(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2010/0081179 A1
`
` ANTHONY et al. (43) Pub. Date: Apr. 1, 2010
`
`
`US 20100081179A1
`
`(54)
`
`INCREASED HETEROLOGOUS FE-S
`ENZYME ACTIVITY IN YEAST
`
`Related US. Application Data
`
`(75)
`
`Inventors:
`
`LARRY CAMERON ANTHONY
`Aston, PA (US); Lori Ann
`Maggio-Hall, Wilmington, DE
`(US); Steven Cary Rothman,
`Wilmington, DE (US);
`Jean-Francois Tomb, Wilmington,
`DE (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:
`
`BUTAMAX(TM) ADVANCED
`BIOFUELS LLC, Wilmington, DE
`(US)
`
`(21) Appl. No.:
`
`12/569,069
`
`(22)
`
`Filed:
`
`Sep. 29, 2009
`
`(60) Provisional application No. 61/ 100, 801, filed on Sep.
`29, 2008, provisional application No. 61/100,806,
`filed on Sep. 29’ 2008‘
`Publication Classification
`
`(51)
`
`Int. Cl'
`CUP ”16
`C12P 7/62
`C12N 1/19
`
`(200601)
`(2006.01)
`(2006. 01)
`
`(52) U.S. Cl. ...................... 435/135; 435/160; 435/254.2;
`435/25421; 435/25422; 435/254 23
`
`ABSTRACT
`(57)
`Yeast strains were engineered that have increased activity of
`heterologous proteins that require binding ofan Feis cluster
`for their activity. The yeast strains have reduced activity of an
`endogenous Feis protein. Activities of heterologous fungal
`or plant 2Fe-2S dihydroxy-acid dehydratases and Feis pro-
`panediol dehydratase reactivase were increased for increased
`production of products made using biosynthetic pathways
`including these enzymes, such as valine, isoleucine, leucine,
`pantothenic acid (vitamin B5), isobutanol, 2-butanone and
`2-butanol.
`
`0
`
`,I
`
`OH \’
`€02
`
`NHz
`
`I
`
`MHZ
`
`0
`
`NHS;
`22.214
`
`. rt
`
`H20
`
`H20 .
`1
`
`NH3;
`23' 2H
`
`2
`
`o
`
`a
`OH?
`002
`
`1»-
`
`OH
`
`‘
`”CH
`
`2e. 2H’
`
`2emug-’JWQLOH—ZJWHB
`(3/0
`H20
`HWOH
`HSCoAf
`
`OH
`
`2 e' 2 H
`+ 9
`2 6'. 2 H \_%AHS—CoA
`
`k
`O
`/\/IK / O
`S—CoA
`
`S-CoA
`
`0”
`
`BUTAMAX 1005
`
`

`

`Patent Application Publication
`
`Apr. 1, 2010 Sheet 1 of2
`
`US 2010/0081179 A1
`
`OI
`
`

`

`Patent Application Publication
`
`Apr. 1, 2010 Sheet 2 0f 2
`
`US 2010/0081179 A1
`
`7310” +
`
`figfi‘orl
`
`
`
`
`
`

`

`US 2010/0081179 A1
`
`Apr. 1,2010
`
`INCREASED HETEROLOGOUS FE-S
`ENZYME ACTIVITY IN YEAST
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application is related to and claims the benefit
`of priority to US. Provisional Application Nos. 61/100,801
`filed Sep. 29, 2008 and 61/100,806 filed Sep. 29, 2008. The
`entirety of each is hereby incorporated by reference.
`
`FIELD OF THE INVENTION
`
`[0002] The invention relates to the field of industrial micro-
`biology and the expression of proteins that require an iron-
`sulfur cluster for activity. More specifically, expression of
`heterologous Feis protein activity in yeast cells is improved
`through specific host gene inactivation.
`
`BACKGROUND OF THE INVENTION
`
`[0003] Engineering of yeast for fermentative production of
`commercial products is an active and growing field. Enzy-
`matic pathways engineered forbiosynthesis of some products
`include enzymes that require binding of an iron-sulfur (Fei
`S) cluster for activity. Dihydroxy-acid dehydratase (DHAD)
`is one example. DHAD is part of naturally occurring biosyn-
`thetic pathways producing valine, isoleucine, leucine and
`pantothenic acid (vitamin B5).
`Increased expression of
`DHAD activity is desired for enhanced microbial production
`of branched chain amino acids or of pantothenic acid. In
`addition, DHAD catalyzed conversion of 2,3-dihydroxyisov-
`alerate to ot-ketoisovalerate is a common step in the multiple
`isobutanol biosynthetic pathways that are disclosed in co-
`pending US Patent Pub No. US 20070092957 A1. Disclosed
`therein is engineering of recombinant microorganisms for
`production of isobutanol, which is useful as a fuel additive
`and whose availability may reduce the demand for petro-
`chemical fuels.
`
`[0004] Diol dehydratase provides an enzyme activity in a
`bio synthetic pathway for production of 2-butanone and 2-bu-
`tanol that is disclosed in co-pending US Patent Pub No. US
`2007-0292927A1. Disclosed in US Patent Pub No.
`
`US20090155870 is a butanediol dehydratase that is useful for
`expression in this pathway due to its coenzyme B-12 inde-
`pendence. A diol dehydratase reactivase that is an Feis
`cluster protein required for activity of the B12-independent
`butanediol dehydratase, is also disclosed in US Patent Pub
`No. US20090155870. 2-Butanone, also referred to as methyl
`ethyl ketone (MEK), is a widely used solvent, extractant and
`activator of oxidative reactions, as well as a substrate for
`chemical synthesis of 2-butanol. 2-butanol is useful as a fuel
`additive, whose availability may reduce the demand for pet-
`rochemical fuels.
`
`For improved production of compounds synthe-
`[0005]
`sized in pathways including an Feis cluster containing
`enzyme,
`it is desirable to provide a host cell capable of
`expressing high levels of this enzymatic activity in the pro-
`duction host of interest. Whereas a number of commercially
`relevant bacteria and yeast can express activity of Feis
`cluster containing proteins, this activity is at levels far below
`what is commercially useful for enhancing introduced bio-
`synthetic pathways. Consequently a need exists for the dis-
`covery of host cells capable of expressing activity of Feis
`cluster containing proteins at levels high enough to enhance
`introduced pathways that have Feis requirements. Obtain-
`
`ing high functional expression of heterologous Feis cluster
`containing enzymes is problematic due to the Feis cluster
`requirement, which involves availability and proper loading
`of the cluster into the apo-protein.
`
`SUMMARY OF THE INVENTION
`
`Provided herein are recombinant yeast host cells
`[0006]
`comprising at least one heterologous Feis cluster protein
`wherein the yeast host has reduced expression of at least one
`endogenous Feis cluster protein.
`[0007] The recombinant yeast cell may be grown under
`suitable conditions for the production of products including
`isobutanol, 2-butanol and 2-butanone.
`[0008]
`In one aspect, the recombinant yeast cell comprises
`a disruption in the gene encoding the at least one endogenous
`Feis cluster protein.
`[0009]
`In another aspect, the endogenous Feis cluster
`protein is selected from the group consisting of dihydroxy-
`acid dehydratase, isopropylmalate dehydratase, sulfite reduc-
`tase, glutamate dehyddrogenase, biotin synthase, aconitase,
`homoaconitase,
`lipoate synthase,
`ferredoxin maturation,
`NADH ubiquinone oxidoreductase, succinate dehydroge-
`nase, ubiquinol-cytochrome-c reductase, ABC protein Rli1,
`NTPase Nbp35, and hydrogenase-like protein.
`[0010]
`In another aspect, the yeast is selected from the
`group consisting of Saccharomyces, Schizosaccharomyces,
`Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia.
`[0011]
`In another aspect, the endogenous Feis protein is
`expressed in the mitochondria, and in another embodiment,
`the endogenous Feis cluster protein has an activity selected
`from the group consisting of: dihydroxy-acid dehydratase
`and isopropylmalate dehydratase activity.
`[0012]
`In another aspect, the host cell is Saccharomyces
`expressing a gene encoding a polypeptide having the amino
`acid sequence as set forth in SEQ ID NO:114.
`[0013]
`In some embodiments, the at least one heterologous
`Feis cluster protein is selected from the group consisting of
`fungal 2Fe-2S dihydroxy-acid dehydratases and plant 2Fe-2S
`dihydroxy-acid dehydratases. In one embodiment, the heter-
`ologous fungal or plant 2Fe-2S cluster dihydroxy-acid dehy-
`dratase is expressed in the cytosol. In one embodiment, the
`heterologous fungal or plant 2Fe-2S cluster dihydroxy-acid
`dehydratase is a polypeptide having an amino acid sequence
`that matches the Profile HMM of table 9 with an E value of
`
`<10"5 wherein the polypeptide additionally comprises all
`three conserved cysteines, corresponding to positions 56,
`129, and 201 in the amino acids sequences of the Streptococ-
`cus mulans DHAD enzyme corresponding to SEQ ID
`NO: 179. In one embodiment, the heterologous fungal orplant
`2Fe-2S cluster dihydroxy-acid dehydratase is a polypeptide
`having an amino acid sequence that has at least about 95%
`sequence identity to an amino acid sequence selected from
`the group consisting of SEQ ID NOs:46, 48, 50, 52, 54, 56,
`58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,
`92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,
`118, 120,122, 124, 126,128, 130, 132,134,136, 138,140,
`142, 144, 146, 148, 150 and 152. In one embodiment, the
`heterologous fungal or plant 2Fe-2S cluster dihydroxy-acid
`dehydratase is a polypeptide having an amino acid sequence
`that is at least about 90% identical to SEQ ID NO: 1 14 using
`the Clustal W method of alignment using the default param-
`eters of GAP PENALTY:10, GAP LENGTH PENALTY:0.
`1, and Gonnet 250 series ofprotein weight matrix over the full
`length of the protein sequence.
`
`

`

`US 2010/0081179 A1
`
`Apr. 1,2010
`
`In another aspect, a method for the conversion of
`[0014]
`2,3-dihydroxyisovalerate to ot-ketoisovalerate is provided,
`said method comprising:
`[0015]
`a) providing (1) a recombinant yeast host cell com-
`prising at least one heterologous gene encoding a 2Fe-2S
`dihydroxy-acid dehydratase wherein the recombinant yeast
`host cell has reduced activity of at least one endogenous
`Feis cluster protein; and (2) a source of 2,3-dihydroxyis-
`ovalerate; and
`[0016]
`b) growing the recombinant host cell of (a) with said
`source of 2,3-dihydroxyisovalerate under conditions where
`the 2,3-dihydroxyisovalerate is converted by the host cell to
`ot-ketoisovalerate.
`
`In another aspect, a method for the conversion of
`[0017]
`2,3-butanediol to 2-butanone is provided, said method com-
`prising:
`a) providing (1) a recombinant yeast host cell com-
`[0018]
`prising at least one heterologus gene encoding a Feis pro-
`panediol dehydratase reactivase wherein the recombinant
`yeast host cell has reduced activity of at least one endogenous
`Feis cluster protein; and (2) a source of 2,3-butanediol; and
`[0019]
`b) growing the recombinant host cell of (a) with said
`source of 2,3-butanediol under conditions where 2,3-butane-
`diol is converted by the hots cell to 2-butanone.
`[0020] Also provided is a method for the production of
`isobutanol comprising growing a recombinant yeast host cell
`disclosed herein under conditions wherein isobutanol is pro-
`duced.
`
`In other embodiments, the at least one heterologous
`[0021]
`Feis cluster protein has Feis propanediol dehydratase
`reactivase activity. In some embodiments, the at least one
`heterologous Feis cluster protein having Feis pro-
`panediol dehydratase reactivase activity is a propanediol dey-
`dratase reactivase having an amino acid sequence that is at
`least about 90% identical to the amino acid sequence as set
`forth in SEQ ID NO:44 using the Clustal W method of align-
`ment using the default parameters of GAP PENALTY:10,
`GAP LENGTH PENALTY:0.1, and Gonnet 250 series of
`protein weight matrix over the full length of the protein
`sequence.
`[0022]
`In some embodiments, the cell produces 2-butanol,
`and in some embodiments the cell produces 2-butanone. In
`some embodiments, the cell comprises a 2-butanol biosyn-
`thetic pathway, and in some embodiments, the cell comprises
`a 2-butanone biosynthetic pathway.
`
`BRIEF DESCRIPTION OF THE FIGURES AND
`
`SEQUENCE DESCRIPTIONS
`
`[0023] The invention can be more fully understood from
`the following detailed description, figures, and the accompa-
`nying sequence descriptions, which form a part of this appli-
`cation.
`
`FIG. 1 shows biosynthetic pathways for isobutanol
`[0024]
`production.
`[0025]
`FIG. 2 shows a biosynthetic pathway for 2-butanone
`and 2-butanol production.
`[0026] Table 9 is a table ofthe Profile HMM for dihydroxy-
`acid dehydratases based on enzymes with assayed function
`prepared as described in Example 1. Table 9 is submitted
`herewith electronically and is incorporated herein by refer-
`ence.
`
`[0027] The following sequences conform with 37 C.F.R.
`1.821-1.825 (“Requirements for Patent Applications Con-
`taining Nucleotide Sequences and/or Amino Acid Sequence
`
`Disclosuresithe Sequence Rules”) and are consistent with
`World Intellectual Property Organization (WIPO) Standard
`ST. 25 (1998) and the sequence listing requirements of the
`EPO and PCT (Rules 5 .2 and 49.5(a-bis), and Section 208 and
`Annex C of the Administrative Instructions). The symbols
`and format used for nucleotide and amino acid sequence data
`comply with the rules set forth in 37 C.F.R. §1.822.
`
`TABLE 1
`
`Inactivation target FeiS protein encoding genes
`
`Organism and gene
`
`Saccharamyces cerevisiae LEUl
`Schizasaccharamyces pambe LEUl
`Candida galbrala CBS 138 LEUl
`Candida albicans SC 5314 LEUl
`Kluyveramyces Zaclis LEUl
`Yarrawia Zipalyrica LEUl
`Pichia sripiris LEUl
`Saccharamyces cerevisiae YJM789 ILV3
`Schizasaccharamyces pambe ILV3
`Candida galbrala CBS 138 ILV3
`Candida albicans SC5314 ILV3
`Kluyveramyces Zaclis ILV3
`Yarrawia Zipalyrica ILV3
`Pichia sripiris CBS 6054 ILV3
`Saccharamyces cerevisiae ACOl
`Schizasaccharamyces pambe
`(chromosome II) ACOl
`Schizasaccharamyces pambe
`(chromosome I) ACOl
`Kluyveramyces Zaclis NRRL Y—1140 ACOl
`Candida albicans SC5314 ACOl
`Yarrawia Zipalyrica CLIB122 ACOl
`Pichia sripiris CBS 6054 ACOl
`Candida glabrala CBS 138
`(chromosome F) ACOl
`Candida glabrala CBS 138
`(chromosome D) ACOl
`Candida glabrala CBS 138
`(chromosome K) ACOl
`
`SEQ ID NO: SEQ ID NO:
`Nucleic Acid
`Peptide
`
`1
`3
`5
`7
`9
`11
`13
`11
`93
`07
`01
`13
`05
`03
`53
`55
`
`57
`
`59
`61
`63
`65
`67
`
`69
`
`71
`
`
`
`2
`4
`6
`8
`10
`12
`14
`12
`94
`08
`02
`14
`06
`04
`54
`56
`
`58
`
`60
`62
`64
`66
`68
`
`70
`
`72
`
`
`
`TABLE 2
`
`Fungal and plant 2Fe72S DHADs in addition to those in Table 1
`
`Description
`
`Chlamydamanas reinhardrii
`Oslreacaccus Zucimarinus CCE9901
`Vitis vinifera (Unnamed protein
`product: CAO71581.1)
`Vilis vinifera (CAN67446.1)
`Arabidapsis lhaZiana
`0ryza saliva (indica cultivar—group)
`Physcamilrellapalens subsp. palens
`Chaelamium glabasum CBS 148.51
`Neuraspara crassa OR74A
`Magnaparlhe grisea 70-15
`Gibberella zeae PH-l
`Aspergillus niger
`Neasarlaryafischeri NRRC 181
`(XP7001266525.1)
`
`Neasarlaryafischeri NRRC 181
`(XP7001262996.1)
`Aspergillus niger (An03g04520)
`Aspergillus niger (Anl4g03280)
`Aspergillus lerreus NIH2624
`Aspergillus CZavalus NRRL 1
`Aspergillus nidulans FGSC A4
`Aspergillus aryzae
`
`SEQ ID NO: SEQ ID NO:
`Nucleic acid
`Peptide
`
`45
`47
`49
`
`51
`53
`55
`57
`59
`61
`63
`65
`67
`69
`
`71
`
`73
`75
`77
`79
`81
`83
`
`46
`48
`50
`
`52
`54
`56
`58
`60
`62
`64
`66
`68
`70
`
`72
`
`74
`76
`78
`80
`82
`84
`
`

`

`US 2010/0081179 A1
`
`Apr. 1,2010
`
`TABLE 2-continued
`
`Fungal and plant 2Fe72S DHADs in addition to those in Table 1
`
`Description
`
`Ajellamyces capsulalus NAIHI
`Caccidiaides immiris RS
`Balryariniafuckeliana B05.10
`Phaeasphaeria nadarum SN15
`Pichia guilliermandii ATCC 6260
`Debaryamyces hansenii CBS767
`Ladderamyces elangisparus NRRL
`YB-4239
`Vanderwallazyma palyspara DSM
`70294
`Ashbya gassypii ATCC 10895
`Laccaria bicalar S238N-H82
`Caprinapsis Cinerea akayama7#130
`Cryplacaccus neafarmans var.
`neoforrnans JEC21
`Uslilaga maydis 521
`Malassezz'a glabasa CBS 7966
`Aspergillus Clavalus NRRu 1
`
`Neasarlaryafischeri NRRu 181
`(Putative)
`Aspergillus aryzae
`Aspergillus niger (An18g04160)
`Aspergillus lerreus NIH2624
`Caccidiaides immiris RS (CIMG704591)
`Paracaccidiaides brasiliensis
`Phaeasphaeria nadarum SN15
`Gibberella zeae PH-1
`Neuraspara crassa OR74A
`Caprinapsis Cinerea Okayama 7#130
`Laccaria bicalar S238N-H82
`Uslilaga maydis 521
`
`SEQ ID NO: SEQ ID NO:
`Nucleic acid
`Peptide
`
`85
`87
`89
`91
`95
`97
`99
`
`09
`
`15
`17
`19
`21
`
`23
`25
`27
`29
`
`31
`33
`35
`37
`39
`41
`43
`45
`47
`49
`51
`
`
`
`86
`88
`90
`92
`96
`98
`00
`
`10
`
`16
`18
`20
`22
`
`24
`26
`28
`30
`
`32
`34
`36
`38
`40
`42
`44
`46
`48
`50
`52
`
`
`
`TABLE 3
`
`Expression genes
`
`Description
`
`Rosebzm'a inulinivarans (RdhtA)
`Rosebzm'a inulinivarans (RdhtB)
`Bacillus subrilis (alsS)
`Vzbria Chalerae (KARI)
`Pseudamanas aeruginasa PAOI (KARI)
`Pseudamanasfluarescens PF5 (KARI)
`Achramabacler xylasaxidans (sadB)
`B 12-independent glycerol dehydratase
`from Claslridium bulyricum
`B-12 independent butanediol
`dehydratase reactivase from
`Claslridium bulyricum
`
`SEQ ID NO: SEQ ID NO:
`Nucleic acid
`Peptide
`
`15
`16
`27
`35
`37
`39
`41
`190
`
`192
`
`43
`44
`28
`36
`38
`40
`42
`191
`
`193
`
`SEQ ID NO: 17 is a synthetic rdhtAB sequence.
`[0028]
`SEQ ID NOs:18-21 and 30-33 are primers for PCR,
`[0029]
`cloning or sequencing analysis used a described in the
`Examples herein.
`[0030]
`SEQ ID NO:22 is a dual terminator sequence.
`[0031]
`SEQ ID NO:23 is the Saccharomyces cerevisiae
`ADH terminator.
`
`SEQ ID NO:24 is the Saccharomyces cerevisiae
`[0032]
`CYC1 terminator.
`
`SEQ ID NO:25 is the Saccharomyces cerevisiae
`[0033]
`FBA promoter.
`[0034]
`SEQ ID NO:26 is the Saccharomyces cerevisiae
`GPM promoter.
`
`SEQ ID NO:29 is the pNY13 vector.
`[0035]
`SEQ ID NO:34 is the Saccharomyces cerevisiae
`[0036]
`CUP1 promoter.
`[0037]
`SEQ ID NO:173 is the codon optimized coding
`region for ILV3 DHAD from Kluyveromyces laclis.
`
`TABLE 4
`
`Functionally verified DHADs used for Profile HMM
`
`Organism
`
`Nilrasamanas eurapaea ATCC 19718
`Synechacysris sp. PCC 6803
`Slreplacaccus mulans UA15 9
`Slreplacaccus lhermaphilus LMG 18311
`Ralslania melallidurans CH34
`Ralslania eulrapha JMP134
`Laclacaccus Zacl‘is subsp. cremaris
`SKI 1
`Flavabacleriumjahnsaniae UW101
`
`SEQ ID NO: SEQ ID NO:
`Nucleic acid
`Peptide
`
`174
`176
`178
`180
`182
`184
`186
`
`188
`
`175
`177
`179
`181
`183
`185
`187
`
`189
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0038] Disclosed herein is the discovery that introduced
`Feis containing proteins in yeast host cells have high activ-
`ity levels when expression of endogenous Feis containing
`proteins is inhibited or disrupted. The present
`invention
`relates to recombinant yeast cells engineered to provide
`expression of at least one heterologous protein that is an
`Feis cluster protein, and engineered for reduced expression
`of at least one endogenous Feis cluster protein. In these
`cells the activity of the heterologous Feis cluster protein is
`improved, such that there is improved production of a product
`made in a biosynthetic pathway that includes the enzyme
`activity. Examples of commercially useful products from a
`pathway including an Feis protein include valine, isoleu-
`cine, leucine, pantothenic acid, iosbutanol, 2-butanone and
`2-butanol.
`
`[0039] The following abbreviations and definitions will be
`used for the interpretation of the specification and the claims.
`[0040] As used herein, the terms “comprises,” “compris-
`ing,” “includes,” “including,” “has,” “having,” “contains” or
`“containing,” or any other variation thereof, are intended to
`cover a non-exclusive inclusion. For example, a composition,
`a mixture, process, method, article, or apparatus that com-
`prises a list ofelements is not necessarily limited to only those
`elements but may include other elements not expressly listed
`or inherent to such composition, mixture, process, method,
`article, or apparatus. Further, unless expressly stated to the
`contrary, “or” refers to an inclusive or and not to an exclusive
`or. For example, a condition A or B is satisfied by any one of
`the following: A is true (or present) and B is false (or not
`present), A is false (or not present) and B is true (or present),
`and both A and B are true (or present).
`[0041] Also, the indefinite articles “a” and “an” preceding
`an element or component of the invention are intended to be
`nonrestrictive regarding the number of instances (i.e. occur-
`rences) of the element or component. Therefore “a” or “an”
`should be read to include one or at least one, and the singular
`word form of the element or component also includes the
`plural unless the number is obviously meant to be singular.
`[0042] The term “invention” or “present invention” as used
`herein is a non-limiting term and is not intended to refer to any
`
`

`

`US 2010/0081179 A1
`
`Apr. 1,2010
`
`single embodiment of the particular invention but encom-
`passes all possible embodiments as described in the specifi-
`cation and the claims.
`
`[0043] As used herein, the term “about” modifying the
`quantity of an ingredient or reactant of the invention
`employed refers to variation in the numerical quantity that
`can occur, for example, through typical measuring and liquid
`handling procedures used for making concentrates or use
`solutions in the real world; through inadvertent error in these
`procedures; through differences in the manufacture, source,
`or purity of the ingredients employed to make the composi-
`tions or carry out the methods; and the like. The term “about”
`also encompasses amounts that differ due to different equi-
`librium conditions for a composition resulting from a particu-
`lar initial mixture. Whether or not modified by the term
`“about”, the claims include equivalents to the quantities. In
`one embodiment, the term “about” means within 10% of the
`reported numerical value, preferably within 5% of the
`reported numerical value
`[0044] The term “Feis cluster protein” is a protein that
`binds an iron-sulfur cluster and requires the binding of the
`cluster for its activity.
`to DHAD
`[0045] The term “2Fe-2S DHAD” refers
`enzymes requiring a bound [2Fe-2S]2+ cluster for activity.
`[0046] The term “Feis propanediol dehydratase reacti-
`vase” refers to propanediol dehydratase reactivases requiring
`a bound Feis cluster for activity.
`[0047] The term “isobutanol biosynthetic pathway” refers
`to an enzyme pathway to produce isobutanol from pyruvate.
`[0048] The term “2-butanol biosynthetic pathway” refers to
`an enzyme pathway to produce 2-butanol from pyruvate.
`[0049] The term “2-butanone biosynthetic pathway” refers
`to an enzyme pathway to produce 2-butanone from pyruvate.
`[0050] There term “Dihydroxy-acid dehydratase”, also
`abbreviated DHAD, will refer to an enzyme that converts
`2,3-dihydroxyisovalerate to ot-ketoisovalerate.
`[0051] The term “butanediol dehydratase”, also known as
`“diol dehydratase” or “propanediol dehydratase” refers to a
`polypeptide (or polypeptides) having an enzyme activity that
`catalyzes the conversion of 2,3-butanediol to 2-butanone.
`Butanediol dehydratases that do not utilize the cofactor
`adenosyl cobalamin (also known as coenzyme B12, or vita-
`min B12; although vitamin B12 may refer also to other forms
`of cobalamin that are not coenzyme B12) are coenzyme B12-
`independent diol dehydratases that require association with a
`diol dehydratase reactivase that is a Feis cluster protein.
`Examples of B12-independent diol dehydratases include
`those from Closlridium glycolicum (Hartmanis et al. (1986)
`Arch. Biochem. Biophys. 245:144-152), Closlridium buzyri—
`cum (protein SEQ ID NO:191; coding region SEQ ID
`NO: 190; O’Brien et al. (2004) Biochemistry 43:4635-4645),
`and Roseburia inulinivorans (coding: SEQ ID NO:15; pro-
`tein: SEQ ID NO:43; disclosed in co-pending US Patent Pub
`No. US20090155870.
`
`[0052] The term “propanediol dehydratase reactivase”,
`also known as “diol dehydratase reactivase” or “butanediol
`dehydratase reactivase” refers to a reactivating factor for diol
`dehydratase, an enzyme which undergoes suicide inactiva-
`tion during catalysis. Diol dehydratase reactivases associated
`with coenzyme B12-independent diol dehydratases may be
`Feis cluster proteins. Examples
`include those from
`Closlridium glycolicum (Hartmanis et al. (1986) Arch. Bio-
`chem. Biophys. 245:144-152), Closlridium buzyricum (pro-
`tein SEQ ID NO:193; coding region SEQ ID NO:192;
`
`O’Brien et al. (2004) Biochemistry 43 :4635-4645), and Rose—
`buria inulinivorans (coding: SEQ ID NO: 16; protein: SEQ ID
`NO:44; disclosed in commonly owned and co-pending US
`Patent Pub No. US20090155870).
`[0053] The term “reduced expression” as it applies to the
`expression of a protein in a cell host will include those situ-
`ations where the activity of the protein is diminished as com-
`pared with a wildtype form (as with antisense technology for
`example) or substantially eliminated as with gene disruption,
`deletion or inactivation for example.
`[0054] The term “carbon substrate” or “fermentable carbon
`substrate” refers to a carbon source capable of being metabo-
`lized by host organisms of the present invention and particu-
`larly carbon sources selected from the group consisting of
`monosaccharides, oligosaccharides, polysaccharides, and
`one-carbon substrates or mixtures thereof.
`
`[0055] The term “gene” refers to a nucleic acid fragment
`that is capable of being expressed as a specific protein,
`optionally including regulatory sequences preceding (5' non-
`coding sequences) and following (3' non-coding sequences)
`the coding sequence. “Native gene” refers to a gene as found
`in nature with its own regulatory sequences. “Chimeric gene”
`refers to any gene that is not a native gene, comprising regu-
`latory and coding sequences that are not found together in
`nature. Accordingly, a chimeric gene may comprise regula-
`tory sequences and coding sequences that are derived from
`different sources, or
`regulatory sequences and coding
`sequences derived from the same source, but arranged in a
`manner different than that found in nature. “Endogenous
`gene” refers to a native gene in its natural location in the
`genome of an organism. A “foreign gene” or “heterologous
`gene” refers to a gene not normally found in the host organ-
`ism, but that is introduced into the host organism by gene
`transfer. “Heterologous gene” includes a native coding
`region, or portion thereof, that is reintroduced into the source
`organism in a form that is different from the corresponding
`native gene. For example, a heterologous gene may include a
`native coding region that is a portion of a chimeric gene
`including non-native regulatory regions that is reintroduced
`into the native host. Also a foreign gene can comprise native
`genes inserted into a non-native organism, or chimeric genes.
`A “transgene” is a gene that has been introduced into the
`genome by a transformation procedure.
`[0056] As used herein the term “coding region” refers to a
`DNA sequence that codes for a specific amino acid sequence.
`“Suitable
`regulatory
`sequences”
`refer
`to
`nucleotide
`sequences located upstream (5' non-coding sequences),
`within, or downstream (3' non-coding sequences) of a coding
`sequence, and which influence the transcription, RNA pro-
`cessing or stability, or translation of the associated coding
`sequence. Regulatory sequences may include promoters,
`translation leader sequences, introns, polyadenylation recog-
`nition sequences, RNA processing site, effector binding site
`and stem-loop structure.
`[0057] The term “promoter” refers to a DNA sequence
`capable of controlling the expression of a coding sequence or
`functional RNA. In general, a coding sequence is located 3' to
`a promoter sequence. Promoters may be derived in their
`entirety from a native gene, or be composed of different
`elements derived from different promoters found in nature, or
`even comprise synthetic DNA segments. It is understood by
`those skilled in the art that different promoters may direct the
`expression of a gene in different tissues or cell types, or at
`different stages of development, or in response to different
`
`

`

`US 2010/0081179 A1
`
`Apr. 1,2010
`
`environmental or physiological conditions. Promoters which
`cause a gene to be expressed in most cell types at most times
`are commonly referred to as “constitutive promoters”. It is
`further recognized that since in most cases the exact bound-
`aries of regulatory sequences have not been completely
`defined, DNA fragments of different lengths may have iden-
`tical promoter activity.
`[0058] The term “operably linked” refers to the association
`ofnucleic acid sequences on a single nucleic acid fragment so
`that the function of one is affected by the other. For example,
`a promoter is operably linked with a coding sequence when it
`is capable of effecting the expression of that coding sequence
`(i.e., that the coding sequence is under the transcriptional
`control of the promoter). Coding sequences can be operably
`linked to regulatory sequences in sense or antisense orienta-
`tion.
`
`[0059] The term “expression”, as used herein, refers to the
`transcription and stable accumulation of sense (mRNA) or
`antisense RNA derived from the nucleic acid fragment of the
`invention. Expression may also refer to translation of mRNA
`into a polypeptide.
`[0060] As used herein the term “transformation” refers to
`the transfer of a nucleic acid fragment into a host organism,
`resulting in genetically stable inheritance. Host organisms
`containing the transformed nucleic acid fragments are
`referred to as “transgenic” or “recombinant” or “trans-
`formed” organisms.
`[0061] The terms “plasmid” and “vector” as used herein,
`refer to an extra chromosomal element often carrying genes
`which are not part of the central metabolism of the cell, and
`usually in the form of circular double-stranded DNA mol-
`ecules. Such elements may be autonomously replicating
`sequences, genome integrating sequences, phage or nucle-
`otide sequences, linear or circular, of a single- or double-
`stranded DNA or RNA, derived from any source, in which a
`number of nucleotide sequences have been joined or recom-
`bined into a unique construction which is capable of intro-
`ducing a promoter fragment and DNA sequence for a selected
`gene product along with appropriate 3' untranslated sequence
`into a cell.
`
`[0062] As used herein the term “codon degeneracy” refers
`to the nature in the genetic code permitting variation of the
`nucleotide sequence without effecting the amino acid
`sequence of an encoded polypeptide. The skilled artisan is
`well aware of the “codon-bias” exhibited by a specific host
`cell in usage of nucleotide codons to specify a given amino
`acid. Therefore, when synthesizing a gene for improved
`expression in a host cell, it is desirable to design the gene such
`that its frequency ofcodon usage approaches the frequency of
`preferred codon usage of the host cell.
`[0063] The term “codon-optimized” as it refers to genes or
`coding regions of nucleic acid molecules for transformation
`of various hosts, refers to the alteration of codons in the gene
`or coding regions of the nucleic acid molecules to reflect the
`typical codon usage of the host organism without altering the
`polypeptide encoded by the DNA.
`[0064] As used herein, an “isolated nucleic acid fragment”
`or “isolated nucleic acid molecule” will be used interchange-
`ably and will mean a polymer of RNA or DNA that is single-
`or double-stranded, optionally containing synthetic, non-
`natural or altered nucleotide bases. An isolated nucleic acid
`
`fragment in the form of a polymer of DNA may be comprised
`of one or more segments of cDNA, genomic DNA or syn-
`thetic DNA.
`
`[0065] A nucleic acid fragment is “hybridizable” to another
`nucleic acid fragment, such as a cDNA, genomic DNA, or
`RNA molecule, when a single-stranded form of the nucleic
`acid fragment can anneal to the other nucleic acid fragment
`under the appropriate conditions of temperature and solution
`ionic strength. Hybridization and washing conditions are well
`known and exemplified in Sambrook, J., Fritsch, E. F. and
`Maniatis, T. Molecular Cloning: A Laboratory Manual, 2"“
`ed., Cold Spring Harbor Laboratory: Cold Spring Harbor,
`NY. (1989), particularly Chapter 11 and Table 11.1 therein
`(entirely incorporated herein by reference). The conditions of
`temperature and ionic strength determine the “stringency” of
`the hybridization. Stringency conditions can be adjusted to
`screen for moderately similar fragments (such as homologous
`sequences from distantly related organisms), to highly similar
`fragments (such as genes that duplicate functional enzymes
`from closely related organisms). Post-hybridization washes
`determine stringency conditions. One set of preferred condi-
`tions uses a series of washes starting with 6><SSC, 0.5% SDS
`at room temperature for 15 min, then repeated with 2><SSC,
`0.5% SDS at 45° C. for 30 min, and then repeated twice with
`0.2><SSC, 0.5% SDS at 50° C. for 30 min. A more preferred
`set of stringent conditions uses higher temperatures in which
`the washes are identical to those above except for the tem-
`perature of the final two 30 min washes in 0.2><SSC, 0.5%
`SDS was increased to 60° C. Another preferred set of highly
`stringent conditions uses two final washes in 0.1><SSC, 0.1%
`SDS at 65° C. An additional set of stringent conditions
`include hybridization at 0.1><SSC, 0.1% SDS, 65° C. and
`washes with 2><SSC, 0.1% SDS followed by 0.1><SSC, 0.1%
`SDS, for example.
`[0066] Hybridization requires that the two nucleic acids
`contain complementary sequences, although depending on
`the stringency of the hybridization, mismatches between
`bases are possible. The appropriate stringency for hybridizing
`nucleic acids depends on the length of the nucleic acids and
`the degree of complementation, variables well known in the
`art. The greater the degree of similarity or homology between
`two nucleotide sequences, the greater the value of Tm for
`hybrids of nucleic acids having those sequences. The relative
`stability (corresponding to higher Tm) ofnucleic acid hybrid-
`izations decreases in the following order: RNA:RNA, DNA:
`RNA, DNA:DNA.