta nol concentra tion rea ches a tox ic
lev el. The buta nol concentra tion ma y be monitored during the
f ermenta tion us ing methods knoWn in the a rt, s uch a s ga s
chroma togra phy or high perf orma nce liq uid chroma togra
phy .
[ 016 4 ] Fermenta tion ma y be run under a erobic conditions
f or a time s u?icient f or the culture to a chiev e a pres elected
lev el of groWth, a s determined by optica l dens ity mea s ure
ment. An inducer ma y then be a dded to induce the ex pres s ion
of the buta nol bios y nthetic pa thWa y in the modi?ed microor
Sep. 2, 2010
ga nis m, a nd f ermenta tion conditions a re s Witched to
microa erobic or a na erobic conditions to s timula te buta nol
production, a s des cribed in deta il in Ex a mple 6 herein beloW.
The ex tra cta nt is a dded a f ter the s Witch to microa erobic or
a na erobic conditions .
[ 016 5 ] Af ter conta cting the f ermenta tion medium With the
orga nic ex tra cta nt, the buta nol product pa rtitions into the
orga nic ex tra cta nt, decrea s ing the concentra tion in the a q ue
ous pha s e conta ining the microorga nis m, thereby limiting the
ex pos ure of the production microorga nis m to the inhibitory
buta nol product. The v olume of the orga nic ex tra cta nt to be
us ed depends on a number of f a ctors , including the v olume of
the f ermenta tion medium, the s iZ e of the f ermentor, the pa r
tition coe?icient of the ex tra cta nt f or the buta nol product, a nd
the f ermenta tion mode chos en, a s des cribed beloW. The v ol
ume of the orga nic ex tra cta nt is a bout 3 % to a bout 6 0% of the
f ermentor Working v olume.
[ 016 6 ] The nex t s tep is s epa ra ting the buta nol-conta ining
orga nic pha s e f rom the a q ueous pha s e us ing methods knoWn
in the a rt, including but not limited to, s iphoning, deca nta tion,
centrif uga tion, us ing a gra v ity s ettler, membra ne-a s s is ted
pha s e s plitting, a nd the like. Recov ery of the buta nol f rom the
buta nol-conta ining orga nic pha s e ca n be done us ing methods
knoWn in the a rt, including but not limited to, dis tilla tion,
a ds orption by res ins , s epa ra tion by molecula r s iev es , per
v a pora tion, a nd the like. Speci?ca lly , dis tilla tion ma y be us ed
to recov er the buta nol f rom the buta nol-conta ining orga nic
pha s e.
[ 016 7 ] Ga s s tripping ma y be us ed concurrently With the
orga nic ex tra cta nt to remov e the buta nol product f rom the
f ermenta tion medium. Ga s s tripping ma y be done by pa s s ing
a ga s s uch a s a ir, nitrogen, or ca rbon diox ide through the
f ermenta tion medium, thereby f orming a buta nol-conta ining
ga s pha s e. The buta nol product ma y be recov ered f rom the
buta nol-conta ining ga s pha s e us ing methods knoWn in the a rt,
s uch a s us ing a chilled Wa ter tra p to condens e the buta nol, or
s crubbing the ga s pha s e With a s olv ent.
[ 016 8] Any buta nol rema ining in the f ermenta tion medium
a f ter the f ermenta tion run is completed ma y be recov ered by
continued ex tra ction us ing f res h or recy cled orga nic ex tra ct
a nt. Alterna tiv ely , the buta nol ca n be recov ered f rom the
f ermenta tion medium us ing methods knoWn in the a rt, s uch a s
dis tilla tion, a Z eotropic dis tilla tion, liq uid-liq uid ex tra ction,
a ds orption, ga s s tripping, membra ne ev a pora tion, perv a pora
tion, a nd the like.
[ 016 9 ] The tWo-pha s e ex tra ctiv e f ermenta tion method ma y
be ca rried out in a continuous mode in a s tirred ta nk f ermen
tor. In this mode, the mix ture of the f ermenta tion medium a nd
the buta nol-conta ining orga nic ex tra cta nt is remov ed f rom
the f ermentor. The tWo pha s es a re s epa ra ted by mea ns knoWn
in the a rt including, but not limited to, s iphoning, deca nta tion,
centrif uga tion, us ing a gra v ity s ettler, membra ne-a s s is ted
pha s e s plitting, a nd the like, a s des cribed a bov e. Af ter s epa
ra tion, the f ermenta tion medium ma y be recy cled to the f er
mentor or ma y be repla ced With f res h medium. Then, the
ex tra cta nt is trea ted to recov er the buta nol product a s
des cribed a bov e. The ex tra cta nt ma y then be recy cled ba ck
into the f ermentor f or f urther ex tra ction of the product. Alter
na tiv ely , f res h ex tra cta nt ma y be continuous ly a dded to the
f ermentor to repla ce the remov ed ex tra cta nt. This continuous
mode of opera tion of f ers s ev era l a dv a nta ges . Beca us e the
product is continua lly remov ed f rom the rea ctor, a s ma ller
v olume of orga nic ex tra cta nt is req uired ena bling a la rger
v olume of the f ermenta tion medium to be us ed. This res ults in
US 2010/0221802 A1
higher production y ields . The v olume of the orga nic ex tra ct
a nt ma y be a bout 3 % to a bout 5 0% of the f ermentor Working
v olume; 3 % to a bout 20% of the f ermentor Working v olume;
or 3 % to a bout 10% of the f ermentor Working v olume. It is
bene?cia l to us e the s ma lles t a mount of ex tra cta nt in the
f ermentor a s pos s ible to ma x imiz e the v olume of the a q ueous
pha s e, a nd theref ore, the a mount of cells in the f ermentor. The
proces s ma y be opera ted in a n entirely continuous mode in
Which the ex tra cta nt is continuous ly recy cled betWeen the
f ermentor a nd a s epa ra tion a ppa ra tus a nd the f ermenta tion
medium is continuous ly remov ed f rom the f ermentor a nd
replenis hed With f res h medium. In this entirely continuous
mode, the buta nol product is not a lloWed to rea ch the critica l
tox ic concentra tion a nd f res h nutrients a re continuous ly pro
v ided s o tha t the f ermenta tion ma y be ca rried out f or long
periods of time. The a ppa ra tus tha t ma y be us ed to ca rry out
thes e modes of tWo-pha s e ex tra ctiv e f ermenta tions a re Well
knoWn in the a rt. Ex a mples a re des cribed, f or ex a mple, by
Kollerup et a l. in Us . Pa t. N o. 4 , 86 5 , 9 7 3 .
[ 017 0] Ba tchWis e f ermenta tion mode ma y a ls o be us ed.
Ba tch f ermenta tion, Which is Well knoWn in the a rt, is a clos ed
s y s tem in Which the compos ition of the f ermenta tion medium
is s et a t the beginning of the f ermenta tion a nd is not s ubjected
to a rti?cia l a ltera tions during the proces s . In this mode, a
v olume of orga nic ex tra cta nt is a dded to the f ermentor a nd the
ex tra cta nt is not remov ed during the proces s . Although this
mode is s impler tha n the continuous or the entirely continu
ous modes des cribed a bov e, it req uires a la rger v olume of
orga nic ex tra cta nt to minimiZ e the concentra tion of the
inhibitory buta nol product in the f ermenta tion medium. Con
s eq uently , the v olume of the f ermenta tion medium is les s a nd
the a mount of product produced is les s tha n tha t obta ined
us ing the continuous mode. The v olume of the orga nic s ol
v ent in the ba tchWis e mode ma y be 20% to a bout 6 0% of the
f ermentor Working v olume; or 3 0% to a bout 6 0% of the
f ermentor Working v olume. It is bene?cia l to us e the s ma lles t
v olume of ex tra cta nt in the f ermentor a s pos s ible, f or the
rea s on des cribed a bov e.
[ 017 1] Fed-ba tch f ermenta tion mode ma y a ls o be us ed.
Fed-ba tch f ermenta tion is a v a ria tion of the s ta nda rd ba tch
s y s tem, in Which the nutrients , f or ex a mple glucos e, a re
a dded in increments during the f ermenta tion. The a mount a nd
the ra te of a ddition of the nutrient ma y be determined by
routine ex perimenta tion. For ex a mple, the concentra tion of
critica l nutrients in the f ermenta tion medium ma y be moni
tored during the f ermenta tion. Alterna tiv ely , more ea s ily
mea s ured f a ctors s uch a s pH, dis s olv ed ox y gen, a nd the pa r
tia l pres s ure of Wa s te ga s es , s uch a s ca rbon diox ide, ma y be
monitored. From thes e mea s ured pa ra meters , the ra te of
nutrient a ddition ma y be determined. The a mount of orga nic
s olv ent us ed in this mode is the s a me a s tha t us ed in the
ba tch-Wis e mode, des cribed a bov e.
[ 017 2] Ex tra ction of the product ma y be done doWns trea m
of the f ermentor, ra ther tha n in s itu. In this ex terna l mode, the
ex tra ction of the buta nol product into the orga nic ex tra cta nt is
ca rried out on the f ermenta tion medium remov ed f rom the
f ermentor. The a mount of orga nic s olv ent us ed is a bout 20%
to a bout 6 0% of the f ermentor Working v olume; or 3 0% to
a bout 6 0% of the f ermentor Working v olume. The f ermenta
tion medium ma y be remov ed f rom the f ermentor continu
ous ly or periodica lly , a nd the ex tra ction of the buta nol prod
uct by the orga nic ex tra cta nt ma y be done With or Without the
remov a l of the cells f rom the f ermenta tion medium. The cells
ma y be remov ed f rom the f ermenta tion medium by mea ns
Sep. 2, 2010
knoWn in the a rt including, but not limited to, ?ltra tion or
centrif uga tion. Af ter s epa ra tion of the f ermenta tion medium
f rom the ex tra cta nt by mea ns des cribed a bov e, the f ermenta
tion medium ma y be recy cled into the f ermentor, dis ca rded,
or trea ted f or the remov a l of a ny rema ining buta nol product.
Simila rly , the is ola ted cells ma y a ls o be recy cled into the
f ermentor. Af ter trea tment to recov er the buta nol product, the
ex tra cta nt ma y be recy cled f or us e in the ex tra ction proces s .
Alterna tiv ely , f res h ex tra cta nt ma y be us ed. In this mode the
s olv ent is not pres ent in the f ermentor, s o the tox icity of the
s olv ent is much les s of a problem. If the cells a re s epa ra ted
f rom the f ermenta tion medium bef ore conta cting With the
s olv ent, the problem of s olv ent tox icity is f urther reduced.
Furthermore, us ing this ex terna l mode there is les s cha nce of
f orming a n emuls ion a nd ev a pora tion of the s olv ent is mini
miZ ed, a llev ia ting env ironmenta l concerns .
[ 017 3 ] A method f or the production of buta nol is prov ided,
Wherein a microorga nis m tha t ha s been genetica lly modi?ed
of being ca pa ble of conv erting a t lea s t one f ermenta ble ca r
bon s ource into buta nol, is groWn in a bipha s ic f ermenta tion
medium. The bipha s ic f ermenta tion medium compris es a n
a q ueous pha s e a nd a Wa ter immis cible orga nic ex tra cta nt, a s
des cribed a bov e, Wherein the bipha s ic f ermenta tion medium
compris es f rom a bout 3 % to a bout 6 0% by v olume of the
orga nic ex tra cta nt. The microorga nis m ma y be groWn in the
bipha s ic f ermenta tion medium f or a time s u?icient to ex tra ct
buta nol into the ex tra cta nt to f orm a buta nol-conta ining
orga nic pha s e. In the ca s e Where the f ermenta tion medium
f urther compris es etha nol, the buta nol-conta ining orga nic
pha s e ma y conta in etha nol. The buta nol-conta ining orga nic
pha s e is then s epa ra ted f rom the a q ueous pha s e, a s des cribed
a bov e. Subs eq uently , the buta nol is recov ered f rom the
buta nol-conta ining orga nic pha s e, a s des cribed a bov e.
[ 017 4 ] Is obuta nol ma y be produced by ex tra ctiv e f ermen
ta tion With the us e of a modi?ed Es cherichia coli or Sa ccha
romy ces cerev is ia e s tra in in combina tion With oley l a lcohol
a s the orga nic ex tra cta nt. Us ing the method des cribed herein
prov ides a high ef f ectiv e titer f or is obuta nol. Ats umi et a l.
( N a ture 4 5 1( 3 ) : 86 -9 0, 2008) report is obuta nol titers up to 22
g/L us ing f ermenta tion With a n Es cherichia coli tha t Wa s
genetica lly modi?ed to conta in a n is obuta nol bios y nthetic
pa thWa y . Buta nol produced by the method dis clos ed herein
ha s a n ef f ectiv e titer of grea ter tha n 22 g per liter of the
f ermenta tion medium. Alterna tiv ely , the buta nol produced by
methods dis clos ed ha s a n ef f ectiv e titer of a t lea s t 25 g per
liter of the f ermenta tion medium. Alterna tiv ely , the buta nol
produced by methods des cribed herein ha s a n ef f ectiv e titer of
a t lea s t 3 0 g per liter of the f ermenta tion medium. Altema
tiv ely , the buta nol produced by methods des cribed herein ha s
a n ef f ectiv e titer of a t lea s t 3 7 g per liter of the f ermenta tion
medium.
[ 017 5 ] Without being held to theory , it is believ ed tha t the
higher buta nol titer obta ined With the ex tra ctiv e f ermenta tion
method dis clos ed herein res ults , in pa rt, f rom the remov a l of
the tox ic buta nol product f rom the f ermenta tion medium,
thereby keeping the lev el beloW tha t Which is tox ic to the
microorga nis m.
[ 017 6 ] The us e of the orga nic ex tra cta nt oley l a lcohol ha s
a n a dditiona l bene?cia l ef f ect tha t is s urpris ing a nd not Well
unders tood a t the time of pres enting this inv ention. Speci?
ca lly , the us e of oley l a lcohol a s the ex tra cta nt in combina tion
With ga s s tripping prov ides s igni?ca ntly higher titers tha n ga s
s tripping a lone, ev en though ga s s tripping a lone is ef f ectiv e in
keeping the buta nol beloW tox ic lev els . Orga nic ex tra cta nts
US 2010/0221802 A1
compris ing or cons is ting es s entia lly of oley l a lcohol ca n pro
v ide improv ed titers in the proces s es des cribed herein.
[ 017 7 ] Ref erring noW to FIG. 3 , there is s hoWn a s chema tic
repres enta tion of one embodiment of proces s es f or producing
a nd recov ering buta nol us ing in s itu ex tra ctiv e f ermenta tion.
An a q ueous s trea m 10 of a t lea s t one f ermenta ble ca rbon
s ource is introduced into a f ermentor 20, Which conta ins a t
lea s t one microorga nis m ( not s hoWn) genetica lly modi?ed to
conv ert the a t lea s t one f ermenta ble ca rbon s ource into
buta nol. A s trea m of the ?rs t Wa ter immis cible ex tra cta nt 12
a nd a s trea m of the optiona l s econd Wa ter immis cible ex tra c
ta nt 14 a re introduced to a v es s el 16 , in Which the ex tra cta nts
a re combined to f orm the ex tra cta nt 18. A s trea m of the
ex tra cta nt 18 is introduced into the f ermentor 20, Whereby
conta ct betWeen the f ermenta tion medium a nd the ex tra cta nt
to f orm a tWo-pha s e mix ture compris ing a n a q ueous pha s e
a nd a buta nol-conta ining orga nic pha s e occurs . A s trea m 26
compris ing both the a q ueous a nd orga nic pha s es is intro
duced into a v es s el 3 8, in Which s epa ra tion of the a q ueous a nd
orga nic pha s es is perf ormed to produce a buta nol-conta ining
orga nic pha s e 4 0 a nd a n a q ueous pha s e 4 2.
[ 017 8] Ref erring noW to FIG. 4 , there is s hoWn a s chema tic
a nd recov ering buta nol us ing in s itu ex tra ctiv e f ermenta tion.
ta nt 14 a re introduced s epa ra tely to the f ermentor 20,
Whereby conta ct betWeen the f ermenta tion medium a nd the
ex tra cta nt to f orm a tWo-pha s e mix ture compris ing a n a q ue
ous pha s e a nd a buta nol-conta ining orga nic pha s e occurs . A
s trea m 26 compris ing both the a q ueous a nd orga nic pha s es is
introduced into a v es s el 3 8, in Which s epa ra tion of the a q ue
ous a nd orga nic pha s es is perf ormed to produce a buta nol
conta ining orga nic pha s e 4 0 a nd a n a q ueous pha s e 4 2.
[ 017 9 ] Ref erring noW to FIG. 5 , there is s hoWn a s chema tic
a nd recov ering buta nol us ing in s itu cx tra ctiv c f crmcnta tion.
s ource is introduced into a ?rs t f ermentor 20, Which conta ins
a t lea s t one microorga nis m ( not s hoWn) genetica lly modi?ed
to conv ert the a t lea s t one f ermenta ble ca rbon s ource into
is introduced to the f ermentor 20, a nd a s trea m 22 compris ing
a mix ture of the ?rs t s olv ent a nd the contents of f ermentor 20
is introduced into a s econd f ermentor 24 . A s trea m of the
optiona l s econd Wa ter immis cible ex tra cta nt 14 is introduced
into the s econd f ermentor 24 , Whereby conta ct betWeen the
f ermenta tion medium a nd the ex tra cta nt to f orm a tWo -pha s e
mix ture compris ing a n a q ueous pha s e a nd a buta nol-conta in
ing orga nic pha s e occurs . A s trea m 26 compris ing both the
a q ueous a nd orga nic pha s es is introduced into a v es s el 3 8, in
Which s epa ra tion of the a q ueous a nd orga nic pha s es is per
f ormed to produce a buta nol-conta ining orga nic pha s e 4 0 a nd
a n a q ueous pha s e 4 2.
[ 0180] Ref erring noW to FIG. 6 , there is s hoWn a s chema tic
a nd recov ering buta nol in Which ex tra ction of the product is
perf ormed doWns trea m of the f ermentor, ra ther tha n in s itu.
Sep. 2, 2010
ta nt 114 a re introduced to a v es s el 116 , in Which the Wa ter
immis cible ex tra cta nts a re combined to f orm the ex tra cta nt
118. At lea s t a portion, s hoWn a s s trea m 122, of the f ermen
ta tion medium in f ermentor 120 is introduced into v es s el 124 .
A s trea m of the ex tra cta nt 118 is a ls o introduced into v es s el
124 , Whereby conta ct betWeen the f ermenta tion medium a nd
the ex tra cta nt to f orm a tWo-pha s e mix ture compris ing a n
a q ueous pha s e a nd a buta nol-conta ining orga nic pha s e
occurs . A s trea m 126 compris ing both the a q ueous a nd
orga nic pha s es is introduced into a v es s el 13 8, in Which
s epa ra tion of the a q ueous a nd orga nic pha s es is perf ormed to
produce a buta nol-conta ining orga nic pha s e 14 0 a nd a n a q ue
ous pha s e 14 2.
[ 0181] Ref erring noW to FIG. 7 , there is s hoWn a s chema tic
ta nt 114 a re introduced s epa ra tely to a v es s el 124 , in Which
the Wa ter immis cible ex tra cta nts a re combined to f orm the
ex tra cta nt 118. At lea s t a portion, s hoWn a s s trea m 122, of the
f ermenta tion medium in f ermentor 120 is a ls o introduced into
v es s el 124 , Whereby conta ct betWeen the f ermenta tion
medium a nd the ex tra cta nt to f orm a tWo -pha s e mix ture com
pris ing a n a q ueous pha s e a nd a buta nol-conta ining orga nic
pha s e occurs . A s trea m 126 compris ing both the a q ueous a nd
orga nic pha s es is introduced into a v es s el 13 8, in Which
s epa ra tion of the a q ueous a nd orga nic pha s es is perf ormed to
produce a buta nol-conta ining orga nic pha s e 14 0 a nd a n a q ue
ous pha s e 14 2.
[ 0182] Ref erring noW to FIG. 8, there is s hoWn a s chema tic
is introduced to a v es s el 128, a nd a t lea s t a portion, s hoWn a s
s trea m 122, of the f ermenta tion medium in f ermentor 120 is
a ls o introduced into v es s el 128. A s trea m 13 0 compris ing a
mix ture of the ?rs t Wa ter immis cible ex tra cta nt a nd the con
tents of f ermentor 120 is introduced into a s econd v es s el 13 2.
A s trea m of the optiona l s econd Wa ter immis cible ex tra cta nt
114 is introduced into the s econd v es s el 13 2, Whereby conta ct
betWeen the f ermenta tion medium a nd the ex tra cta nt to f orm
a tWo-pha s e mix ture compris ing a n a q ueous pha s e a nd a
buta nol-conta ining orga nic pha s e occurs . A s trea m 13 4 com
pris ing both the a q ueous a nd orga nic pha s es is introduced into
a v es s el 13 8, in Which s epa ra tion of the a q ueous a nd orga nic
pha s es is perf ormed to produce a buta nol-conta ining orga nic
pha s e 14 0 a nd a n a q ueous pha s e 14 2.
[ 0183 ] The ex tra ctiv e proces s es des cribed herein ca n be run
a s ba tch proces s es or ca n be run in a continuous mode Where
US 2010/0221802 A1
f res h ex tra cta nt is a dded a nd us ed ex tra cta nt is pumped out
s uch tha t the a mount of ex tra cta nt in the f ermentor rema ins
cons ta nt during the entire f ermenta tion proces s . Such con
tinuous ex tra ction of products a nd by products f rom the f er
menta tion ca n increa s e ef f ectiv e ra te, titer a nd y ield.
[ 0184 ] In y et a nother embodiment, it is a ls o pos s ible to
opera te the liq uid-liq uid ex tra ction in a ?ex ible co-current or,
a lterna tiv ely , counter-current Wa y tha t a ccounts f or the dif
f erence in ba tch opera ting pro?les When a s eries of ba tch
f ermentors a re us ed. In this s cena rio the f ermentors a re ?lled
With f ermenta ble ma s h Which prov ides a t lea s t one f erment
a ble ca rbon s ource a nd microorga nis m in a continuous f a s h
ion one a f ter a nother f or a s long a s the pla nt is opera ting.
Ref erring to FIG. 9 , once Fermentor F100 ?lls With ma s h a nd
microorga nis m, the ma s h a nd microorga nis m f eeds a dv a nce
to Fermentor F101 a nd then to Fermentor F102 a nd then ba ck
to Fermentor P100 in a continuous loop. The f ermenta tion in
a ny one f ermentor begins once ma s h a nd microorga nis m a re
pres ent together a nd continues until the f ermenta tion is com
plete. The ma s h a nd microorga nis m ?ll time eq ua ls the num
ber of f ermentors div ided by the tota l cy cle time ( ?ll, f erment,
empty a nd clea n) . If the tota l cy cle time is 6 0 hours a nd there
a re 3 f ermentors then the ?ll time is 20 hours . If the tota l cy cle
time is 6 0 hours a nd there a re 4 f ermentors then the ?ll time is
15 hours .
[ 0185 ] Ada ptiv e co-current ex tra ction f olloWs the f ermen
ta tion pro?le a s s uming the f ermentor opera ting a t the higher
broth pha s e titer ca n utiliZ e the ex tra cting s olv ent s trea m
riches t in buta nol concentra tion a nd the f ermentor opera ting
a t the loWes t broth pha s e titer Will bene?t f rom the ex tra cting
s olv ent s trea m lea nes t in buta nol concentra tion. For ex a mple,
ref erring a ga in to FIG. 9 , cons ider the ca s e Where Fermentor
F100 is a t the s ta rt of a f ermenta tion a nd opera ting a t rela
tiv ely loW buta nol broth pha s e ( B) titer, Fermentor F101 is in
the middle of a f ermenta tion opera ting a t rela tiv ely modera te
buta nol broth pha s e titer a nd Fermentor F102 is nea r the end
of a f ermenta tion opera ting a t rela tiv ely high buta nol broth
pha s e titer. In this ca s e, lea n ex tra cting s olv ent ( S) , With
minima l or no ex tra cted buta nol, ca n be f ed to Fermentor
F100, the s olv ent out s trea m ( S' ) f rom Fermentor F100
ha v ing a n ex tra cted buta nol component ca n then be f ed to
Fermentor F101 a s its s olv ent in s trea m a nd the s olv ent out
s trea m f rom F101 ca n then e f ed to Fermentor F102 a s its
s olv ent in s trea m. The s olv ent out s trea m f rom F102 ca n then
be s ent to be proces s ed to recov er the buta nol pres ent in the
s trea m. The proces s ed s olv ent s trea m f rom Which mos t of the
buta nol is remov ed ca n be returned to the s y s tem a s lea n
ex tra cting s olv ent a nd Would be the s olv ent in f eed to Fer
mentor F100 a bov e.
[ 0186 ] As the f ermenta tions proceed in a n orderly f a s hion
the v a lv es in the ex tra cting s olv ent ma nif old ca n be repos i
tioned to f eed the lea nes t ex tra cting s olv ent to the f ermentor
opera ting a t the loWes t buta nol broth pha s e titer. For ex a mple,
a s s ume ( a ) Fermentor F102 completes its f ermenta tion a nd
ha s been reloa ded a nd f ermenta tion begins a neW, ( b) Fermen
tor F100 is in the middle of its f ermenta tion opera ting a t
modera te buta nol broth pha s e titer a nd ( c) Fermentor F101 is
nea r the end of its f ermenta tion opera ting a t rela tiv ely higher
buta nol broth pha s e titer. In this s cena rio the lea nes t ex tra ct
ing s olv ent Would f eed F102, the ex tra cting s olv ent lea v ing
F102 Would f eed Fermentor F100 a nd the ex tra cting s olv ent
lea v ing Fermentor F100 Would f eed Fermentor F101.
[ 0187 ] The a dv a nta ge of opera ting this Wa y ca n be to ma in
ta in the broth pha s e buta nol titer a s loW a s pos s ible f or a s long
Sep. 2, 2010
a s pos s ible to rea liZ e improv ements in productiv ity . Addition
a lly , it ca n be pos s ible to drop the tempera ture in the other
f ermentors tha t ha v e progres s ed f urther into f ermenta tion tha t
a re opera ting a t higher buta nol broth pha s e titers . The drop in
tempera ture ca n a lloW f or improv ed tolera nce to the higher
buta nol broth pha s e titers .
EXAMPLES
[ 0188] The pres ent inv ention is f urther de?ned in the f ol
loWing Ex a mples . It s hould be unders tood tha t thes e
Ex a mples , While indica ting pref erred embodiments of the
inv ention, a re giv en by Wa y of illus tra tion only . From the
a bov e dis cus s ion a nd thes e Ex a mples , one s killed in the a rt
ca n a s certa in the es s entia l cha ra cteris tics of this inv ention,
a nd Without depa rting f rom the s pirit a nd s cope thereof , ca n
ma ke v a rious cha nges a nd modi?ca tions of the inv ention to
a da pt it to v a rious us es a nd conditions .
[ 0189 ] All s olv ents ( tha t is , ex tra cta nts ) Were obta ined f rom
Sigma -Aldrich ( St. Louis , Mo. ) a nd Were us ed Without f ur
ther puri?ca tion. The oley l a lcohol us ed Wa s technica l gra de,
Which conta ined a mix ture of oley l a lcohol ( 6 5 %) a nd higher
a nd loWer f a tty a lcohols . The purity of the other s olv ents us ed
Wa s a s f olloWs : oleic a cid, 6 5 to 88%; octa noic a cid, 9 8%;
nona nol, 9 8%; l-dodeca nol, 9 8%; l-nona na l, 9 5 %, a nd l-de
ca nol, 9 8%. Is obuta nol Wa s obta ined f rom Sigma -Aldrich
a nd Wa s us ed Without f urther puri?ca tion.
Genera l Methods
[ 019 0] Cons truction of Recombina nt Es cherichia coli
Stra in N GCI-03 l
[ 019 1] A recombina nt Es cherichia coli s tra in compris ing
a n is obuta nol bios y nthetic pa thWa y a nd deletions of the f ol
loWing genes , p?B ( SEQ ID N Oz 23 , encoding f or py ruv a te
f orma te ly a s e) , IdhA ( SEQ ID N O: 24 , encoding f or la cta te
dehy drogena s e) , a dhE ( SEQ ID N O: 25 , encoding f or a lcohol
dehy drogena s e) , a nd f rdB ( SEQ ID N O: 27 , encoding a s ub
unit of f uma ra te reducta s e) , Wa s cons tructed a s des cribed
beloW. The genes in the is obuta nol bios y nthetic pa thWa y Were
budB f rom Klebs iella pneumonia e ( giv en a s SEQ ID N O: 1) ,
ilv C f rom Es cherichia coli ( giv en a s SEQ ID N O: 3 ) , ilv D
f rom Es cherichia coli ( giv en a s SEQ ID N O: 5 ) , kiv D f rom
La clococcus la ctis ( giv en a s SEQ ID N O: 7 ) , a nd s a dB f rom
Achromoba cler x y los ox ida ns ( giv en a s SEQ ID N O: 9 ) . The
cons truction of the recombina nt s tra in Wa s done in tWo s teps .
Firs t, a n Es cherichia coli s tra in ha v ing the a f orementioned
gene deletions Wa s cons tructed. Then, the genes encoding the
is obuta nol bios y nthetic pa thWa y Were introduced into the
s tra in.
[ 019 2] Cons truction of Recombina nt Es cherichia coli
Stra in Ha v ing Deletions of p?B, IdhA, a dhE a nd f rdB Genes
[ 019 3 ] The Keio collection of E. coli s tra ins ( Ba ba et a l. ,
Ma l. Sy s l. Bi0l. , 211-11, 2006 ) Wa s us ed f or the production of
the E. coli s tra in ha v ing the intended gene deletions , Which is
ref erred to herein a s the f our-knock out E. coli s tra in. The
Keio collection is a libra ry of s ingle gene knockouts crea ted
in s tra in E. coli BW25 113 by the method of Da ts enko a nd
Wa nner ( Da ts enko, K. A. & Wa nner, B. L. , Proc. N a tl. Aca d.
Sci, USA. 9 7 6 6 4 0-6 6 4 5 , 2000) . In the collection, ea ch
deleted gene Wa s repla ced With a FRT-?a nked ka na my cin
ma rker tha t Wa s remov a ble by Flp recombina s e. The f our
knock out E. coli s tra in Wa s cons tructed by mov ing the knock
out-ka na my cin ma rker f rom the Keio donor s tra in by P1
tra ns duction to a recipient s tra in. Af ter ea ch Pl tra ns duction

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