CL4842USPRV
`
`IMPROVED ACTIVITY OF FE—S CLUSTER REQUIRING PROTEINS
`
`BACKGROUND OF THE INVENTION
`
`Field of the Invention
`
`[0001]
`
`This invention relates generally to the fields of microbiology and biochemistry.
`
`Specifically, 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 specific activity of the dihydroxy-acid
`
`dehydratase polypeptide as
`
`a result of increased expression of the polypeptide,
`
`modulation of the Fe-S cluster biosynthesis 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 flux in an Fe—S cluster biosynthesis pathway
`
`in a host cell.
`
`Background of the Invention
`
`[0002]
`
`Iron-sulfur (Fe-S) clusters serve as cofactors or prosthetic groups essential for the
`
`normal function of the class of proteins that contain them. In the class of Fe-S cluster
`
`containing proteins, the Fe-S clusters have been found to play several roles. When
`
`proteins of this class are first synthesized by the cell, they lack the Fe-S clusters required
`
`for their proper function and are referred to as apoproteins. Fe—S clusters are made in a
`
`series of reactions by proteins involved in Fe—S cluster biosynthesis and are transferred to
`
`the apo-proteins to form the filnctional Fe-S cluster containing holoproteins.
`
`[0003]
`
`One such protein that requires F e-S clusters for proper function is dihydroxy-acid
`
`dehydratase (DHAD)
`
`(EC 4.2.1.9).
`
`DHAD catalyzes
`
`the conversion of 2,3-
`
`dihydroxyisovalerate to u-ketoisovalerate, and of 2,3-dihydroxymethylvalerate to 0.-
`
`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 conversion of 2,3-
`
`dihydroxyisovalerate to a—ketoisovalerate is also a common step in the multiple
`
`isobutanol biosynthetic pathways that are disclosed in US. Patent Appl. Pub. No. US
`
`20070092957 Al,
`
`incorporatcd by rcfcrcncc hcrcin. Discloscd thcrcin is, e.g.,
`
`thc
`
`engineering of recombinant microorganisms for the production of isobutanol.
`
`BUTAMAX 1004
`
`

`

`-2-
`
`[0004]
`
`High levels of DHAD activity are desired for increased production of products
`
`from biosynthetic pathways that include this enzyme activity, including, e. g, enhanced
`
`microbial production of branched chain amino acids, pantothenic acid, and isobutanol.
`
`Isobutanol, in particular, is useful as a fuel additive, and its ready availability may rcducc
`
`the demand for petrochemical fuels. However, since all known DHAD enzymes require a
`
`Fe-S cluster for their function, they must be expressed in a host having the genetic
`
`machinery to provide the Fe-S clusters required by these proteins.
`
`In yeast, most of the
`
`steps in Fe-S Cluster biosynthesis take place in mitochondria.
`
`If the DHAD is to be
`
`functionally expressed in yeast cytosol, a system to transport the requisite Fe-S precursor
`
`from mitochondria and assemble the Fe—S cluster on the cytosolic apoprotein is required.
`
`Prior to the work of the present inventors, it was previously unknown whether yeast could
`
`provide Fe-S 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
`
`[0005]
`
`Under certain conditions the rate of synthesis of Fe-S cluster requiring apo-
`
`proteins may exceed the cell's ability to synthesize and assemble Fe-S clusters for them.
`
`Cluster-less apo-proteins that accumulate under these conditions cannot carry out their
`
`normal function. Such conditions can include 1) the expression of a heterologous Fe-S
`
`cluster requiring protein especially in high amounts, 2) the expression of a native Fe—S
`
`cluster biosynthesis protein at higher levels than normal, or 3) a state where the host cell's
`
`[0006]
`
`[0007]
`
`ability to synthesize Fe-S clusters is debilitated.
`
`BRIEF SUMMARY OF THE INVENTION
`
`Disclosed herein is the surprising discovery that recombinant host cells expressing
`
`a high level of a heterologous Fe-S cluster requiring protein can supply the complement
`
`of Fe-S clusters for that protein if the level(s) of at least one Fe uptake, utilization, and/or
`
`Fe-S cluster biosynthesis protein are altered.
`
`[0008]
`
`Provided herein are recombinant host cells comprising at least one heterologous
`
`polynucleotide encoding a polypeptide having dihydroxy—acid dehydratase activity
`
`wherein said at least one heterologous polynucleotide comprises a high copy number
`
`plasmid or a plasmid with a copy number that can bc rcgulatcd. Also providcd arc
`
`recombinant host cells comprising at least one heterologous polynucleotide encoding a
`
`polypeptide having dihydroxy-acid dehydratase activity wherein said at
`
`least one
`
`

`

`-3-
`
`heterologous polynucleotide is integrated at least once in the recombinant host cell DNA.
`
`Also provided are recombinant host cells comprising at
`
`least one heterologous
`
`polynucleotide encoding a polypeptide having dihydroxy-acid dehydratase activity,
`
`wherein said host cell comprises at least one dclction, mutation, and/or substitution in an
`
`endogenous gene encoding a polypeptide affecting Fe-S cluster biosynthesis. Also
`
`provided are recombinant host cells comprising at least one heterologous polynucleotide
`
`encoding a polypeptide having dihydroxy-acid dehydratase activity and at
`
`least one
`
`heterologous polynucleotide encoding a polypeptide affecting Fe-S cluster biosynthesis.
`
`[0009]
`
`In embodiments,
`
`said heterologous polynucleotide encoding a polypeptide
`
`affecting Fe—S 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 Fe-S cluster biosynthesis is selected from the group consisting of
`
`AFTl, AFT2, PSEl, FRA2, GRX3, MSNS, and combinations thereof.
`
`ln embodiments,
`
`polypeptide is encoded by a polynucleotide that is constitutive mutant.
`
`In embodiments,
`
`said constitutive mutant is selected from the group consisting of AFTl L99A, AFTl
`
`LlOZA, AFTl C29lF, AFTl C293F, and combinations thereof.
`
`In embodiments said
`
`polypeptide affecting Fe-S cluster biosynthesis
`
`is encoded by a polynucleotide
`
`comprising a high copy number plasmid or a plasmid With a copy number that can be
`
`regulated.
`
`In embodiments, said polypeptide affecting Fe—S cluster biosynthesis is
`
`encoded by a polynucleotide integrated at least once in the recombinant host cell DNA.
`
`In embodiments, the at least one deletion, mutation, and/or substitution in an endogenous
`
`gene encoding a polypeptide affecting Fe-S cluster biosynthesis is selected from the
`
`group consisting of FRA2, GRX3, MSNS, and combinations thereof.
`
`In embodiments,
`
`the at least one heterologous polynucleotide encoding a polypeptide affecting Fe-S cluster
`
`biosynthesis is
`
`selected from the group consisting of AFTl, AFT2, PSEl, and
`
`combinations thereof.
`
`[0010]
`
`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
`
`

`

`-4-
`
`host cell DNA.
`
`In embodiments, said Fe—S cluster biosynthesis is increased compared to
`
`a recombinant host cell having endogenous Fe—S cluster biosynthesis.
`
`[0011]
`
`In embodiments, said host cell is a yeast host cell.
`
`In embodiments, said yeast
`
`host cell is selected from thc group consisting of Saccharomyces, Schizosaccharomyces,
`
`Hansenula, Candida, Kluyveromyces, Yarrcwia, [ssatchenkia and Pichia.
`
`[0012]
`
`In
`
`embodiments,
`
`said
`
`heterologous
`
`polypeptide
`
`having
`
`dihydroxy-acid
`
`dehydratase activity is expressed in the cytosol of the host cell.
`
`In embodiments, said
`
`heterologous polypeptide having dihydroxy-acid dehydratase activity has an amino acid
`
`sequence that matches the Profile HMM of Table 12 with an E value of < 10'5 wherein
`
`the polypeptide filrther 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, 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 specific activity
`
`selected from the group consisting of:
`
`greater than about 5-fold with respect to the
`
`control host cell comprising 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
`
`encoding a polypeptide having dihydroxy—acid dehydratase activity, or greater than about
`
`lO-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 specific
`
`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.
`
`[0013]
`
`In cmbodimcnts
`
`said recombinant host ccll produccs
`
`isobutanol, and in
`
`embodiments, said recombinant host cell comprises an isobutanol biosynthetic pathway.
`
`

`

`-5-
`
`[0014]
`
`Also provided herein are methods of making a product comprising: providing a
`
`recombinant host cell; and contacting the recombinant host cell of with a fermentable
`
`carbon substrate in a fermentation medium under conditions wherein said product is
`
`produccd; and rccovcring said product, whcrcin thc product is sclcctcd from the group
`
`consisting of branched chain amino acids, pantothenic acid, 2-methyl-l -butanol, 3-
`
`methyl-l-butanol, isobutanol, and combinations thereof.
`
`[0015]
`
`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; and
`
`recovering said isobutanol.
`
`[0016]
`
`Also provided are methods for the conversion of 2,3—dihydroxyisovalerate to (X—
`
`ketoisovalerate comprising: providing a recombinant host cell; growing the recombinant
`
`host cell of under conditions where the 2,3-dihydroxyisovalerate is converted to (1-
`
`ketoisovalerate.
`
`[0017]
`
`Also provided are methods for increasing the specific activity of a heterologous
`
`polypeptide having dihydroxy-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
`
`dehydratase activity is expressed in functional form having a specific activity greater than
`
`the same host cell lacking said heterologous polypeptide.
`
`[0018]
`
`Also provided are methods for increasing the flux in an Fe-S cluster biosynthesis
`
`pathway in a host cell comprising: providing a recombinant host cell; and growing the
`
`recombinant host cell under conditions whereby the flux in the Fe-S cluster biosynthesis
`
`pathway in the host cell is increased.
`
`[0019]
`
`Also provide are methods of increasing the activity of an Fe-S cluster requiring
`
`protein in a recombinant host cell comprising: providing a recombinant host cell
`
`comprising an Fe-S cluster requiring protein; changing the expression or activity of a
`
`polypeptide affecting Fe—S cluster biosynthesis in said host cell; and growing the
`
`recombinant host cell under conditions whereby the activity of the Fe—S cluster requiring
`
`protcin is incrcascd. In cmbodimcnts, said incrcasc in activity is an amount sclcctcd 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%;
`
`

`

`—6—
`
`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;
`
`grcatcr than about 10 fold.
`
`[0020]
`
`A method for identifying polypeptides that increase the flux in an Fe-S cluster
`
`biosynthesis pathway in a host cell comprising: changing the expression or activity of a
`
`polypeptide affecting Fe-S cluster biosynthesis; measuring the activity of a heterologous
`
`Fe-S cluster requiring protein; and comparing the activity of the heterologous Fe-S Cluster
`
`requiring protein measured in the presence of the change in expression or activity of a
`
`polypeptide to the activity of the heterologous Fe—S cluster requiring protein measured in
`
`the absence of the change in expression or activity of a polypeptide, wherein an increase
`
`in the activity of the heterologous Fe-S cluster requiring protein indicates an increase in
`
`the flux in said F e-S cluster biosynthesis pathway.
`
`[0021]
`
`Provided herein are methods for identifying polypeptides that increase the flux in
`
`an Fe-S cluster biosynthesis pathway in a host cell comprising: changing the expression
`
`or activity of a polypeptide affecting Fe-S cluster biosynthesis; measuring the activity of a
`
`polypeptide having dihydroxy-acid dehydratase activity; and comparing the activity of the
`
`polypeptide having dihydroxy-acid dehydratase activity measured in the presence of the
`
`change to the activity of the polypeptide having dihydroxy—acid dehydratase activity
`
`measured in the absence of change, wherein an increase in the activity of the polypeptide
`
`having dihydroxy-acid dehydratase activity indicates an increase in the flux in said Fe-S
`
`cluster biosynthesis pathway.
`
`[0022]
`
`In embodiments, said changing the expression or activity of a polypeptide
`
`affecting Fe-S 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 Fe-S cluster requiring protein has
`
`dihydroxy-acid dehydratase activity and wherein said Fe-S cluster requiring protein
`
`having dihydroxy—acid dehydratase activity has an amino acid sequence that matches the
`
`Profile HMM of Table 12 with an E value of < 10'5 wherein the polypeptide further
`
`compriscs all thrcc conscrvcd cystcincs, corrcsponding to positions 56, 129, and 201 in
`
`the amino acids sequences of the Streptococcus mutans DHAD enzyme corresponding to
`
`

`

`-7-
`
`SEQ ID NO: 168.
`
`In embodiments, the polypeptide affecting Fe—S cluster biosynthesis is
`
`selected from the group consisting of the genes in Tables 7, 8 and 9.
`
`[0023]
`
`Also provided are recombinant host cells comprising at least one polynucleotide
`
`encoding a polypeptide identified by the methods providcd hcrcin.
`
`In cmbodimcnts, said
`
`host cell
`
`further comprises at
`
`least one heterologous polynucleotide 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.
`
`[0024]
`
`In embodiments, said host cell is a yeast host cell.
`
`In embodiments, said yeast
`
`host cell is selected from the group consisting of Sacckarcmyces, Schizcsacchammyces,
`
`Hansenula, Candida, Kluyvercmyces, Yarrcwz'a,
`
`lssatchenkia,
`
`and Pichia.
`
`In
`
`embodiments, said heterologous polypeptide having dihydroxy-acid dehydratase activity
`
`is expressed in the cytosol of the host cell.
`
`In embodiments, said heterologous
`
`polypeptide having dihydroxy-acid dehydratase activity has an amino acid sequence that
`
`matches the Profile HMM of Table 12 with an E 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, said recombinant host cell produces
`
`a product selected from the group consisting of branched chain amino acids, pantothenic
`
`acid, 2-methyl-l-butanol, 3-methyl-l-butanol, isobutanol, and combinations thereof.
`
`In
`
`embodiments,
`
`recombinant host cell produces isobutanol.
`
`In embodiments,
`
`said
`
`recombinant host cell comprises 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 isobutanol
`
`biosynthetic pathway comprises at least two polypeptides encoded by polynucleotides
`
`heterologous to the host cell.
`
`BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
`
`Figure 1A depicts a vector map of a vector for overexpression of the IlvD gene
`
`from S. mutans.
`
`[0025]
`
`[0026]
`
`

`

`—8—
`
`[0027]
`
`Figure 1B depicts a vector map of an integration vector for overexpression of the
`
`IlvD gene from S. mutans in the chromosome.
`
`[0028]
`
`Figure 2 depicts a vector map of a centromere vector used to clone AFTI or AFTI
`
`mutants and useful for other gcncs of intcrcst.
`
`[0029]
`
`[0030]
`
`[0031]
`
`[0032]
`
`[0033]
`
`[0034]
`
`[0035]
`
`[0036]
`
`[0037]
`
`Figure 3 depicts a UV-Vis absorbance spectrum of purified S. mutans DHAD.
`
`Figure 4 depicts an EPR spectrum of purified S. mutans DHAD.
`
`Figure 5 depicts a biosynthetic pathway for biosynthesis of isobutanol.
`
`Figure 6A depicts a schematic of Azotobacter vinelandz'z' nif genes.
`
`Figure 6B depicts a schematic of additional Azotobacter vinelandz'z' nz‘f genes.
`
`Figure 6C depicts a schematic of the equation in which NFU acts as a persulfide
`
`reductase.
`
`Figure 7 depicts a schematic of Helicobacter pylori nzf genes.
`
`Figure 8 depicts a schematic of E. 0012' 2'30 genes.
`
`Figure 9 depicts a schematic of E. (3011' sufgenes.
`
`Table 12 is a table of the Profile HMM for dihydroxy-acid dehydratases based on
`
`enzymes with assayed function prepared as described in US. Patent Appl. No.
`
`12/569,636, filed Sept. 29, 2009. Table 12 is submitted herewith electronically and is
`
`incorporated herein by reference.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0038]
`
`Described herein is a method to increase the fraction of the Fe-S cluster requiring
`
`proteins that are loaded with Fe—S clusters. Also described are recombinant host cells that
`
`express functional Fe—S cluster requiring proteins, such as DHAD enzymes, and at least
`
`one heterologous Fe uptake, utilization, or Fe-S cluster biosynthesis protein, recombinant
`
`host cells that express functional DHAD enzymes and comprise at least one deletion,
`
`mutation, and/or substitution in a native Fe-S cluster biosynthesis protein, or recombinant
`
`host cells comprising combinations thereof.
`
`In addition, the present invention describes a
`
`method to identify polypeptides that increase the flux in an Fe-S cluster biosynthesis
`
`pathway in a host cell. Also described is a method to identify polypeptides that alter the
`
`activity of an Fe-S cluster requiring protein.
`
`[0039]
`
`Definitions
`
`

`

`-9-
`
`[0040]
`
`Unless defined otherwise, all technical and scientific 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 conflict, the present application including the definitions
`
`will control. Also, unless otherwise required by contcxt, singular terms shall include
`
`pluralities and plural terms shall include the singular. All publications, patents and other
`
`references mentioned herein are incorporated by reference in their entireties for all
`
`purposes.
`
`[0041]
`
`In order to further define this invention, the following terms and definitions are
`
`herein provided.
`
`[0042]
`
`As used herein,
`
`the terms "comprises,
`
`comprising,
`
`includes,
`
`including,"
`
`"has," "having," "contains" or "containing," or any other variation thereof, will be
`
`understood 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
`
`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).
`
`[0043]
`
`As used herein,
`
`the term "consists of," or variations such as "consist of" or
`
`"consisting of,” as used throughout the specification 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 specified method, structure, or composition.
`
`[0044]
`
`As used herein, the term "consists essentially of," or variations such as “consist
`
`essentially of” or "consisting essentially of," as used throughout the specification 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 specified method, structure or composition.
`
`[0045]
`
`Also, the indefinite articles "a" and "an" prcccding an element or component of
`
`the invention are intended to be nonrestrictive regarding the number of instances, i.e.,
`
`occurrences of the element or component. Therefore "a" or "an" should be read to include
`
`

`

`-10-
`
`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.
`
`[0046]
`
`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
`
`encompasses all possible embodiments as described in the application.
`
`[0047]
`
`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 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 compositions 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 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.
`
`[0048]
`
`The term "isobutanol biosynthetic pathway" refers to an enzyme pathway to
`
`produce isobutanol from pyruvate.
`
`[0049]
`
`The term "a facultative anaerobe" refers to a microorganism that can grow in both
`
`aerobic and anaerobic environments.
`
`[0050]
`
`The term "carbon substrate" or "fermentable carbon substrate” refers to a carbon
`
`source capable of being metabolized by host organisms of the present invention and
`
`particularly carbon sources selected from the group consisting of monosaccharides,
`
`oligosaecharides, polysaccharides, and one-carbon substrates or mixtures thereof
`
`[0051]
`
`The term "Fe-S cluster biosynthesis” refers to biosynthesis of Fe-S clusters,
`
`including, e.g.,
`
`the assembly and loading of Fe-S clusters. The term "Fe-S cluster
`
`biosynthesis genes", "Fe-S cluster biosynthesis proteins" or ”Fe-S cluster biosynthesis
`
`pathway" refers to those polynucleotides/genes and the encoded polypeptides that are
`
`involved in the biosynthesis of Fe—S clusters, including, e. g, the assembly and loading of
`
`Fe-S clusters.
`
`[0052]
`
`The term “Fe uptake and utilization” refers to processes which can effect Fe-S
`
`cluster biosynthesis such as Fe sensing, uptake, utilization, and homeostasis. “Fe uptake
`
`

`

`-11-
`
`and utilization genes” refers to those polynucleotides/genes and the encoded polypeptides
`
`that are involved in Fe uptake, utilization, and homeostasis.
`
`Some of these
`
`polynucleotides/genes are contained in the "Fe Regulon" that has been described in the
`
`literature and is further described hcrcaftcr. As used herein, Fc uptake and utilization
`
`genes and Fe-S cluster biosynthesis genes can encode a polypeptide affecting Fe-S cluster
`
`biosynthesis.
`
`[0053]
`
`The term "specific activity" as used herein is defined as the units of activity in a
`
`given amount of protein. Thus, the specific activity is not directly measured but is
`
`calculated by dividing l) the activity in units/ml of the enzyme sample by 2) the
`
`concentration of protein in that sample, so the specific activity is expressed as units/mg.
`
`The specific activity of a sample of pure, fully active enzyme is a characteristic of that
`
`enzyme. The specific 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
`
`interest. The specific activity of a polypeptide of the invention may be selected from
`
`greater than about 0.25 U/mg; greater than about 0.3 U/mg; greater than about 0.4 U/mg;
`
`greater than about 0.5 U/mg; greater than about 0.6 U/mg; greater than about 0.7 U/mg;
`
`
`
`
`
`greater than about 6.5 L/mg; greater than about 7.0 L'/mg; greater than about 7.5 L/mg;
`
`
`
`greater than about 8.0 L/mg; greater than about 8.5 L/mg; greater than about 9.0 L/mg;
`
`greater than about 9.5 U/mg; greater than about 10.0 U/mg; greater than about 20.0 U/mg;
`
`or greater than about 50.0 U/mg.
`
`In one embodiment,
`
`the specific activity of a
`
`polypeptide of the invention is greater than about 0.25 U/mg. In another embodiment, the
`
`specific activity is greater than about 1.0 U/mg.
`
`In yet another embodiment, the specific
`
`activity is greater than about 2.0 U/mg or greater than about 3.0 U/mg.
`
`[0054]
`
`The term ”polynucleotide" is intended to encompass a singular nucleic acid as
`
`well as plural nucleic acids, and refers to a nucleic acid molecule or construct, e.g.,
`
`mcsscgcr RNA (mRNA) or plasmid DNA (pDNA). A polynuclcotidc can contain the
`
`nucleotide sequence of the full-length cDNA sequence, or a fragment thereof, including
`
`the untranslated 5' and 3' sequences and the coding sequences. The polynucleotide can be
`
`

`

`-12-
`
`composed of any polyribonucleotide or polydeoxyribonucleotide, which may be
`
`unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can
`
`be composed of single- and double-stranded DNA, DNA that is a mixture of single- and
`
`double-stranded rcgions, singlc- and double-stranded RNA, and RNA that is mixture of
`
`single- and double-stranded regions, hybrid molecules comprising DNA and RNA that
`
`may be single-stranded or, more typically, double-stranded or a mixture of single- and
`
`double-stranded regions.
`
`"Polynucleotide" embraces chemically, enzymatically, or
`
`metabolically modified forms.
`
`[0055]
`
`A polynucleotide sequence may be referred to as "isolated," in which it has been
`
`removed from its native environment.
`
`For example, a heterologous polynucleotide
`
`encoding a polypeptide or polypeptide fragment having dihydroxy—acid dehydratase
`
`activity contained in a vector is considered isolated for the purposes of the present
`
`invention.
`
`Further examples of an isolated polynucleotide include recombinant
`
`polynucleotides maintained in heterologous host cells or purified (partially or
`
`substantially) polynucleotides in solution.
`
`Isolated polynucleotides or nucleic acids
`
`according to the present invention further include such molecules produced synthetically.
`
`An isolated polynucleotide fragment in the form of a polymer of DNA may be comprised
`
`of one or more segments of cDNA, genomic DNA or synthetic DNA.
`
`[0056]
`
`The term "gene" refers to a polynucleotide 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 regulatory and coding sequences
`
`that are not found together in nature. Accordingly, a chimeric gene may comprise
`
`regulatory 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.
`
`[0057]
`
`As used herein, a "coding region" is a portion of nucleic acid which consists of
`
`codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not
`
`translated into an amino acid, it may bc considered to be part of a coding region, but any
`
`flanking sequences,
`
`for example promoters,
`
`ribosome binding sites,
`
`transcriptional
`
`terminators, introns, and the like, are not part of a coding region. Two or more coding
`
`

`

`-13-
`
`regions of the present invention can be present in a single polynucleotide construct, e.g.,
`
`on a single vector, or in separate polynucleotide constructs, e.g., on separate (different)
`
`vectors. Furthermore, any vector may contain a single coding region, or may comprise
`
`two or more coding rcgions.
`
`In addition, a vector, polynucleotide, or nuclcic acid of the
`
`invention may encode heterologous coding regions.
`
`[0058]
`
`The term "endogenous," when used in reference to a polynucleotide, a gene, or a
`
`polypeptide refers to a native polynucleotide or gene in its natural location in the genome
`
`of an organism, or for a native polypeptide,
`
`is transcribed and translated from this
`
`location in the genome.
`
`[0059]
`
`The term "heterologous" when used in reference to a polynucleotide, a gene, or a
`
`polypeptide refers to a polynucleotide, gene, or polypeptide not normally found in the
`
`host organism.
`
`"Heterologous" also 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, e.g., not in its natural location in the organism's genome. The
`
`heterologous polynucleotide or gene may be introduced into the host organism by, eg,
`
`gene transfer. A heterologous gene may include a native coding region with non-native
`
`regulatory regions that is reintroduced into the native host. A "transgene" is a gene that
`
`has been introduced into the genome by a transformation procedure.
`
`[0060]
`
`The term "recombinant genetic expression elemen " refers to a nucleic acid
`
`fragment that expresses one or more specific proteins, including regulatory sequences
`
`preceding (5' non-coding sequences) and following (3' termination sequences) coding
`
`sequences for the proteins. A chimeric gene is a recombinant genetic expression element.
`
`The coding regions of an operon may form a recombinant genetic expression element,
`
`along with an operably linked promoter and termination region.
`
`[0061]
`
`"Regulatory sequences" refers 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 processing or stability, or
`
`translation of the associated coding sequence. Regulatory s