(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2014/0038263 A1
`FLINT et al.
`(43) Pub. Date:
`Feb. 6, 2014
`
`US 2014003 8263A1
`
`(54) ACTIVITY OF FE-S CLUSTER REQUIRING
`PROTEINS
`
`(71) Applicant: ButamaX(TM) Advanced Biofuels
`LLC, (US)
`
`(72) Inventorsl Dennis FLINT, Newark, DE (US);
`Brian James Paul, Wilmington, DE
`(US); Rick W. Ye, Hockessin, DE (US)
`
`(73) Assignee: BUTAMAX(TM) ADVANCED
`BIOFUELS LLC, Wilmington, DE (U S)
`
`(21) App1_ NO; 13/837,921
`
`(22) Filed;
`
`Mar, 15, 2013
`
`Related US. Application Data
`.
`.
`.
`.
`(63) 5;? 11171123301111 of apphcanon NO' 13/029558’ ?led on
`'
`’
`'
`(60) Provisional application No. 61/305,333, ?led on Feb.
`17, 2010.
`
`Publication Classi?cation
`
`(51) Int. Cl.
`C12N 9/88
`(52) U.S. Cl.
`CPC ...................................... .. C12N 9/88 (2013.01)
`USPC ........................................................ .. 435/232
`
`(2006.01)
`
`(57)
`
`ABSTRACT
`
`_
`_
`_
`_
`_
`The present 1nvent1on is related to a recomblnant host cell, in
`particular a yeast cell, comprising a dihydroXy-acid dehy
`dratase polypeptide. The invention is also related to a recom
`binant host cell having increased speci?c activity of the dihy
`droxy-acid dehydratase polypeptide as a result of increased
`expression of the polypeptide, modulation of the FeiS clus
`ter biosynthesis of the cell, or a combination thereof. The
`present invention also includes methods of using the host
`cells, as Well as, methods for identifying polypeptides that
`increase the ?ux in an FeiS cluster biosynthesis pathWay in
`a host cell.
`
`

`
`Patent Application Publication
`
`Feb. 6, 2014 Sheet 1 0f 11
`
`US 2014/0038263 A1
`
`Figure 1A
`
`END (Strep mutans UA159)“
`
`pHR81 FBAp um (Sm)
`11382 hp
`
`FBA pmmoierf
`
`/'/
`2 micron
`
`\Defective LEUZ
`
`Figure 1
`
`p tKDeita(s)~Leu2~FBApmiivmsm)"
`8354 hp
`
`mmgm)
`
`Ascl (576:5) ./
`{3932a
`
`Defec?ve 2.5m"
`
`'
`
`'FBAp
`
`

`
`Patent Application Publication
`
`Feb. 6, 2014 Sheet 2 0f 11
`
`US 2014/0038263 A1
`
`Figure 2
`
`(ARSA
`’
`[cans
`
`Kpni (MW-LN»
`
`pRS4 1 1 —aft1 +fianking
`898.’; hp
`
`\_\ ‘Sac promoter
`Sac} (2793)
`
`AFTf
`
`

`
`Patent Application Publication
`
`Feb. 6, 2014 Sheet 3 0f 11
`
`US 2014/0038263 A1
`
`Figure 3
`
`mm
`
`m"
`
`m~~
`
`5,335:
`
`$5
`
`5,13
`
`aés
`
`sés
`4w
`Waveieragth {rim}
`
`aén
`
`m
`
`

`
`Patent Application Publication
`
`Feb. 6, 2014 Sheet 4 0f 11
`
`US 2014/0038263 A1
`
`Figure 4
`
`

`
`Patent Application Publication
`
`Feb. 6, 2014 Sheet 5 0f 11
`
`US 2014/0038263 A1
`
`+2 m 5 N X
`
`m Emma
`
`

`
`Patent Application Publication
`
`Feb. 6, 2014 Sheet 6 0f 11
`
`US 2014/0038263 A1
`
`Figure 6A’
`
`Figure é
`
`Figure 6C
`
`.2 jimii-iiysé?“ +~ mm“
`
`

`
`Patent Application Publication
`
`Feb. 6, 2014 Sheet 7 0f 11
`
`US 2014/0038263 A1
`
`

`
`Patent Application Publication
`
`Feb. 6, 2014 Sheet 8 0f 11
`
`US 2014/0038263 A1
`
`Figure 3
`
`

`
`Patent Application Publication
`
`Feb. 6, 2014 Sheet 9 0f 11
`
`US 2014/0038263 A1
`
`

`
`Patent Application Publication
`
`Feb. 6, 2014 Sheet 10 0f 11
`
`US 2014/0038263 A1
`
`

`
`Patent Application Publication
`
`Feb. 6, 2014 Sheet 11 0f 11
`
`US 2014/0038263 A1
`
`Figure 11
`
`ADH ierminatnr_
`
`iEvmLJactis)
`
`FBA promoter
`
`Bgi iElBam Hi fusion"
`
`HR81 FBA—IEVD(LE)~ADHt
`1024 7 hp
`
`APR
`pBR322 sequence
`
`pBR322 ori
`
`2 micron
`
`“um”, coding
`
`LEUZd
`
`

`
`US 2014/003 8263 A1
`
`Feb. 6, 2014
`
`ACTIVITY OF FE-S CLUSTER REQUIRING
`PROTEINS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application claims the bene?t of US. Provi
`sional Appl. No. 61/305,333, ?led Feb. 17, 2010, Which is
`incorporated by reference in its entirety.
`
`SEQUENCE LISTING INFORMATION
`
`[0002] The content of the electronically submitted
`sequence listing inASCII text ?le CL4842sequencelisting.txt
`?led With the application is incorporated herein by reference
`in its entirety.
`
`BACKGROUND OF THE INVENTION
`
`[0003] 1. Field of the Invention
`[0004] This invention relates generally to the ?elds of
`microbiology and biochemistry. Speci?cally, the present
`invention is related to a recombinant host cell, in particular a
`yeast cell, comprising a dihydroxy-acid dehydratase
`polypeptide. The invention is also related to a recombinant
`host cell having increased speci?c activity of the dihydroxy
`acid dehydratase polypeptide as a result of increased expres
`sion of the polypeptide, modulation of the FeiS cluster
`bio synthesis activity of the cell, or a combination thereof. The
`present invention also includes methods of using the host
`cells, as Well as methods for identifying polypeptides that
`increase the ?ux in an FeiS cluster biosynthesis pathWay in
`a host cell.
`[0005] 2. Background of the Invention
`[0006] Iron-sulfur (FeiS) clusters serve as cofactors or
`prosthetic groups essential for the normal function of the
`class of proteins that contain them. In the class of FeiS
`cluster containing proteins, the FeiS clusters have been
`found to play several roles. When proteins of this class are
`?rst synthesiZed by the cell, they lack the FeiS clusters
`required for their proper function and are referred to as apo
`proteins. FeiS clusters are made in a series of reactions by
`proteins involved in FeiS cluster biosynthesis and are trans
`ferred to the apo-proteins to form the functional FeiS cluster
`containing holoproteins.
`[0007] One such protein that requires FeiS clusters for
`proper function is dihydroxy-acid dehydratase (DHAD)
`(E.C. DHAD catalyZes the conversion of 2,3-dihydroxyisov
`alerate to ot-ketoisovalerate, and of 2,3-dihydroxymethylval
`erate to ot-ketomethylvalerate. The DHAD enZyme is part of
`naturally occurring biosynthetic pathWays producing the
`branched chain amino acids, (i .e., valine, isoleucine, leucine),
`and pantothenic acid (vitamin B5). DHAD catalyZed conver
`sion of 2,3-dihydroxyisovalerate to ot-ketoisovalerate is also a
`common step in the multiple isobutanol biosynthetic path
`Ways that are disclosed in US. Patent Appl. Pub. No. US
`20070092957 A1, incorporated by reference herein. Dis
`closed therein is, e. g., the engineering of recombinant micro
`organisms for the production of isobutanol.
`[0008] High levels of DHAD activity are desired for
`increased production of products from bio synthetic pathWays
`that include this enZyme activity, including, e.g., enhanced
`microbial production of branched chain amino acids, pan
`tothenic acid, and isobutanol. Isobutanol, in particular, is
`useful as a fuel additive, and its ready availability may reduce
`the demand for petrochemical fuels. HoWever, since all
`
`knoWn DHAD enZymes require a FeiS cluster for their
`function, they must be expressed in a host having the genetic
`machinery to provide the FeiS clusters required by these
`proteins. In yeast, mitochondria play an essential role in
`FeiS cluster biosynthesis. If the DHAD is to be functionally
`expressed in yeast cytosol, a system to transport the requisite
`FeiS precursor or signal from mitochondria and assemble
`the FeiS cluster on the cytosolic apoprotein is required.
`Prior to the Work of the present inventors, it Was previously
`unknown Whether yeast could provide FeiS clusters for any
`DHAD located in the cytoplasm (since native yeast DHAD is
`located in the mitochondria) and more importantly When the
`DHAD is expressed at high levels in the cytoplasm
`[0009] Under certain conditions the rate of synthesis of
`FeiS cluster requiring apo-proteins may exceed the cell’s
`ability to synthesiZe and assemble FeiS clusters for them.
`Cluster-less apo-proteins that accumulate under these condi
`tions cannot carry out their normal function. Such conditions
`can include I) the expression of a heterologous FeiS cluster
`requiring protein especially in high amounts, 2) the expres
`sion of a native FeiS cluster biosynthesis protein at higher
`levels than normal, or 3) a state Where the host cell’ s ability to
`synthesiZe FeiS clusters is debilitated.
`
`BRIEF SUMMARY OF THE INVENTION
`
`[0010] Disclosed herein is the surprising discovery that
`recombinant host cells expressing a high level of a heterolo
`gous FeiS cluster requiring protein can supply the comple
`ment of FeiS clusters for that protein if the level(s) of at least
`one Fe uptake, utiliZation, and/or FeiS cluster biosynthesis
`protein are altered.
`[001 1] Provided herein are recombinant ho st cells compris
`ing at least one heterologous polynucleotide encoding a
`polypeptide having dihydroxy-acid dehydratase activity
`Wherein said at least one heterologous polynucleotide com
`prises a high copy number plasmid or a plasmid With a copy
`number that can be regulated. Also provided are recombinant
`host cells comprising at least one heterologous polynucle
`otide encoding a polypeptide having dihydroxy-acid dehy
`dratase activity Wherein said at least one heterologous poly
`nucleotide is integrated at least once in the recombinant host
`cell DNA. Also provided are recombinant host cells compris
`ing at least one heterologous polynucleotide encoding a
`polypeptide having dihydroxy-acid dehydratase activity,
`Wherein said host cell comprises at least one deletion, muta
`tion, and/or substitution in an endogenous gene encoding a
`polypeptide affecting iron metabolism or FeiS cluster bio
`synthesis. Also provided are recombinant host cells compris
`ing at least one heterologous polynucleotide encoding a
`polypeptide having dihydroxy-acid dehydratase activity and
`at least one heterologous polynucleotide encoding a polypep
`tide affecting iron metabolism or FeiS cluster biosynthesis.
`[0012] In embodiments, said heterologous polynucleotide
`encoding a polypeptide affecting FeiS cluster biosynthesis
`is selected from the group consisting of the genes in Tables 7,
`8 and 9. In embodiments, said heterologous polynucleotide
`encoding a polypeptide affecting FeiS cluster biosynthesis
`is selected from the group consisting of AFT2, CCCl, FRA2,
`and GRX3, and combinations thereof. In embodiments,
`polypeptide is encoded by a polynucleotide that is constitu
`tive mutant. In embodiments, said constitutive mutant is
`selected from the group consisting of AFTl L99A, AFTl
`L102A, AFTl C291F, AFTl C293F, and combinations
`thereof. In embodiments said polypeptide affecting FeiS
`
`

`
`US 2014/003 8263 A1
`
`Feb. 6, 2014
`
`cluster biosynthesis is encoded by a polynucleotide compris
`ing a high copy number plasmid or a plasmid With a copy
`number that can be regulated. In embodiments, said polypep
`tide affecting FeiS cluster biosynthesis is encoded by a
`polynucleotide integrated at least once in the recombinant
`host cell DNA.
`[0013] In embodiments, the at least one deletion, mutation,
`and/or substitution in an endogenous gene encoding a
`polypeptide affecting FeiS cluster biosynthesis is selected
`from the group consisting of CCCl, FRA2, and GRX3, and
`combinations thereof. In embodiments, the at least one het
`erologous poly-nucleotide encoding a polypeptide affecting
`FeiS cluster biosynthesis is selected from the group con
`sisting of AFTl, AFT2, their mutants, and combinations
`thereof.
`[0014] In embodiments, said at least one heterologous
`polynucleotide encoding a polypeptide having dihydroxy
`acid dehydratase activity is expressed in multiple copies. In
`embodiments, said at least one heterologous polynucleotide
`comprises a high copy number plasmid or a plasmid With a
`copy number that can be regulated. In embodiments, said at
`least one heterologous polynucleotide is integrated at least
`once in the recombinant host cell DNA. In embodiments, said
`FeiS cluster biosynthesis is increased compared to a recom
`binant host cell having endogenous FeiS cluster biosynthe
`s1s.
`[0015] In embodiments, said host cell is a yeast host cell. In
`embodiments, said yeast host cell is selected from the group
`consisting of Saccharomyces,
`Schizosaccharomyces,
`Hansenula, Candida, Kluyveromyces, Yarrowia, Issalchenkia
`and Pichia.
`[0016] In embodiments, said heterologous polypeptide
`having dihydroxy-acid dehydratase activity is expressed in
`the cytosol of the host cell. In embodiments, said heterolo
`gous polypeptide having dihydroxy-acid dehydratase activity
`has an amino acid sequence that matches the Pro?le HMM of
`Table 12 With an E value of <10 Wherein the polypeptide
`further comprises all three conserved cysteines, correspond
`ing to positions 56, 129, and 201 in the amino acids sequences
`of the Streptococcus mutans DHAD enZyme corresponding
`to SEQ ID NO:168. In embodiments, said heterologous
`polypeptide having dihydroxy-acid dehydratase activity has
`an amino acid sequence With at least about 90% identity to
`SEQ ID NO: 168 or SEQ ID NO: 232. In embodiments said
`polypeptide having dihydroxy-acid dehydratase activity has a
`speci?c activity selected from the group consisting of: greater
`than about 5-fold With respect to the control host cell com
`prising at least one heterologous polynucleotide encoding a
`polypeptide having dihydroxy-acid dehydratase activity,
`greater than about 8-fold With respect to the control host cell
`comprising at least one heterologous polynucleotide encod
`ing a polypeptide having dihydroxy-acid dehydratase activ
`ity, or greater than about 10-fold With respect to the control
`host cell comprising at least one heterologous polynucleotide
`encoding a polypeptide having dihydroxy-acid dehydratase
`activity. In embodiments said polypeptide having dihydroxy
`acid dehydratase activity has a speci?c activity selected from
`the group consisting of: greater than about 3-fold With respect
`to a control host cell comprising at least one heterologous
`polynucleotide encoding a polypeptide having dihydroxy
`acid dehydratase activity and greater than about 6-fold With
`respect to the control host cell comprising at least one heter
`ologous polynucleotide encoding a polypeptide having dihy
`droxy-acid dehydratase activity. In embodiments, said
`
`polypeptide having dihydroxy-acid dehydratase activity has a
`speci?c activity selected from the group consisting of: greater
`than about 0.25 U/mg; greater than about 0.3 U/mg; greater
`than about 0.5 U/mg; greater than about 1.0 U/mg; greater
`than about 1.5 U/mg; greater than about 2.0 U/mg; greater
`than about 3.0 U/mg; greater than about 4.0 U/mg; greater
`than about 5.0 U/mg; greater than about 6.0 U/mg; greater
`than about 7.0 U/mg; greater than about 8.0 U/mg; greater
`than about 9.0 U/mg; greater than about 10.0 U/mg; greater
`than about 20.0 U/mg; and greater than about 50.0 U/mg.
`[0017] In embodiments said recombinant host cell pro
`duces isobutanol, and in embodiments, said recombinant ho st
`cell comprises an isobutanol biosynthetic pathWay.
`[0018] Also provided herein are methods of making a prod
`uct comprising: providing a recombinant host cell; and con
`tacting the recombinant host cell of With a fermentable carbon
`substrate in a fermentation medium under conditions Wherein
`said product is produced; Wherein the product is selected
`from the group consisting of branched chain amino acids,
`pantothenic acid, 2-methyl-!1-butanol, 3-methyl-1-butanol,
`isobutanol, and combinations thereof. In embodiments, the
`methods further comprise optionally recovering said product.
`In embodiments, the methods further comprise recovering
`said product.
`[0019] Also provided are methods of making isobutanol
`comprising: providing a recombinant host cell; contacting the
`recombinant host cell With a fermentable carbon substrate in
`a fermentation medium under conditions Wherein isobutanol
`is produced. In embodiments, the methods further comprise
`optionally recovering said isobutanol. In embodiments, the
`methods further comprise recovering said isobutanol.
`[0020] Also provided are methods for the conversion of
`2,3-dihydroxyisovalerate to ot-ketoisovalerate comprising:
`providing a recombinant host cell; groWing the recombinant
`host cell of under conditions Where the 2,3-dihydroxyisoval
`erate is converted to ot-ketoisovalerate. In embodiments, the
`conversion of 2,3-dihydroxyisovalerate to ot-ketoisovalerate
`compared to a control host cell comprising at least one het
`erologous polynucleotide encoding a polypeptide having
`dihydroxy-acid dehydratase activity is increased in an
`amount selected from the group consisting of: (a) at least
`about 5%; (b) at least about 10%; (c) at least about 15%; (d)
`at least about 20%; (e) at least about 25%; (f) at least about
`30%; (g) at least about 35%; (h) at least about 40%; (i) at least
`about 45%; (0 at least about 50%; (k) at least about 60%; (1)
`at least about 70%; (m) at least about 80%; (n) at least about
`90%; and (0) at least about 95%.
`[0021] Also provided are methods for increasing the spe
`ci?c activity of a heterologous polypeptide having dihy
`droxy-acid dehydratase activity in a recombinant host cell
`comprising: providing a recombinant host cell; and groWing
`the recombinant host cell of under conditions Whereby the
`heterologous polypeptide having dihydroxy-acid dehy
`dratase activity is expressed in functional form having a spe
`ci?c activity greater than the same host cell lacking said
`heterologous polypeptide.
`[0022] Also provided are methods for increasing the ?ux in
`an FeiS cluster biosynthesis pathWay in a host cell compris
`ing: providing a recombinant host cell; and groWing the
`recombinant host cell under conditions Whereby the ?ux in
`the FeiS cluster biosynthesis pathWay in the host cell is
`increased.
`[0023] Also provide are methods of increasing the activity
`of an FeiS cluster requiring protein in a recombinant host
`
`

`
`US 2014/003 8263 A1
`
`Feb. 6, 2014
`
`cell comprising: providing a recombinant host cell compris
`ing an FeiS cluster requiring protein; changing the expres
`sion or activity of a polypeptide affecting FeiS cluster bio
`synthesis in said host cell; and groWing the recombinant host
`cell under conditions Whereby the activity of the FeiS clus
`ter requiring protein is increased, in embodiments, said
`increase in activity is an amount selected from the group
`consisting of: greater than about 10%; greater than about
`20%; greater than about 30%; greater than about 40%; greater
`than about 50%; greater than about 60%; greater than about
`70%; greater than about 80%; greater than about 90%; and
`greater than about 95%, 98%, or 99%. In embodiments, the
`increase in activity is in an amount selected from the group
`consisting of: greater than about 5-fold; greater than about
`8-fold; greater than about 10-fold. In embodiments, the
`increase in activity is in an amount selected from the group
`consisting greater than about 3-fold and greater than about
`6-fold.
`[0024] A method for identifying polypeptides that increase
`the ?ux in an FeiS cluster biosynthesis pathWay in a host
`cell comprising: changing the expression or activity of a
`polypeptide affecting FeiS cluster biosynthesis; measuring
`the activity of a heterologous FeiS cluster requiring protein;
`and comparing the activity of the heterologous FeiS cluster
`requiring protein measured in the presence of the change in
`expression or activity of a polypeptide to the activity of the
`heterologous FeiS cluster requiring protein measured in the
`absence of the change in expression or activity of a polypep
`tide, Wherein an increase in the activity of the heterologous
`FeiS cluster requiring protein indicates an increase in the
`?ux in said FeiS cluster biosynthesis pathWay.
`[0025] Provided herein are methods for identifying
`polypeptides that increase the ?ux in an FeiS cluster bio
`synthesis pathWay in a host cell comprising: changing the
`expression or activity of a polypeptide affecting FeiS clus
`ter biosynthesis; measuring the activity of a polypeptide hav
`ing dihydroxy-acid dehydratase activity; and comparing the
`activity of the polypeptide having dihydroxy-acid dehy
`dratase activity measured in the presence of the change to the
`activity of the polypeptide having dihydroxy-acid dehy
`dratase activity measured in the absence of change, Wherein
`an increase in the activity of the polypeptide having dihy
`droxy-acid dehydratase activity indicates an increase in the
`?ux in said FeiS cluster biosynthesis pathWay.
`[0026] In embodiments, said changing the expression or
`activity of a polypeptide affecting FeiS cluster biosynthesis
`comprises deleting, mutating, substituting, expressing, up
`regulating, doWn-regulating, altering the cellular location,
`altering the state of the protein, and/or adding a cofactor. In
`embodiments, the FeiS cluster requiring protein has dihy
`droxy-acid dehydratase activity and Wherein said FeiS clus
`ter requiring protein having dihydroxy-acid dehydratase
`activity has an amino acid sequence that matches the Pro?le
`HMM of Table 12 With an P value of <10“5 Wherein the
`polypeptide further comprises all three conserved cysteines,
`corresponding to positions 56, 129, and 201 in the amino
`acids sequences of the Streptococcus mutans DHAD enZyme
`corresponding to SEQ ID NO: 168, in embodiments, the
`polypeptide affecting FeiS cluster biosynthesis is selected
`from the group consisting of the genes in Tables 7, 8 and 9.
`[0027] Also provided are recombinant host cells compris
`ing at least one polynucleotide encoding a polypeptide iden
`ti?ed by the methods provided herein. In embodiments, said
`host cell further comprises at least one heterologous poly
`
`nucleotide encoding a polypeptide having dihydroxy-acid
`dehydratase activity. In embodiments, said heterologous
`polynucleotide encoding a polypeptide having dihydroxy
`acid dehydratase activity is expressed in multiple copies. In
`embodiments, said heterologous polynucleotide comprises a
`high copy number plasmid or a plasmid With a copy number
`that can be regulated. In embodiments, said heterologous
`polynucleotide is integrated at least once in the recombinant
`host cell DNA.
`[0028] In embodiments, said host cell is a yeast host cell. In
`embodiments, said yeast host cell is selected from the group
`consisting of Saccharomyces,
`Schizosaccharomyces,
`Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchen
`kia, and Pichia. In embodiments, said heterologous polypep
`tide having dihydroxy-acid dehydratase activity is expressed
`in the cytosol of the host cell. In embodiments, said heterolo
`gous polypeptide having dihydroxy-acid dehydratase activity
`has an amino acid sequence that matches the Pro?le HMM of
`Table 12 With an E value of <10“5 Wherein the polypeptide
`further comprises all three conserved cysteines, correspond
`ing to positions 56, 129, and 201 in the amino acids sequences
`of the Streptococcus mutans MAD enZyme corresponding to
`SEQ ID NO: 168. In embodiments, said recombinant host cell
`produces a product selected from the group consisting of
`branched chain amino acids, pantothenic acid, 2-methyl-1
`butanol, 3-methyl-1-butanol, isobutanol, and combinations
`thereof. In embodiments, recombinant host cell produces
`isobutanol. In embodiments, said recombinant host cell com
`prises an isobutanol biosynthetic pathWay. In embodiments
`said isobutanol biosynthetic pathWay comprises at least one
`polypeptide encoded by a polynucleotide heterologous to the
`host cell. In embodiments, said isohutanol biosynthetic path
`Way comprises at least tWo polypeptides encoded by poly
`nucleotides heterologous to the host cell.
`[0029] In embodiments, monomers of the polypeptides of
`the invention having dihydroxy-acid dehydratase activity
`have an FeiS cluster loading selected from the group con
`sisting of: (a) at least about 10%; (b) at least about 15%; (c) at
`least about 20%; (d) at least about 25%; (e) at least about
`30%; (f) at least about 35%; (g) at least about 40%; (h) at least
`about 45%; (i) at least about 50%; (0 at least about 60%; 10%
`(k) at least about 70%; (l) at least about 80%; (m) at least
`about 9O % and (n) at least about 95%.
`
`BRIEF DESCRIPTION OF THE
`DRAWINGS/FIGURES
`
`[0030] FIG. 1A depicts a vector map of a vector tier over
`expression of the IND gene from S.
`[0031] FIG. 1B depicts a vector map of an integration vec
`tor for overexpression of the IlvD gene from S. mutans in the
`chromosome.
`[0032] FIG. 2 depicts a vector map of a centromere vector
`used to clone AFT1 or AFT1 mutants and useful for other
`genes of interest.
`[0033] FIG. 3 depicts a UV-Vrs absorbance spectrum of
`puri?ed S. mutans DHAD.
`[0034] FIG. 4 depicts an EPR spectrum of puri?ed S.
`mutans DHAD.
`[0035] FIG. 5 depicts a biosynthetic pathWay for biosyn
`thesis of isobutanol.
`[0036] FIG. 6A depicts a schematic of Azotobacter vine
`landli nif genes.
`[0037] FIG. 6B depicts a schematic of additional Azoto
`bacter vinelandli nif genes.
`
`

`
`US 2014/003 8263 A1
`
`Feb. 6, 2014
`
`[0038] FIG. 6C depicts a schematic of the equation in
`Which NFU acts as a persui?de reductase.
`[0039] FIG. 7 depicts a schematic of Helicobacler pylori nif
`genes.
`[0040] FIG. 8 depicts a schematic ofE. coli isc genes.
`[0041] FIG. 9 depicts a schematic ofE. coli sufgenes.
`[0042] FIG. 10 depicts a schematic of the cytosolic [2Fe
`2S] biosynthesis and assembly system.
`[0043] FIG. 11 depicts a vector map of a vector for overex
`pression of the IlvD gene from L. laclis.
`[0044] Table 12 is a table of the Pro?le HMM for dihy
`droxy-acid dehydratases based on enZymes With assayed
`function prepared as described in Us. patent application Ser.
`No. 12/569,636, ?led Sep. 29, 2009. Table 12 is submitted
`hereWith electronically and is incorporated herein by refer
`ence.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0045] Described herein is a methodto increase the fraction
`of the FeiS cluster requiring proteins that are loaded With
`FeiS clusters.Also described are recombinant host cells that
`express functional FeiS cluster requiring proteins, such as
`DHAD enzymes, and at least one heterologous Fe uptake,
`utiliZation, or FeiS cluster biosynthesis protein, recombi
`nant host cells that express functional DHAD enZymes and
`comprise at least one deletion, mutation, and/ or substitution
`in a native protein involved in Fe utiliZation or FeiS cluster
`biosynthesis, or recombinant host cells comprising combina
`tions thereof. In addition, the present invention describes a
`method to identify polypeptides that increase the ?ux in an
`FeiS cluster biosynthesis pathWay in a host cell. Also
`described is a method to identify polypeptides that alter the
`activity of an FeiS cluster requiring protein.
`
`DEFINITIONS
`
`[0046] Unless de?ned otherWise, all technical and scien
`ti?c terms used herein have the same meaning as commonly
`understood by one of ordinary skill in the art to Which this
`invention belongs. In case of con?ict, the present application
`including the de?nitions Will control. Also, unless otherWise
`required by context, singular terms shall include pluralities
`and plural terms shall include the singular. All publications,
`patents and other references mentioned herein are incorpo
`rated by reference in their entireties for all purposes.
`[0047] In order to further de?ne this invention, the folloW
`ing terms and de?nitions are herein provided.
`[0048] As used herein, the terms “comprises, compris
`ing,” “includes,” “including,” “has,” “having,” “contains” or
`“containing,” or any other variation thereof, Will be under
`stood to imply the inclusion of a stated integer or group of
`integers but not the exclusion of any other integer or group of
`integers. For example, a composition, a mixture, a process, a
`method, an article, or an apparatus that comprises a list of
`elements 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 appa
`ratus. 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 satis?ed 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).
`
`[0049] As used herein, the term “consists of,” or variations
`such as “consist of’ or “consisting of,” as used throughout the
`speci?cation and claims, indicate the inclusion of any recited
`integer or group of integers, but that no additional integer or
`group of integers may be added to the speci?ed method,
`structure, or composition.
`[0050] As used herein, the term “consists essentially of,” or
`variations such as “consist essentially of’ or “consisting
`essentially of,” as used throughout the speci?cation and
`claims, indicate the inclusion of any recited integer or group
`of integers, and the optional inclusion of any recited integer or
`group of integers that do not materially change the basic or
`novel properties of the speci?ed method, structure or compo
`sition. See M.P.E.P. §2111.03.
`[0051] Also, the inde?nite 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.
`[0052] The term “invention” or “present invention” as used
`herein is a non-limiting term and is not intended to refer to any
`single embodiment of the particular invention but encom
`passes all possible embodiments as described in the applica
`tion.
`[0053] 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 solu
`tions 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 to carry out the methods; and the like. The term
`“about” also encompasses amounts that differ due to different
`equilibrium conditions for a composition resulting from a
`particular initial mixture. Whether or not modi?ed 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.
`[0054] The term “isobutanol biosynthetic pathWay” refers
`to an enZyme pathWay to produce isobutanol from pyruvate.
`[0055] The term “a facultative anaerobe” refers to a micro
`organism that can groW in both aerobic and anaerobic envi
`ronments.
`[0056] 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.
`[0057] The term “FeiS cluster biosynthesis” refers to bio
`synthesis of FeiS clusters, including, e.g., the assembly and
`loading of FeiS clusters. The term “FeiS cluster biosyn
`thesis genes”, “FeiS cluster biosynthesis proteins” or
`“FeiS cluster biosynthesis pathWay” refers to those poly
`nucleotides/ genes and the encoded polypeptides that are
`involved in the biosynthesis of FeiS clusters, including, e. g.,
`the assembly and loading of FeiS clusters. The term “Fe
`uptake and utiliZation” refers to processes Which can effect
`FeiS cluster biosynthesis such as Fe sensing, uptake, utili
`Zation, and homeostasis. “Fe uptake and utiliZation genes”
`
`

`
`US 2014/003 8263 A1
`
`Feb. 6, 2014
`
`refers to those polynucleotides/ genes and the encoded
`polypeptides that are involved in Fe uptake, utilization, and
`homeostasis. Some of these polynucleotides/genes are con
`tained in the “Fe Regulon” that has been described in the
`literature and is further described hereafter. As used herein.
`Fe uptake and utiliZation genes and FeiS cluster biosynthe
`sis genes can encode a polypeptide affecting FeiS cluster
`biosynthesis.
`[0058] The term “speci?c activity” as used herein is de?ned
`as the units of activity in a given amount of protein. Thus, the
`speci?c activity is not directly measured but is calculated by
`dividing 1) the activity in units/ml of the enzyme sample by 2)
`the concentration of protein in that sample, so the speci?c
`activity is expressed as units/mg. The speci?c activity of a
`sample of pure, fully active enZyme is a characteristic of that
`enZyme. The speci?c activity of a sample of a mixture of
`proteins is a measure of the relative fraction of protein in that
`sample that is composed of the active enZyme of intere