recombinant yeast host cell, method for making a product, method for converting 2,3-dihydroxyisovale

专利摘要:
RECOMBINANT ACCOMMODATION CELLS, METHODS FOR MANUFACTURING A PRODUCT AND FOR MANUFACTURING ISOBUTANOL, METHOD FOR CONVERTING 2,3-DIIDROXYISOVALERATE IN A-CETOISOVALERATE ACTIVITY, METHODS OF INCREASING A SPECIFIC PHARMACEUTICAL EMOTIONAL ACTIVITY , METHODS FOR IDENTIFYING POLYPEPTIDES AND METHOD FOR MEASURING THE CONCENTRATION OF POLYPEPTIDE FORMS. The present invention relates to a recombinant host cell, in particular a yeast cell, which comprises a dihydroxy acid dehydratase polypeptide. The invention also relates to a recombinant host cell that has increased specific activity of the dihydroxy acid polypeptide dehydratase as a result of increased expression of the polypeptide, modulation of the cell's Fe-S cluster biosynthesis or a combination thereof. The present invention also includes methods of using host cells, as well as methods for identifying polypeptides that increase flow in a Fe-S cluster biosynthetic pathway in a host cell.
公开号:BR112012020589B1
申请号:R112012020589-7
申请日:2011-02-17
公开日:2020-12-29
发明作者:Dennis Flint；Brian James Paul；Rick W. Ye
申请人:Butamax Advanced Biofuels Llc；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[001] This application claims the benefit of Provisional Application No. U.S. 61 / 305,333, filed on February 17, 2010, the entirety of which is hereby incorporated by reference. SEQUENCE LISTING INFORMATION
[002] The content of the sequence listing submitted electronically in the ASCII text file CL4842sequencelisting.txt deposited with the application is incorporated in this document as a reference in its entirety. BACKGROUND OF THE INVENTION FIELD OF THE INVENTION
[003] This invention generally refers to the fields of microbiology and biochemistry. Specifically, the present invention relates to a recombinant host cell, in particular a yeast cell, which comprises a dihydroxy acid dehydratase polypeptide. The invention is also related to a recombinant host cell that has increased specific activity of the dihydroxy acid polypeptide dehydratase as a result of increased expression of the polypeptide, modulation of the cell's Fe-S cluster biosynthesis activity, or a combination thereof. The present invention also includes methods of using host cells, as well as methods for identifying polypeptides that increase flow in a Fe-S cluster biosynthetic pathway in a host cell. BACKGROUND OF THE INVENTION
[004] Ferro-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 Fe-S cluster class containing proteins, Fe-S clusters have been revealed to play several roles. When proteins of this class are first synthesized by the cell, they need 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 apoproteins to form the functional Fe-S cluster containing holoproteins.
[005] Such a protein that requires Fe-S clusters for proper function is dihydroxy acid dehydratase (DHAD) (E.C. 4.2.1.9). DHAD catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate, and 2,3-dihydroxymethylvalerate to α-cetomethylvalerate. The DHAD enzyme is part of naturally occurring biosynthetic pathways that produce branched-chain amino acids, (ie, valine, isoleucine, leucine), and pantothenic acid (vitamin B5). DHAD catalyzed conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate is also a common step in the multiple isobutanol biosynthetic pathways that are disclosed in US Patent Application Publication No. 20070092957 A1, incorporated by reference in this document. . Revealed in it is, for example, the engineering of recombinant microorganisms for the production of isobutanol.
[006] High levels of DHAD activity are desired for increased production of products from biosynthetic pathways that include this enzyme activity, which includes, for example, 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 can reduce 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 that has the genetic machinery to supply the Fe-S clusters required by these proteins. In yeast, mitochondria play an essential role in Fe-S cluster biosynthesis. If DHAD is functionally expressed in yeast cytosol, a system to transport the Fe-S precursor or signal requirement from the mitochondria and mount the Fe-S cluster on the cytosolic apoprotein is required. Before the work of the present inventors, it was previously unknown whether yeast could provide clusters of Fe-S for any DHAD located in the cytoplasm (since native yeast DHAD is located in the mitochondria) and more importantly when DHAD is expressed in high levels in the cytoplasm.
[007] Under certain conditions, the rate of synthesis of apoproteins requiring Fe-S clusters may exceed the cell's ability to synthesize and assemble Fe-S clusters for them. Less clustered apoproteins that accumulate under these conditions cannot perform their normal function. Such conditions may include 1) the expression of a protein that requires heterologous Fe-S cluster especially in high amounts, 2) the expression of a native Fe-S cluster biosynthesis protein at higher than normal levels, or 3) a state in which the host cell capacity synthesizes Fe-S clusters is impaired. BRIEF DESCRIPTION OF THE INVENTION
[008] Revealed in this document is a surprising revelation that the recombinant host cell that expresses a high level of a protein requires a heterologous Fe-S cluster can supply the complement of Fe-S clusters for such a protein if hair levels minus Fe absorption, utilization, and / or cluster biosynthesis of Fe-S protein are altered.
[009] Provided herein are recombinant host cells that comprise at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity wherein said at least one heterologous polynucleotide comprises a high copy number plasmid or a plasmid with a number of copies that can be adjusted. Also provided are recombinant host cells that comprise at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity in which said at least one heterologous polynucleotide is integrated at least once in the recombinant host cell DNA. Also provided are recombinant host cells that comprise at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity, wherein said host cell comprises at least one deletion, mutation, and / or substitution in an endogenous gene that encodes a polypeptide that affects iron metabolism or Fe-S cluster biosynthesis. Also provided are recombinant host cells that comprise at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity and at least one heterologous polynucleotide that encodes a polypeptide that affects iron metabolism or Fe-S cluster biosynthesis.
[010] In the embodiments, said heterologous polynucleotide that encodes a polypeptide that affects the Fe-S cluster biosynthesis is selected from the group consisting of the genes in Tables 7, 8 and 9. In embodiments, said heterologous polynucleotide that encodes a polypeptide that affects Fe-S cluster biosynthesis is selected from the group consisting of AFT1, AFT2, CCC1, FRA2, and GRX3, and combinations thereof. In embodiments, the polypeptide is encoded by a polynucleotide that is a constitutive mutant. In embodiments, said constitutive mutant is selected from the group consisting of AFT1 L99A, AFT1 L102A, AFT1 C291F, AFT1 C293F, and combinations thereof. In embodiments, said polypeptide that affects 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 that affects 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 that affects the Fe-S cluster biosynthesis is selected from the group consisting of CCC1, FRA2, and GRX3, and combinations thereof. In embodiments, the at least one heterologous polynucleotide that encodes a polypeptide that affects the Fe-S cluster biosynthesis is selected from the group consisting of AFT1, AFT2, its mutants, and combinations thereof.
[011] In embodiments, said at least one heterologous polynucleotide that encodes a polypeptide that has 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 Fe-S cluster biosynthesis is increased compared to a recombinant host cell that has endogenous Fe-S cluster biosynthesis.
[012] 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, Issatchenkia and Pichia.
[013] In embodiments, said heterologous polypeptide that has dihydroxy acid dehydratase activity is expressed in the cytosol of the host cell. In embodiments, said heterologous polypeptide that has dihydroxy acid dehydratase activity has an amino acid sequence that is compatible with the HMM Profile of Table 12 with an E value of <10-5 where the polypeptide still comprises all three conserved cysteines, corresponding to positions 56, 129, and 201 in the amino acid sequences of the DHAD enzyme of Streptococcus mutans corresponding to SEQ ID NO: 168. In embodiments, said heterologous polypeptide which has dihydroxy acid dehydratase activity has an amino acid sequence with at least about 90% identity in SEQ ID NO: 168 or SEQ ID NO: 232. In embodiments, said polypeptide that has dihydroxy acid dehydratase activity has a specific activity selected from the group consisting of: more than about 5 times in relation to the control host cell comprising at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity, greater than about 8 times s relative to the control host cell that comprises at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity, or more than about 10 times relative to the control host cell that comprises at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity. In embodiments, said polypeptide which has dihydroxy acid dehydratase activity has a specific activity selected from the group consisting of: more than about 3 times in relation to a control host cell comprising at least one heterologous polynucleotide that encodes a polypeptide that it has dihydroxy acid dehydratase activity and more than about 6 times in relation to the control host cell that comprises at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity. In embodiments, said polypeptide which has dihydroxy acid dehydratase activity has a specific activity selected from the group consisting of: more than about 0.25 U / mg; more than about 0.3 U / mg; more than about 0.5 U / mg; more than about 1.0 U / mg; more than about 1.5 U / mg; more than about 2.0 U / mg; more than about 3.0 U / mg; more than about 4.0 U / mg; more than about 5.0 U / mg; more than about 6.0 U / mg; more than about 7.0 U / mg; more than about 8.0 U / mg; more than about 9.0 U / mg; more than about 10.0 U / mg; more than about 20.0 U / mg; and more than about 50.0 U / mg.
[014] In embodiments, said recombinant host cell produces isobutanol, and in embodiments, said recombinant host cell comprises an isobutanol biosynthetic pathway.
[015] Methods of manufacturing a product are also provided in this document which comprises: providing a recombinant host cell; and contacting the recombinant host cell with a fermentable carbon substrate in a fermentable medium under conditions in which 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 still comprise recovering said product.
[016] Methods of making isobutanol are also provided which comprise: providing a recombinant host cell; contacting the recombinant host cell with a fermentable carbon substrate in a fermentable medium under conditions in which isobutanol is produced. In embodiments, the methods further comprise optionally recovering said isobutanol. In embodiments, the methods further comprise recovering said isobutanol.
[017] Methods are also provided for the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate which comprises: providing a recombinant host cell; culturing the recombinant host cell under conditions where 2,3-dihydroxyisovalerate is converted to α-ketoisovalerate. In embodiments, the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate compared to a control host cell comprising at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity is increased by an amount selected from the group consisting of in: (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%; j) 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 (o) at least about 95%.
[018] Methods are also provided to increase the specific activity of a heterologous polypeptide that has dihydroxy acid dehydratase activity in a recombinant host cell comprising: providing a recombinant host cell; and cultivating the recombinant host cell under conditions in which the heterologous polypeptide having dihydroxy acid dehydratase activity is expressed in functional form that has a specific activity greater than the same host cell without said heterologous polypeptide.
[019] Methods are also provided to increase flow in a Fe-S cluster biosynthetic pathway in a host cell comprising: providing a recombinant host cell; and culturing the recombinant host cell under conditions where the flow in the Fe-S cluster biosynthetic pathway in the host cell is increased.
[020] Methods are also provided to increase the activity of a protein that requires Fe-S cluster in a recombinant host cell that comprises: providing a recombinant host cell that comprises a protein that requires Fe-S cluster; alter the expression or activity of a polypeptide that affects the biosynthesis of the Fe-S cluster in said host cell; and cultivating the recombinant host cell under conditions where the activity of the protein that requires Fe-S cluster is increased. In realizations, said increase in activity is a quantity selected from the group consisting of: more than about 10%; more than about 20%; more than about 30%; more than about 40%; more than about 50%; more than about 60%; more than about 70%; more than about 80%; more than about 90%; and more than about 95%, 98%, or 99%. In achievements, the increase in activity is in an amount selected from the group consisting of: more than about 5 times; greater than about 8 times; more than about 10 times. In achievements, the increase in activity is in an amount selected from the group consisting of: more than about 3 times and more than about 6 times.
[021] A method for identifying polypeptides that increase flow in a Fe-S cluster biosynthetic pathway in a host cell comprises: changing the expression or activity of a polypeptide that affects Fe-S cluster biosynthesis; measure the activity of a protein that requires a heterologous Fe-S cluster; and compare the activity of the protein that requires heterologous Fe-S cluster measured in the presence of the change in expression or activity of a polypeptide to the activity of the protein that requires heterologous Fe-S cluster measured in the absence of the change in expression or activity of a polypeptide. polypeptide, in which an increase in protein activity that requires heterologous Fe-S cluster indicates an increase in flow in said Fe-S cluster biosynthetic pathway.
[022] Methods are provided in this document to identify polypeptides that increase flow in a Fe-S cluster biosynthetic pathway in a host cell comprising: altering the expression or activity of a polypeptide that affects Fe-S cluster biosynthesis S; measuring the activity of a polypeptide that has dihydroxy acid dehydratase activity; and comparing the activity of the polypeptide that has dihydroxy acid dehydratase activity measured in the presence of the change to the activity of the polypeptide that has dihydroxy acid dehydratase activity in the absence of change, where an increase in activity of the polypeptide that has dihydroxy acid dehydratase activity indicates an increase in flow in said Fe-S cluster biosynthetic pathway.
[023] In embodiments, said alteration of the expression or activity of a polypeptide that affects Fe-S cluster biosynthesis comprises deletion, mutation, substitution, expression, upward regulation, downward regulation, alteration of cell location, alteration of the state of protein, and / or adding a cofactor. In embodiments, the protein that requires Fe-S cluster has dihydroxy acid dehydratase activity and said protein that requires Fe-S cluster that has dihydroxy acid dehydratase activity has an amino acid sequence that is compatible with the HMM Profile of Table 12 with an E value of <10-5 where the polypeptide still comprises all three conserved cysteines, corresponding to positions 56, 129, and 201 in the amino acid sequences of the DHAD enzyme of Streptococcus mutans corresponding to SEQ ID NO: 168. In embodiments, the polypeptide that affects Fe-S cluster biosynthesis is selected from the group consisting of the genes in Tables 7, 8 and 9.
[024] Recombinant host cells are also provided that comprise at least one polynucleotide that encodes a polypeptide identified by the methods provided herein. In embodiments, said host cell further comprises at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity. In embodiments, said heterologous polynucleotide that encodes a polypeptide that has 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.
[025] 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, Issatchenkia, and Pichia. In embodiments, said heterologous polypeptide which has dihydroxy acid dehydratase activity is expressed in the cytosol of the host cell. In embodiments, said heterologous polypeptide that has dihydroxy acid dehydratase activity has an amino acid sequence that is compatible with the HMM Profile of Table 12 with an E value of <10-5 where the polypeptide still comprises all three conserved cysteines, corresponding to positions 56, 129, and 201 in the amino acid sequences of the DHAD enzyme of Streptococcus mutans 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, acid pantothenic, 2-methyl-1-butanol, 3-methyl-1-butanol, isobutanol, and combinations thereof. In embodiments, the 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 heterologous polynucleotides for the host cell.
[026] In embodiments, monomers of the polypeptides of the invention that have dihydroxy acid dehydratase activity have a Fe-S cluster load selected from the group consisting 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%; (j) at least about 60%; (k) at least about 70%; (1) at least about 80%; (m) at least about 90%; and (n) at least about 95%. BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES
[027] Figure 1A represents a vector map of a vector for overexpression of the IlvD gene from S. mutans.
[028] Figure 1B represents a vector map of an integration vector for overexpression of the IlvD gene from S. mutans on the chromosome.
[029] Figure 2 represents a vector map of a centromeric vector used to clone AFTI or AFTI mutants and useful for other genes of interest.
[030] Figure 3 represents the UV-Vis absorbance spectrum of purified DHAD S. mutans.
[031] Figure 4 represents a purified DHAD S. mutans EPR spectrum.
[032] Figure 5 represents a biosynthetic pathway for isobutanol biosynthesis.
[033] Figure 6A represents a schematic of the Azotobacter vinelandii nif genes.
[034] Figure 6B represents a schematic of the additional Azotobacter vinelandii nif genes.
[035] Figure 6C represents a schematic of the equation in which NFU acts as a disulfide reductase.
[036] Figure 7 represents a scheme of the Helicobacter pylori nif genes.
[037] Figure 8 represents a schematic of the E. coli isc genes.
[038] Figure 9 represents a schematic of the E. coli suf genes.
[039] Figure 10 represents a schematic of the biosynthesis system and cytosolic set [2Fe-2S].
[040] Figure 11 represents a vector map of a vector for overexpression of the IlvD gene from Z. lactis.
[041] Table 12 is a table of the HMM Profile for enzyme-based dihydroxy acid dehydratases prepared as described in Patent Application No. US 12 / 569,636, filed on September 29, 2009. Table 12 is presented in attached electronically and is incorporated by reference in this document. DETAILED DESCRIPTION OF THE INVENTION
[042] Described in this document is a method for increasing the fraction of proteins that require Fe-S clusters that are loaded with Fe-S clusters. Also described are recombinant host cells that express functional proteins that require Fe-S clusters, such as DHAD enzymes, and at least one heterologous Fe absorption, utilization, or Fe-S cluster biosynthesis protein, recombinant host cells that express enzymes DHADs are functional and comprise at least one deletion, mutation, and / or replacement in the native protein involved in the use of Fe or Fe-S cluster biosynthesis, or recombinant host cells comprising combinations thereof. In addition, the present invention describes a method for identifying polypeptides that increase flow in a Fe-S cluster biosynthetic pathway in a host cell. Also described is a method for identifying polypeptides that alter the activity of a protein that requires a Fe-S cluster. DEFINITIONS
[043] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as the common understanding of elements versed in the technique to which this invention belongs. In the event of a conflict, this application includes the settings that will control you. In addition, unless otherwise required, in the context, singular terms must include pluralities and plural terms must include the singular. All publications, patents and other references mentioned in this document are incorporated by reference in their entirety for all purposes.
[044] To further define this invention, the following terms and definitions are provided in this document.
[045] As used herein, the terms "comprises", "which comprises", "includes", "which includes", "has", "has", "contains" or "contains" or any other variation of themselves, will be understood to imply the inclusion of a declared whole number or group of whole numbers, but not the exclusion of any other whole number or group of whole numbers. For example, a composition, a mixture, a process, a method, an article, or an apparatus comprising a list of elements is not necessarily limited to just such elements, but may include other elements not expressly listed or inherent in such a composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly stated otherwise, "or" refers to an inclusive or not to an exclusive or exclusive. For example, a condition A or B is satisfied by any 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).
[046] As used herein, the term "consists of", or variations such as "consists of" or "that consists of", as used throughout the specification and claims, indicates the inclusion of any quoted integer or group of integers, but no additional integers or groups of integers can be added to the specified method, structure, or composition.
[047] 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, indicates the inclusion of any integer cited or group of whole numbers, and the optional inclusion of any cited whole number or group of whole numbers that do not materially alter the basic and new properties of the specified method, structure or composition, see MPEP $ 2111.03.
[048] In addition, the indefinite articles "one" and "one" that precede an element or component of the invention are intended to be non-restrictive in relation to the number of cases, that is, occurrences of the element or component. Thus "one" or "one" must be read to include one or at least one, and the word form in the singular of the element or component also includes the plural, unless the number is obviously denoted to be singular.
[049] 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 request.
[050] As used in this document, the term "about" which modifies the amount of an ingredient or reagent of the invention employed refers to the variation in the numerical amount that can occur, for example, through typical measurements and handling procedures of liquids used to manufacture concentrates or solutions in the real world; through involuntary error in these procedures; through differences in the manufacture, source, or purity of the ingredients used to make the compositions or to execute the methods; and the like. The term "about" also encompasses amounts that are different because of the different equilibrium conditions for a composition resulting from a particular initial blend. Whether or not modified by the term "about", the claims include equivalences to the quantities. In one embodiment, the term "about" means 10% coverage of the reported numerical value, preferably 5% of the reported numerical value.
[051] The term "isobutanol biosynthetic pathway" refers to an enzyme pathway to produce isobutanol from pyruvate.
[052] The term "an optional anaerobic" refers to a microorganism that can grow in both aerobic and anaerobic environments.
[053] 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, oligosaccharides, polysaccharides, and substrates of a carbon or mixtures thereof.
[054] The term "Fe-S cluster biosynthesis" refers to the biosynthesis of Fe-S clusters, which includes, for example, the assembly and loading of Fe-S clusters. The term "Fe-S cluster biosynthesis genes", "Fe-S cluster biosynthesis proteins" or "Fe-S cluster biosynthetic pathway" refer to those polynucleotides / genes and the encoded polypeptides that are involved in the biosynthesis of Fe-S clusters, which includes, for example, the assembly and loading of Fe-S clusters.
[055] The term "Fe absorption and utilization" refers to processes that can effect Fe-S cluster biosynthesis such as Fe detection, absorption, utilization, and homeostasis. "Fe absorption and utilization genes" refer to those polynucleotides / genes and the encoded polypeptides that are involved in the absorption, utilization, and homeostasis of Fe. Some of these polynucleotides / genes are contained in the "Fe regulation" that has been described in the literature and is further described below. As used herein, Fe absorption and utilization genes and Fe-S cluster biosynthesis genes can encode a polypeptide that affects Fe-S cluster biosynthesis.
[056] The term "specific activity", as used herein, is defined as the units of activity in a given amount of protein. Therefore, the specific activity is not directly measured, but is calculated by dividing 1) the activity in units / ml of the enzyme sample by 2) the protein concentration in such a sample, so the specific activity is expressed as units / mg. The specific activity of a sample of completely pure active enzyme is a characteristic of such an enzyme. The specific activity of a sample of a protein mixture is a measure of the relative fraction of protein in such a sample that is composed of the active enzyme of interest. The specific activity of a polypeptide of the invention can be selected from more than about 0.25 U / mg; more than about 0.3 U / mg; more than about 0.4 U / mg; more than about 0.5 U / mg; more than about 0.6 U / mg; more than about 0.7 U / mg; more than about 0.8 U / mg; more than about 0.9 U / mg; more than about 1.0 U / mg; more than about 1.5 U / mg; more than about 2.0 U / mg; more than about 2.5 U / mg; more than about 3.0 U / mg; more than about 3.5 U / mg; more than about 4.0 U / mg; more than about 5.5 U / mg; more than about 5.0 U / mg; more than about 6.0 U / mg; more than about 6.5 U / mg; more than about 7.0 U / mg; more than about 7.5 U / mg; more than about 8.0 U / mg; more than about 8.5 U / mg; more than about 9.0 U / mg; more than about 9.5 U / mg; more than about 10.0 U / mg; more than about 20.0 U / mg; or more than about 50.0 U / mg. In one embodiment, the specific activity of a polypeptide of the invention is more than about 0.25 U / mg. In another embodiment, the specific activity is more than about 1.0 U / mg. In yet another embodiment, the specific activity is more than about 2.0 U / mg or more than about 3.0 U / mg.
[057] The term "polynucleotide" is intended to encompass singular nucleic acid as well as plural nucleic acids, and refers to a nucleic acid molecule or construct, for example, messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may contain the nucleotide sequence of the full-length cDNA sequence, or a fragment thereof, which includes the 5 'and 3' untranslated sequences and the coding sequences. The polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be RNA or unmodified DNA or RNA or modified DNA. For example, polynucleotides can be composed of single or double stranded DNA, DNA that is a mixture of single or double stranded regions, single or double stranded RNA, and RNA that is a mixture of single or double stranded regions, molecules hybrids comprising DNA and RNA which can be single stranded or, more typically, double stranded or a mixture of single or double stranded regions. "Polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms.
[058] A polynucleotide sequence can be referred to as "isolated", as it has been removed from its native environment. For example, a heterologous polynucleotide that encodes a polypeptide or polypeptide fragment that has dihydroxy acid dehydratase activity contained in a vector is considered isolated for the purposes of the present invention. Additional examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified polynucleotides (partially or substantially) in solution. Isolated polynucleotides or nucleic acids according to the present invention still include such synthetically produced molecules. An isolated polynucleotide fragment in the form of a DNA polymer can be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
[059] The term "gene" refers to a polynucleotide that is capable of being expressed as a specific protein, which optionally includes regulatory sequences that precede (5 'non-coding sequences) and then (3' non-coding sequences) the sequence encoder. "Native gene" refers to a gene as revealed in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, which comprises regulatory and coding sequences that are not revealed together in nature. Consequently, a chimeric gene can 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 different way than was revealed in nature.
[060] As used herein, a "coding region" is a portion of nucleic acid that consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example, promoters, ribosome binding sites , transcription terminators, introns, and the like, are not part of a coding region. Two or more coding regions of the present invention can be present in a single polynucleotide construct, for example, in a single vector, or in separate polynucleotide constructs, for example, in separate (different) vectors. In addition, any vector can contain a single coding region, or it can comprise two or more coding regions. In addition, a vector, polynucleotide, or nucleic acid of the invention can encode heterologous coding regions.
[061] The term "endogenous", when used in reference to a polynucleotide, gene or polypeptide, refers to a polynucleotide or native gene in its natural location in the genome of an organism, or to a native polypeptide, is transcribed and translated from that location in the genome.
[062] The term "heterologous" when used in reference to a polynucleotide, gene, or polypeptide, refers to a polynucleotide, gene, or polypeptide not normally found in the host organism. "Heterologist" 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, for example, not at that natural location in the organism's genome. The heterologous polynucleotide or gene can be introduced into the host organism by, for example, gene transfer. A heterologous gene can include a native coding region with non-native regulatory regions that is reintroduced into the native host. A "transgene" is a gene that was introduced into the genome by a transformation procedure.
[063] The term "recombinant gene expression element" refers to a fragment of nucleic acid that expresses one or more specific proteins, which include regulatory sequences that precede (non-coding sequences 5 ') and that follow (termination sequences 3 ') the coding sequences for the proteins. A chimeric gene is an element of recombinant gene expression. The coding regions of an operon can form a recombinant gene expression element, along with an operationally linked promoter and termination region.
[064] "Regulatory sequences" refers to nucleotide sequences located upstream (5 'non-coding sequences), in, or downstream (3' non-coding sequences) of a coding sequence, and which influence a transcription, processing or RNA stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, enhancers, operators, repressors, transcription termination signals, leading translation sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and semicircular structure.
[065] The term "promoter" refers to a nucleic acid sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located from 3 'to a promoter sequence. Promoters can 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 segments of synthetic nucleic acid. It is understood by elements versed in the technique that different promoters can direct the expression of a gene in different types of tissues or cells, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types are most often referred to as "constitutive promoters". "Inducible promoters", on the other hand, cause a gene to be expressed when the promoter is induced or transformed by a specific promoter signal or molecule. It is further recognized that, since in most cases, the exact limits of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
[066] The term "operably linked" refers to the association of nucleic acid sequences in a single fragment of nucleic acid 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 able to effect the expression of such a coding sequence (i.e., that coding sequence is under the transcriptional control of the promoter). Coding sequences can be operationally linked to regulatory sequences in sense or antisense orientation.
[067] The term "expression", as used herein, refers to the transcription and accumulation of sense RNA (mRA) or antisense derived from the nucleic acid fragment of the invention. The expression can also refer to the translation of mRNA into a polypeptide. The process includes any manifestation of the functional presence of the expressed polynucleotide, gene, or polypeptide in the cell that includes, without limitation, gene inactivation as well as, both transient and stable expression.
[068] The term "overexpression", as used herein, refers to the expression that is higher than endogenous expression of the same polynucleotide or related gene. A heterologous polynucleotide or gene is also overexpressed if its expression is higher than that of a comparable endogenous gene, or if its expression is higher than that of the same polynucleotide or gene introduced by a medium that does not overexpress the polynucleotide or gene. For example, a polynucleotide can be expressed in a host cell from a low copy number plasmid, which is present in few or only limited copies, and the same polynucleotide can be overexpressed in a host cell from a plasmid high copy number or a plasmid with a regulable number of copies, which is present in multiple copies. Any medium can be used to overexpress a polynucleotide, as long as it increases the copies of the polynucleotide in the host cell. In addition to the use of a high copy number plasmid, or a plasmid with a regulable copy number, a polynucleotide can be overexpressed by multiple chromosomal integrations.
[069] The expression or overexpression of a polypeptide of the invention in a recombinant host cell can be quantified according to any number of methods known to the person skilled in the art and can be represented, for example, by a percentage of the cell's total protein . The percentage of total protein can be an amount selected from more than about 0.001% of the total protein in the cell; more than about 0.01% of the cell's total protein; more than about 0.1% of the cell's total protein; more than about 0.5% of the cell's total protein; more than about 1.0% of the cell's total protein; more than about 2.0% of the cell's total protein; more than about 3% of the cell's total protein; more than about 4.0% of the cell's total protein; more than about 5% of the cell's total protein; more than about 6.0% of the cell's total protein; more than about 7.0% of the cell's total protein; more than about 8.0% of the cell's total protein; more than about 9.0% of the cell's total protein; more than about 10% of the cell's total protein; or more than about 20% of the cell's total protein. In one embodiment, the amount of polypeptide expressed is more than about 0.5% of the cell's total protein. In another embodiment, the amount of polypeptide expressed is more than about 1.0% of the cell's total protein or more than about 2.0% of the cell's total protein.
[070] As used herein, the term "transformation" refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance with or without selections. The host organisms that contain the transformed nucleic acid fragments are called "transgenic" or "recombinant" or "transformed" organisms.
[071] The terms "plasmid" and "vector", as used in this document, refer to an extrachromosomal element that often carry genes that are not part of the cell's central metabolism, and generally in the form of filament DNA molecules double circular. Such elements can be autonomous replication sequences, genomic integration sequences, phage or nucleotide sequences, linear or circular, from a single or double stranded RNA or DNA, derived from any source, in which several nucleotide sequences have been joined or recombined in a unique construct that is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with an appropriate 3 'untranslated sequence into a cell.
[072] As used herein, the term "codon degeneration" refers to nature in the genetic code that allows variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The person skilled in the art is aware of the "codon bias" exhibited by a specific host cell in the use of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for enhanced expression in a host cell, it is desirable to design the gene in such a way that its codon usage frequency approximates the host cell's preferred codon usage frequency.
[073] The term "codon enhanced", since it refers to genes or coding regions for nucleic acid molecules for the transformation of multiple hosts, refers to the change of codons in the gene or coding regions for nucleic acid molecules to reflect the codon usage typical of the host organism without altering the polypeptide encoded by DNA. Such an improvement includes replacing at least one, or more than one, or a significant number of codons with one or more codons that are most frequently used from that organism.
[074] Deviations in the nucleotide sequence comprising the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence encoding the gene. Since each codon consists of three nucleotides, and the nucleotides that comprise DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals that complete the translation). The "genetic code" that shows which codons encode which amino acids is reproduced in this document as Table 1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are encoded by four triplets, serine and arginine by six, while tryptophan and methionine are encoded by only one triplet. This degeneration allows the base composition of DNA to vary over a wide range without changing the amino acid sequence of proteins encoded by DNA. TABLE 1. THE STANDARD GENETIC CODE

[075] Many organisms exhibit a bias towards the use of particular codons to code for the insertion of a particular amino acid in a growing peptide chain. Codon preference, or codon bias, differences in codon usage between organisms, are provided by degeneracy of the genetic code, and are well documented among many organisms. Codon bias often correlates with the effectiveness of messenger RNA (mRNA) translation, which in turn is believed to be dependent on, among others, the properties of the codons that are translated and the availability of transfer RNA molecules ( tRNA). The predominance of selected tRNAs in a cell is usually a reflection of the codons most frequently used in peptide synthesis. Consequently, genes can be adapted to express the ideal gene in a given organism based on codon enhancement.
[076] Given the large number of gene sequences available for a wide variety of animal species, plants and microbes, it is possible to calculate the relative frequencies of codon use. The codon use tables are readily available, for example, in the "Codon Use Database" available at http://www.kazusa.ou.jp/códon/(visited on March 20, 2008), and these tables can be adapted in several ways. See Nakamura, Y., et al. Nucl. Acids Res. 28: 292 (2000). The codon usage tables for yeasts, calculated from GenBank version 128.0 [February 15, 2002], are reproduced below as Table 2. This table uses mRNA nomenclature instead of thymine (T) which is found in DNA, the tables use uracil (U) which is found in RNA. Table 2 was adapted in such a way that the frequencies are calculated for each amino acid, instead of for all 64 codons. TABLE 2. CODE USE TABLE FOR SACCHAROMYCES GENES



[077] Using this table or similar, an element with common skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment from a codon-enhanced coding region encoding the polypeptide, but which uses ideal codons for a particular species.
[078] The random assignment of codons at an improved frequency to encode the given polypeptide sequence can be done manually by calculating the codon frequencies for each amino acid, and then the codons are assigned to the polypeptide sequence at random. In addition, several algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the "EditSeq" function in Lasergene Package, available from DNAstar, Inc., Madison, WI, the back-translation function in VectorNTI Suite, available from InforMax, Inc., Bethesda, MD, and the "back-translation function "in the GCG-Wisconsin Package, available from Accelrys, Inc., San Diego, CA. In addition, several resources are publicly available for codon-enhanced coding region sequences, for example, the "back-translation" function at http: //www.entelechon om / bioinformatics / backtranslation.php Lang = eng (visited on April 15 2008) and the "backtranseq" function available at http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited on July 9, 2002). The construction of a rudimentary algorithm to assign codons based on a given frequency can also be easily achieved with basic mathematical functions by an element with common skill in the technique.
[079] Codon-enhanced coding regions can be designed by various methods known to those skilled in the art including software packages such as "synthetic gene designer" (http://phenotvpe.biosci.umbc.edu/codon/sgd/index. php).
[080] As used herein, the term "polypeptide" is intended to encompass a "polypeptide" in the singular as well as "polypeptides" in the plural, and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein", "chain of amino acids" or any other term used to refer to a chain or chains of two or more amino acids, are included in the definition of "polypeptide", and the term "polypeptide" can be used instead of, or interchangeably with, any of those terms. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but it is not necessarily translated from a designated nucleic acid sequence. It can be generated in any way, including by chemical synthesis.
[081] By an "isolated" polypeptide, fragment, variant, or derivative of it, is meant a polypeptide that is not in its natural environment. No particular level of purification is needed. For example, an isolated polypeptide can be removed from its natural or native environment. Recombinantly produced proteins and polypeptides expressed in host cells are considered isolated for the purposes of the invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.
[082] As used herein, the term "variant" refers to a polypeptide that differs from a specifically cited polypeptide of the invention, such as DHAD, by insertions, deletions, mutations and substitutions of amino acids, created using, for example, recombinant DNA techniques, such as mutagenesis. Guidance for determining that amino acid residues can be substituted, added or deleted without abolishing the activities of interest, can be found by comparing the sequence of the particular polypeptide with that of homologous polypeptides, for example, yeast or bacteria, and minimizing it whether the number of amino acid sequence changes made in regions of high homology (conserved regions) or replacing the amino acids with consensus sequences.
[083] Alternatively, recombinant polynucleotide variants that encode these same or similar polypeptides can be synthesized or selected using "redundancy" in the genetic code. Various codon substitutions, such as silent changes that produce multiple restriction sites, can be introduced to improve cloning into a plasmid or viral vector for expression. Mutations in the polynucleotide sequence can be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide. For example, mutations can be used to reduce or eliminate the expression of a target protein and include, but are not limited to, deletion of the entire gene or a portion of the gene by inserting a DNA fragment into the gene (in the region promoter or coder) so that the protein is not expressed or expressed at low levels, introducing a mutation in the coding region that adds a stop codon or frame change in a way that a functional protein is not expressed, and introducing one or more mutations are made in the coding region to alter amino acids in a way that a non-functional or less enzymatically active protein is expressed.
[084] Amino acid "substitutions" may be the result of replacing one amino acid with another amino acid that has similar structural and / or chemical properties, that is, conservative amino acid substitutions, or they may be the result of replacing an amino acid with an amino acid that has different structural and / or chemical properties, that is, substitutions for non-conservative amino acids. Substitutions for "conservative" amino acids can be made on a basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged amino acids (acids) include aspartic acid and glutamic acid. Alternatively, "non-conservative" amino acid substitutions can be made by selecting differences in polarity, charge, solubility, hydrophobicity, hydrophilicity or the amphipathic nature of any of these amino acids. The "insertions" or "deletions" can be within the range as tolerated structurally or functionally by the recombinant proteins. The allowed variation can be experimentally determined by systematically inserting, deleting or replacing amino acids in a polypeptide molecule using recombinant DNA techniques and testing the resulting recombinant variants for activity.
[085] A "substantial portion" of an amino acid or nucleotide sequence is that portion which comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify which polypeptide or gene, by manual evaluation of the sequence by the person skilled in the art, or by identification and comparison of automated computer sequences using algorithms such as BLAST (Altschul, SF, et al., J. Mol. Biol., 215: 403 to 410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is required in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. In addition, for nucleotide sequences, gene-specific oligonucleotide probes comprising 20 to 30 contiguous nucleotides can be used in methods dependent on gene identification sequence (for example, Southern type hybridization) and isolation (for example, hybridization in situ of bacterial colonies or bacteriophage plaques). In addition, short 12 to 15 base oligonucleotides can be used as PCR amplification primers to obtain a particular nucleic acid fragment comprising the primers. Therefore, the "substantial portion" of a nucleotide sequence comprises enough of the sequence to specifically identify and / or isolate a nucleic acid fragment that comprises the sequence. The present specification teaches the complete nucleotide and amino acid sequence that encodes particular proteins. The person skilled in the art, who has the benefit of the sequences, as reported in this document, can now use all or a substantial portion of the sequences described for the purposes known to those skilled in the art. Accordingly, the present invention comprises the complete sequences as reported in the attached Sequence Listing, as well as substantial portions of those sequences as defined above.
[086] The term "complementary" is used to describe the relationship between nucleotide bases that are able to hybridize to each other. For example, in relation to DNA, adenine is complementary to thymine and cytosine is complementary to guanine, and in relation to RNA, adenine is complementary to uracil and cytosine is complementary to guanine.
[087] The term "percentage of identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relationship between polypeptide or polynucleotide sequences, as may be the case, as determined by the compatibility between the strands of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including, but not limited to, those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).
[088] The preferred methods for determining identity are designed to provide the best compatibility between the sequences tested. The methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations can be performed using the MegAlign ™ program from LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple sequence alignments are performed using the "Clustal alignment method" which covers several varieties of the algorithm including the "Clustal V alignment method" corresponding to the alignment method identified as Clustal V (described by Higgins and Sharp, CABIOS. 5 : 151 to 153 (1989); Higgins, DG et al, Comput. Appl. Biosci., 8: 189 to 191 (1992)) and found in the MegAlign ™ program of LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY = 10 and GAP LENGTH PENALTY = 10. The standard parameters for pairwise alignments and percent protein identity sequence calculations using the Clustal method are KTUPLE = 1, GAP PENALTY = 3, WINDOW = 5 and DIAGONALS SAVED = 5. For nucleic acids, these parameters are KTUPLE = 2, GAP PENALTY = 5, WINDOW = 4 and DIAGONALS SAVED = 4. After aligning the sequences using the Clustal V program, it is possible to obtain a "percentage of identity" by looking at the "sequence distances" table in the same program. Additionally, the "Clustal W alignment method" is available and corresponds to the alignment method identified as Clustal W (described by Higgins and Sharp, CABIOS. 5: 151 to 153 (1989); Higgins, DG et al, Comput. Appl. Biosci 8: 189 to 191 (1992)) and found in the MegAlign v6.1 program of LASERGENE bioinformatics computing suite (DNASTAR Inc.). Standard parameters for multiple alignments (GAP PENALTY = 10, GAP LENGTH PENALTY = 0.2, Divergen Delay Seqs (%) = 30, DNA Transition Weight = 0.5, Protein Weight Matrix = Gonnet series, Matrix DNA weight = IUB). After aligning the sequences using the Clustal W program, it is possible to obtain the "percentage of identity" by looking at the "sequence distances" table in the same program.
[089] The skilled person understands well that many levels of sequence identity are useful in the identification of polypeptides, from other species, in which such polypeptides have the same or similar activity or function, or in the description of the corresponding polynucleotides. Useful examples of percentages of identity include, but are not limited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100%) may be useful in describing the present invention, such as 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 %. Suitable polynucleotide fragments not only have the above homologies, but typically comprise a polynucleotide that has at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, or at least 250 nucleotides. In addition, suitable polynucleotide fragments having the above homologies encode a polypeptide that has at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, or at least 250 amino acids.
[090] The term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. "Sequence analysis software" can be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215: 403 to 410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, WI); 4.) SEQUENCHER (Gene Codes Corporation, Ann Arbor, MI); and 5.) the FASTA program that incorporates the Smith-Waterman algorithm (WR Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111 to 20. Editor (s): Suhai, Sandor, Plenum: New York, NY). In the context of this application it will be understood that where the sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the referenced program, unless otherwise specified. As used in this document, "default values" will mean any set of values or parameters that are originally loaded with the software when initialized for the first time.
[091] The standard recombinant DNA and molecular cloning techniques used in this document are well known in the art and are described by Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) (hereinafter "Maniatis"); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1984); and by Ausubel, F. M. et al, Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). THE FUNCTIONS OF PROTEINS THAT REQUIRE FE-S CLUSTER
[092] The functions of proteins that contain Fe-S clusters are diverse. One of the most complete efforts to classify these functions is provided in the following table, which is adapted from Johnson, DC, et al., Structure, function, and formation of biological iron-sulfur clusters. Annu. Rev. Biochem., 2005. 74: p. 247 to 281. TABLE 3. BIOLOGICAL CLUSTER FUNCTIONS

the abbreviations used are SAM, S-adenosylmethionine; acetyl-CoA, acetyl coenzymeA; FNR, reduction of fumarate and nitrate; IRP, iron regulatory protein; IscR, iron-sulfur cluster assembly regulatory protein; PRPP, phosphoribosylpyrophosphate.
[093] It is believed that an increase in the supply and loading efficiency of Fe-S clusters in one or more of the members of the above classes will have commercial and / or medical benefits. Of the many possibilities that will be verified by the person skilled in the art, three examples are given. 1) When an Fe-S cluster containing enzyme is used in a pathway for a fermentation product and needs to be expressed at high levels to maintain a high flow in the pathway for the product (for example, dihydroxy acid dehydratase in the pathway for isobutanol) . 2) When an Fe-S cluster containing enzyme is used in a pathway for a fermentation product and the Fe-S cluster undergoes a renewal during catalysis (eg biotin synthase in the commercial glucose to biotin fermentation ). 3) In a sick state, so that the normal concentration of a Fe-S cluster containing protein important for good health is low (for example, in cases of Friedreich's ataxia). DHAD AND DHAD TESTS
[094] DHAD is a Fe-S cluster that requires protein of the class dehydratase (more appropriately hydro-lyase). A gene encoding a DHAD enzyme can be used to provide expression of DHAD activity in a recombinant host cell. DHAD catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate and 2,3-dihydroxymethyl valerate to α-cetomethylvalerate and is classified as EC 4.2.1.9. The coding sequences for DHADs that are suitable for use in a recombinant host cell can be derived from bacterial, fungal or plant sources. DHADs that can be used can have a cluster of [4Fe-4S] or [2Fe-2S]. Tables 4a, 4b, 5 and 6 list SEQ ID NOs for coding regions and representative DHAD proteins that can be used in the present invention. Proteins with at least about 95% identity to certain listed sequences have been omitted for simplicity, but proteins, including those omitted for simplicity, are understood to have at least about 95% sequence identity to any of the proteins listed in Tables 4a, 4b, 5, and 6 and which have DHAD activity can be used as described in this document. Additional DHAD proteins and their coding sequences can be identified by BLAST searching public databases, as is well known to the person skilled in the art. Typically the BLAST search (described above) of publicly available databases with known DHAD sequences, such as those provided herein, is used to identify DHADs and the coding sequences thereof that can be expressed in the present cells. For example, DHAD proteins that have amino acid sequence identities of at least about 80 to 85%, at least about 85 to 90%, at least about 90 to 95%, or at least about 98% sequence identity to any of the DHAD proteins in Table 3 can be expressed in the present cells. The identities are based on the Clustal W alignment method using the standard parameters of GAP PENALTY = 10, GAP LENGTH PENALTY = 0.1, and Gonnet 250 series of protein weight matrix. TABLE 4A. DHAD PROTEINS SEQ ID NO [2FE-2S] REPRESENTATIVE BACTERIALS AND ENCODING SEQUENCES











TABLE 6. DHAD PROTEIN SEQ ID NO. [4FE-4S] REPRESENTATIVES AND ENCODING SEQUENCES

[095] [2Fe-2S] Additional DHADs can be identified through the use of the analysis described in US patent application 12 / 569,636, filed on September 29, 2009, which is incorporated by reference in this document. The analysis is as follows: A Hidden Markov Model in profile (HMM) was prepared based on the amino acid sequences of eight DHADs verified in a functional way. The application of the profile HMM has been described. See, for example, Krogh et al., J. Mol. Biol. 255: 1501 to 1531 (1994) and Durbin et al., "Markov chains and hidden Markov models", in Biological Sequence Analysis: Probabilistic Models of Proteins and nucleic acids, Cambridge University Press (1998). A profile HMM is a statistical model constructed of multiple sequence alignments that can be used to determine whether or not a test sequence belongs to a particular sequence family. See the same. A profiled HMM can be built primarily by generating a sequence alignment verified in a functional way through the use of conventional sequence alignment tools. Next, sequence alignment is used to build the profile HMM through the use of publicly available software programs (for example, HMMER) that use a specific position scoring system to capture information about the degree of conservation in various positions amino acid in the multiple alignment of the input sequences. More specifically, the amino acid residue scores in a “compatible” state (that is, the compatibility state emission scores), or in an “insertion” state (that is, the insertion state emission scores) are captured that are proportional to the expression: Log 2 (p_x) / (null_x). See the same. In this expression, the term "p_x" is the probability of an amino acid residue, in a particular position in the alignment, according to the profile HMM, and the term "null_x" is the probability according to the null model. See the same. The null model is a simple probabilistic model of a state with a pre-calculated set of emission probabilities for each of the amino acids derived from the distribution of amino acids. See the same. The “state” transition scores are also calculated as parameters of log odds and are proportional to Log 2 (t_x). See the same. In this expression, the term "t_x" is the probability of moving to an emitting or non-emitting state. See the same. Additional details regarding the particular statistical analyzes to generate a profile HMM are available from Krogh et al, J. Mol. Biol. 255: 1501 to 1531 (1994) and Durbin et al., "Markov chains and hidden Markov models", in Biological Sequence Analysis: Probabilistic Models of Proteins and nucleic acids, Cambridge University Press (1998), and in the US patent application 12 / 569,636.
[096] A Hidden Markov Model in profile (HMM) was prepared based on the amino acid sequences of eight functionally verified DHADs that are Nitrosomonas europaea (DNA SEQ ID NO: 309; protein SEQ ID NO: 310), Synechocystis sp. PCC6803 (DNA SEQ ID: 297; protein SEQ ID NO: 298), Streptococcus mutans (DNA SEQ ID NO: 167; protein SEQ ID NO: 168), Streptococcus thermophilus (DNA SEQ ID NO: 163; SEQ ID No: 164) , Ralstonia metallidurans (DNA SEQ ID NO: 345; protein SEQ ID NO: 346), Ralstonia eutropha (DNA SEQ ID NO: 343; protein SEQ ID NO: 344), and Lactococcus lactis (DNA SEQ ID NO: 231; protein SEQ ID NO: 232). In addition, the DHAD of Flavobacterium johnsoniae (DNA SEQ ID NO: 229; protein SEQ ID NO: 230) was found to have dihydroxy-acid dehydratase activity when expressed in E. coli and was used in the fabrication of the profile. Profile HMM is prepared using the HMMER software package (The theory on which profile HMMs are based is described in R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998; Krogh et al, 1994; J. Mol. Biol. 235: 1501 to 1531), following the user guide which is available from HMMER (Janelia Farm Research Campus , Ashburn, VA). An output of the HMMER software program is a profile Markov Hidden Model (HMM) that characterizes the input strings. The profile HMM prepared for the eight DHAD proteins is provided in the order in US 12 / 569,636, deposited on September 29, 2009 and in Table 12.
[097] The first line in Table 12 for each position reports the probability for each amino acid to be in that "state" (compatibility status emission scores). The second line reports the insertion status emission scores, and the third line reports the state transition scores. The highest probability is highlighted for each position. These scores can be converted into "E values" (expectation values), which are the number of occurrences or compatibilities like the profile HMM that a person would expect to obtain just by luck. A protein that has an E value of <10-5 compatible with the profile HMM, indicates that the protein shares significant sequence similarity with the seed proteins used to build the profile HMM and whose protein belongs to the family represented by the HMM of profile.
[098] Any protein that is compatible with HMM in profile with an E value of <10-5 DHAD-related protein, which includes [4Fe-4S] DHADs, [2Fe-2S] DHADs, arabonate dehydratases, and phosphogluconate dehydratases. In the embodiments, sequences compatible with the profile HMM are then analyzed for the presence of the three conserved cysteines, corresponding to positions 56, 129, and 201 in the DHAD of Streptococcus mutans. The presence of all three conserved cysteines is characteristic of proteins that have a [2Fe-2S] cluster. The proteins that have the three conserved cysteines include arabonate dehydratases and [2Fe-2S] DHADs. The [2Fe-2S] DHADs can be differentiated from arabonate dehydratases by analysis with conserved signature amino acids found to be present in the [2Fe-2S] DHADs or arabonate dehydratases at the positions corresponding to the following positions in the DHAD amino acid sequence of Streptococcus mutans . These signature amino acids are in [2Fe-2S] DHADs or arabonate dehydratases, respectively, in the following positions (with more than 90% occurrence): 88 asparagine vs. glutamic acid; 113 not preserved vs. glutamic acid; 142 arginine or asparagine vs. not preserved; 165 not preserved vs. glycine; 208 asparagine vs. not preserved; 454 leucine vs. not preserved; 477 phenylalanine or tyrosine vs. not preserved; and 487 glycine vs. not preserved.
[099] Additionally, the DHAD coding region sequences provided in this document can be used to identify other homologues in nature. Such methods are well known in the art, and several methods that can be used to isolate genes that encode homologous proteins are described in the application in US 12 / 569,636, filed September 29, 2009, the methods of which are incorporated herein by way of reference.
[0100] The presence of DHAD activity in a cell engineered to express a heterologous DHAD can be confirmed through the use of methods known in the art. As an example, and as demonstrated in the Examples herein, raw extracts of cells engineered to express a bacterial DHAD can be used in a DHAD assay as described by Flint and Emptage (J. Biol. Chem. (1988) 263 (8) : 3558 to 64) through the use of dinitrophenylhydrazine. In another example, DHAD activity can be assayed by expressing a heterologous DHAD identifiable by the methods presented in this document in a yeast strain that lacks endogenous DHAD activity. If DHAD activity is present, the yeast strain will proliferate in the absence of branched chain amino acids. DHAD activity can also be confirmed by more indirect methods, such as by testing a product downstream on a pathway that requires DHAD activity. Any product that has a-ketoisovalerate or a-cetomethylvalerate as an intermediate route can be measured in an assay for DHAD activity. A list of such products includes, but is not limited to, valine, isoleucine, leucine, pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol, and isobutanol. DHAD ACTIVITY OVEREXPRESSION
[0101] Depositors have found that the expression of a heterologous DHAD can provide DHAD activity when expressed in a host cell. The expression of a DHAD that can be identified as described herein can provide DHAD activity for a biosynthetic pathway that includes the conversion of 2,3-dihydroxyisovalerate to a-ketoisovalerate or 2,3-dihydroxymethylvalerate to a-cetometyl valerate. In addition, S. mutans [2Fe-2S] DHAD was shown in the related application in US 12 / 569,636, filed on September 29, 2009, incorporated by reference in this document, to have higher stability in the air when compared to air sensitivity of E. coli [4Fe-4S] DHAD, which is desirable to obtain better activity in a heterologous host cell.
[0102] Furthermore, as described in this document, it has been found that expressing a heterologous DHAD protein at higher levels can provide increased DHAD activity when expressed in a host cell. Elevated expression of a recombinant polynucleotide can be accomplished in at least two ways: 1) by increasing the copy number of a plasmid comprising the recombinant polynucleotide; or 2) by integrating multiple copies of the gene of interest into the host cell's chromosome. As exemplified herein, the expression of multiple copies of heterologous DHAD provides an increase in specific activity of heterologous DHAD.
[0103] Recombinant polynucleotides are typically cloned to express themselves using the coding sequence as part of a chimeric gene used for transformation, which includes a promoter operably linked to the coding sequence as well as a ribosome binding site and a region termination control. The coding region can be from the host cell for transformation and combined with regulatory sequences that are not native to the gene encoding DHAD. Alternatively, the coding region can be from another host cell.
[0104] Vectors useful for the transformation of a variety of host cells are common and described in the literature. Typically the vector contains a selectable marker and sequences that allow autonomous replication or chromosomal integration in the desired host. In addition, suitable vectors may comprise a promoter region that houses the transcriptional initiation controls and a transcriptional termination control region, among which such coding region DNA fragment may be insertion, to provide expression of the inserted coding region. Both control regions can be derived from genes homologous to the transformed host cell, although it should be understood that such control regions can also be derived from genes that are not native to the specific species chosen as a production host.
[0105] Yeast cells that can host the expression or overexpression of a heterologous bacterial DHAD are any yeast cells that are receptive to genetic manipulation and include, but are not limited to, Saccharomyces, Schizosaccharomyces, Hansenula, Cândida, Kluyveromyces , Yarrowia, Issatchenkia, and Pichia. Suitable strains include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces thermotolerans, Candida glabrata, Candida albicans, Pichia stipitis and Yarrowia lipolytica. In one embodiment, the host is Saccharomyces cerevisiae.
[0106] Expression is achieved by transforming a host cell with a gene that comprises a sequence encoding DHAD, for example, a DHAD listed in Tables 4a, 4b, 5 or 6, or identified using screening methods in the order listed in US 12 / 569,636, filed September 29, 2009, incorporated by reference in this document. The coding region for DHAD to be expressed can be codon optimized for the target host cell, as is well known to a person skilled in the art. Methods for gene expression in yeast are known in the art (see, for example, Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds .)), Elsevier Academic Press, San Diego, CA). Gene expression in yeast typically requires a promoter, operably linked to the coding region of interest, and a transcriptional terminator. Various yeast promoters can be used in the construction of expression cassettes for the genes in yeast, including, but not limited to, promoters derived from the following genes: CYC1, HIS3, GAL1, GAL 10, ADH1, PGK, PH05, GAPDH, ADC1 , TRP1, URA3, LEU2, ENO, TPI, CUP1, FBA, GPD, GPM and AOXl. Suitable transcriptional terminators include, but are not limited to, FBAt, GPDt, GPMt, ERGlOt, GAL It, CYC1, and ADH1.
[0107] Suitable promoters, transcriptional terminators, and DHAD coding regions can be cloned into alternating vectors of E. coli and yeast, and transformed into yeast cells. These vectors allow strain propagation in both E. coli and yeast strains. In one embodiment, the vectors used contain a selectable marker and sequences that allow autonomous replication or chromosomal integration in the desired host. Examples of plasmids used in yeast are alternating vectors pRS423, pRS424, pRS425, and pRS426 (American Type Culture Collection, Manassas, VA), which contains an E. coli origin of replication (eg, pMBl), an origin of replication of 2 microns of yeast, and a marker for nutritional selection. The selection markers for these four vectors are His3 (vector pRS423), Trpl (vector pRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426). The construction of expression vectors with a chimeric gene encoding the described DHADs can be performed either by standard molecular cloning techniques in E. coli or by the yeast gap repair recombination method.
[0108] The gap repair cloning approach has the advantage of highly efficient homologous recombination in yeast. For example, a yeast vector DNA is digested (for example, at its multiple cloning site) to create a "gap" in its sequence. Several inserted DNAs of interest are generated that contain a> 21 bp sequence at both the 5 'and 3' ends which overlap sequentially with each other, and with the 5 'and 3' termination of the vector DNA. For example, to construct a yeast expression vector for "Gene X", a yeast promoter and a yeast terminator are selected for the expression cassette. The promoter and terminator are amplified from yeast genomic DNA, and Gene X is either amplified by PCR from its source organism or obtained from a cloning vector that comprises the sequence of Gene X. There is at least one sequence 21 bp overlap between the 5 'end of the linearized vector and the promoter sequence, between the promoter and Gene X, between Gene X and the terminator sequence, and between the terminator and the 3' end of the linearized vector. The "gap" vector and the insertion DNAs are then co-transformed into a yeast strain and plated in a medium that contains the appropriate compound mixtures that allow the complementation of the nutritional selection markers on the plasmids. The presence of correct insertion combinations can be confirmed by PCR mapping through the use of plasmid DNA prepared from the selected cells. Plasmid DNA isolated from yeast (often in low concentration) can be transformed into an E. coli strain, for example, TOP 10, followed by minipreparations and restriction mapping to further verify the plasmid construct. Finally, the construct can be verified through sequence analysis.
[0109] Like the gap repair technique, integration into the yeast genome also benefits from the homologous recombination system in yeast. For example, a cassette containing a coding region plus control elements (promoter and terminator) and auxotropic marker is amplified by PCR with a high-fidelity DNA polymerase through the use of primers that hybridize to the cassette and contains 40 to 70 base pairs of sequence homology for the 5 'and 3' regions of the genomic area where insertion is desired. The PCR product is then made into yeast and plated in a medium containing the appropriate compound mixtures that allow selection for the integrated auxotropic marker. For example, to integrate "Gene X" into the "Y" chromosomal location, the regionX terminator construct that encodes promoter is amplified by PCR of a plasmid construct DNA and joined to an autotrophic marker (such as URA3) or by PCR of SOE or through digestion and cloning of common restriction. The entire cassette, which contains the regionX-terminator-i7ft43 region encoding the promoter, is amplified by PCR with primers containing 40 to 70 bp of homology with the 5 'and 3' regions of the "Y" location on the yeast chromosome . The PCR product is transformed into yeast and selected in a growth medium that lacks uracil. Transformants can be verified either by colony PCR or by chromosomal chromosomal DNA sequencing.
[0110] In addition to the above materials and methods that can be used to express a heterologous DHAD, those same materials and methods or the like, can be used to overexpress a heterologous DHAD through the use of modifications known to a person skilled in the art. For example, when using a plasmid-based system to overexpress the recombinant polynucleotide, a high copy number vector, or a vector with a regulable copy number, can be constructed. Such an adjustable or inducible system is described herein in Example 1; however, other systems are known to a person skilled in the art and can be used to construct another high copy number or adjustable copy number vectors. Alternatively, when using an integration-based system to overexpress the recombinant polypeptide, an integration vector is needed to target multiple integration sites. A multiple system based on integration is described in the present document in Example 2; however, systems based on multiple integration are known to a person skilled in the art and can be used to target multiple integrations of a recombinant polypeptide, for example, integration into regions of rDNA.
[0111] The expression of heterologous DHAD in the recombinant host cell can be quantified, for example, by a percentage of total cell protein. Such overexpression can be quantified in an amount selected from the group consisting of: (a) greater than about 0.001% of total cell protein; (b) greater than about 0.01% total cell protein; (c) greater than about 0.1% of total cell protein; (d) greater than about 0.5% total cell protein; (e) greater than about 1.0% of total cell protein; (f) greater than about 2.0% of total cell protein; (g) greater than about 5% total cell protein; (h) greater than about 10% total cell protein; and (i) greater than about 20% total cell protein.
[0112] The specific activity of heterologous DHAD produced in a recombinant host cell can be quantified, for example, as u / mg. The specific heterologous DHAD activity can be selected from the group consisting of: (a) greater than about 0.25 u / mg; (b) greater than about 0.3 u / mg; (c) greater than about 0.5 u / mg; (d) greater than about 1.0 u / mg; (e) greater than about 1.5 u / mg; (f) greater than about 2.0 u / mg; (g) greater than about 3.0 u / mg; (h) greater than about 4.0 u / mg; (i) greater than about 5.0 u / mg; (j) greater than about 6.0 u / mg; (k) greater than about 7.0 u / mg; (1) greater than about 8.0 u / mg; (m) greater than about 9.0 u / mg; (n) greater than about 10.0 u / mg; (o) greater than about 20.0 u / mg; and (p) greater than about 50.0 u / mg.
[0113] The specific heterologous DHAD activity can also be quantified, for example, as a percentage comparison with a specific endogenous DHAD activity or with some other specific control DHAD activity. An example of a specific "control" DHAD activity is that of a heterologous DHAD expressed in a recombinant host cell through the use of a low copy number plasmid or a plasmid that is not otherwise inducible or regulable. Such control establishes a baseline that compares the specific activity of the same heterologous DHAD expressed in a recombinant host cell through the use of a high copy number plasmid or a plasmid with copy number that can be regulated, or coexpressed with polynucleotides that encode polypeptides that affect Fe-S cluster biosynthesis or the absorption and utilization of Fe, as described below. Thus, the increase in specific activity of heterologous DHAD when compared to specific control DHAD activity can be in an amount selected from the group consisting of: greater than about 10% increase; greater than about 20% increase; greater than about 30% increase; greater than about 40% increase; greater than about 50% increase; greater than about 60% increase; greater than about 70% increase; greater than about 80% increase; greater than about 90% increase; greater than about 95% increase; greater than about 98% increase; and greater than about 99% increase. The specific heterologous DHAD activity can also be expressed by the "increase in times" above the control. Thus, the increase in specific activity can be selected from the group consisting of: (a) greater than about 2 times greater, (b) greater than about 5 times greater, (c) greater than about 8 times greater, or (d) greater than about 10 times greater than the control. FE-S CLUSTER FORMATION PROTEINS AND FE REGULATION, USE AND HOMEOSTASIS
[0114] As described above, DHAD enzymes need Fe-S clusters to function, so they need to be expressed in a host that has the genetic machinery to produce and load Fe-S clusters in the apoprotein if they will be to be expressed in functional form. As described elsewhere in this document, in normal yeast, mitochondria play an important role in Fe-S cluster biosynthesis. The flow in the formation and movement of Fe-S cluster precursors from the mitochondria to the Fe-S cluster that require proteins in the normal yeast cytosol is considered to be limited. For example, after a period, an additional increase in the expression of the heterologous DHAD protein in the cytosol does not result in a corresponding increase in DHAD activity. Without sticking to the theory, it is believed that this is due to the increased amounts of heterologous DHAD not being loaded with the necessary Fe-S cluster for the activity since a cell is not able to supply the increased demand for Fe-S clusters that arise under the conditions described above. It is demonstrated in this document that yeast cells can be genetically modified in 2 ways (separately or at the same time) which will result in an increased fraction of the heterologous DHAD expressed in the cytosol that is loaded with its necessary Fe-S cluster. One way is to modify the expression of yeast genes involved in the formation of Fe-S clusters, such as genes from the Fe-S cluster biosynthetic pathway or the absorption and utilization of Fe genes. The other way is to express the heterologous genes involved in Fe-S cluster biosynthesis or in the absorption and use of Fe in the yeast cytoplasm.
[0115] Yeast genes encoding expression modification. In the embodiments, the results of the modification in the increased function of a selected Fe-S cluster that requires protein.
[0116] As an example, Aftl was found to act as a transcriptional activator for genes in iron regulation (Kumanovics, et al. J. Biol. Chem., 2008. 283, p. 10276 to 10286; Li, H. , et al., The Yeast Iron Regulatory Proteins Grx3 / 4 is Fra2 form Heterodimeric Complexes Containing a [2Fe-2S] Cluster with Cysteinyl is Histidyl Ligation. Biochemistry, 2009. 48 (40): p. 9569 to 9581. As exemplified in the present document, the deletion of known Aftl translation inhibitors, results in an increase in the specific activity of a Fe-S cluster that requires protein since it leads to an increase in Fe-S cluster protein loading. if you stick to the theory, then it is believed that altering the expression of certain Fe-regulating genes, either directly or through the deletion or upward regulation of inhibitors, will likewise increase Fe loading and cluster function -S that requires proteins. For example, genes that play a role in, or are part of, use of Fe and homeostasis in yeast, such as Fe-regulating genes, can be targeted for altered expression. such genes are known in the art, and examples of these genes are listed in Table 7. (The list in Table 7 is taken from Rutherford, JC, et al., Activation of the Iron Regulon by the Yeast Aftl / Aft2 Transcription Factors Depends on Mitochondrial but Not Citosolic Ferro-Sulfur Protein Biogenesis., J. Biol. Chem., 2005. 280 (11): p. 10135 to 10140; Foury, F. and D. Talibi, Mitochondrial control of Iron homeostasis. gene expression in a yeast frataxin-deficient strain. J. Biol. Chem., 2001. 276 (11): p. 7762 to 7768; and Shakoury-Elizeh, M., et al., Transcriptional remodeling in response to deprivation by iron in Saccharomyces cerevisiae. Mol. Biol. Cell, 2004. 15 (3): p. 1233 to 1243.) TABLE 7. EXAMPLES OF YEAST GENES ASSOCIATED WITH THE ABSORPTION AND USE OF FE




[0117] Based on their functions and the association with the absorption and utilization of Fe, the proteins encoded by the genes presented in Table 7 are candidates for affecting the Fe-S cluster biosynthesis. The additional yeast genes associated with the absorption and use of Fe or Fe-S cluster biosynthesis include those listed in Table 8. TABLE 8. GENES ASSOCIATED WITH ABSORPTION AND USE OF YEAST OR CLUSTERFE-S BIOSYNTHESIS








[0118] Additional genes encoding polypeptides that affect Fe-S cluster biosynthesis of other host cells have been identified and include, but are not limited to, those genes listed in Table 9. TABLE 9. GENES DIRECTLY INVOLVED IN CLUSTERDE BIOSYNTHESIS MULTIPLE CELL FE-S





[0119] Fe absorption and metabolism genes and / or Fe-S cluster biosynthesis, including, but not limited to, those listed in Tables 7, 8 or 9 can potentially be deleted, mutated, expressed, regulated in a way upward or downwardly regulated to increase flow in a Fe-S cluster biosynthetic pathway and enhance the specific activity of proteins that require Fe-S clustering such as DHAD. Additionally, cofactors can be added to change the activity of polypeptides that have Fe-S cluster regulatory activity to increase flow in a Fe-S cluster biosynthetic pathway and enhance specific DHAD activity.
[0120] For example, genes that increase the flow of a Fe-S cluster biosynthetic pathway can be expressed to enhance DHAD activity by providing an adequate amount of Fe-S cluster for the apoenzyme. Any gene, or a combination thereof, such as one or more genes listed in Tables 7, 8, or 9, can be cloned and expressed in a pRS411 plasmid as described in Example 4. The resulting constructs, together with the expression vector DHAD pHR81 FBA ilvD (Sm), can then be transformed into wild-type BY4741. As a control, pRS411 without any gene of interest and the pHR81 FBA ilvD (Sm) vector are transformed into a wild type strain. Transformants are selected on agar plates with SD medium without uracil and methionine to maintain both plasmids as described in Example 4. The enzyme activity for DHAD in the crude extract of different strains of the transformation can be measured. The results can be compared with the specific activity obtained from the pRS411 control without any gene of interest and the transformed pHR81 FBA ilvD (Sm) vector and a wild type coat. An increase in specific activity indicates a gene that can be used to increase flow in a Fe-S cluster biosynthetic pathway.
[0121] In addition, strains without deletions in more than one of the genes involved in the Fe-S cluster regulatory activity can be created to provide additive effects in enhancing the enzymes or proteins that contain the Fe-S cluster (s) S. For example, double mutants with deletions in both the FRA2 and GXR3 genes can be used to transform the pHR81 vector FBA-IlvD (sm), and the DHAD activity in the raw extract of the transformants can be measured.
[0122] Another alternative is to alter the expression, for example, of the PSE1 gene (SEQ ID NO: 777), which encodes a protein involved in the import of AFT1p into the nucleus (Fukunaka, et al, 2003, J. Biological Chem., Volume 278, pages 50,120 to 50,127). The expression of this gene can be accompanied by cloning it into the vector pRS411 as described above.
[0123] Accordingly, recombinant host cells are provided in the present invention that comprise a change in the expression of any polypeptide encoded by a Fe absorption and use gene or Fe-S cluster biosynthesis. Recombinant host cells are included which comprise at least one heterologous polynucleotide from any of the Fe-S cluster biosynthesis genes referred to above. Also included are recombinant host cells in which the host cell comprises at least one deletion, mutation, and / or replacement in an endogenous gene of any of the Fe absorption and utilization genes or Fe-S cluster biosynthesis referred to above. . Also provided are recombinant host cells that comprise at least one heterologous polynucleotide than any of the Fe absorption and use genes or Fe-S cluster biosynthesis referred to above, wherein the host cell comprises at least one deletion, mutation, and / or replacement in an endogenous gene of any of the Fe absorption and utilization genes or Fe-S cluster biosynthesis referred to above.
[0124] These recombinant host cells may also comprise at least one protein that requires a heterologous Fe-S cluster. For example, a recombinant host cell comprising at least one heterologous DHAD and at least one heterologous polynucleotide encoding a polypeptide that affects Fe-S cluster biosynthesis is provided in the present invention. Also provided is a recombinant host cell comprising at least one heterologous DHAD, wherein the host cell comprises at least one deletion, mutation, and / or substitution in an endogenous gene that encodes a polypeptide that affects Fe-S cluster biosynthesis . A recombinant host cell is also provided that comprises at least one heterologous DHAD and at least one heterologous polynucleotide that encodes a polypeptide that affects Fe-S cluster biosynthesis, wherein the host cell comprises at least one deletion, mutation, and / or substitution in an endogenous gene that encodes a polypeptide that affects Fe-S cluster biosynthesis.
[0125] Host cells that can be used in the present invention include yeast host cells including, but not limited to, Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia and Pichia. Bacterial host cells can also be used to create recombinant host cells that comprise at least one heterologous polynucleotide that encodes a polypeptide that has DHAD activity and at least one heterologous polynucleotide that encodes a polypeptide that affects Fe-S cluster biosynthesis. For example, lactic acid bacteria comprising recombinant DHAD and at least one recombinant gene expression element encoding Fe-S cluster-forming proteins are the subject of Application No. US 12 / 569,103, filed on September 29, 2009 that is incorporated herein as a reference. The present recombinant host cells that comprise at least one heterologous polynucleotide that encodes a polypeptide that has DHAD activity and at least one heterologous polynucleotide that encodes a polypeptide that affects Fe-S cluster biosynthesis do not include the lactic bacteria described in Order n US 12 / 569,103, deposited on September 29, 2009, which is hereby incorporated by reference.
[0126] The polypeptide that affects the Fe-S cluster biosynthesis can be selected from the group consisting of the Fe absorption and utilization genes or the Fe-S cluster biosynthetic pathway in Tables 7, 8 and 9. In a In this embodiment, the polypeptide that affects Fe-S cluster biosynthesis is encoded by ARN1, ARN2, ATX1, CCC2, COT1, ENB1, FET3, FET5, FIT1, FIT2, FIT3, FKE1, FRE2, FRE3, FRE4, FRE5, FRE6 , FTH1, FTR1, HMX1, SIT1, SMF3, TIS11. VHT1, AFT1, AFT2, AIM1, ARH1, ATM1, BUD32, CADI, CCC1, CFD1, CIA1, CMK1, CTH1, CTI6, CYC8, DAP1, DRE2, ERV1, ESA1, FET4, FRA1, FRA2, GEF1, GGC1, GF1 GRX2, GRX4, GRX5, HDA1, IBA57, ISA1, ISA2, ISU1, ISU2, JAC1, MGE1, MRS3, MRS4, MSN5, NAR1, NFS1, NFU1, NHP6a, NHP6b, PSE1, SMF1, SNF1, SNF2, SNF3 SSQ1, TIM12, TUP1, NP_011911.1, VPS41, YAP5, YFH1, YRA1, ZPR1, iscAnif, nifU, nifS, cysE1, cysE2, iscS, iscU, iscA, hscB, hscA, Fdx, sufS, sufE, cysE iscA2, Nfu, nfuA, nfuV, nfu, sufA, sufB, sufC, sufD, sufE1, sufS2, or sufE2. In one embodiment, the polypeptide that affects Fe-S cluster biosynthesis is AFT1, AFT2, PSE1, FRA2, GRX3, or MSN5. In one embodiment, the polypeptide that affects Fe-S cluster biosynthesis is selected from the group consisting of AFT1, AFT2, PSE1, FRA2, GRX3, MSN5, and combinations thereof. In one embodiment, the polypeptide that affects Fe-S cluster biosynthesis is selected from the group consisting of AFT1, AFT2, PSE1, FRA2, MSN5, and combinations thereof. In another embodiment, the polypeptide that affects Fe-S cluster biosynthesis is selected from the group consisting of AFT1, AFT2, PSE1, FRA2, GRX3, MSN5, and combinations thereof, and the polypeptide that affects cluster biosynthesis of Fe-S is encoded by a polynucleotide that comprises a plasmid. In some embodiments, DHAD is coexpressed with AFT1, AFT2, PSE1, and combinations thereof. The polypeptide that affects Fe-S cluster biosynthesis can be a constitutive mutant, such as, but not limited to, AFT1 L99A, AFT1 L102A, AFT1 C291F, AFT1 C293F, and combinations thereof. The deletion, mutation, and / or substitution in the endogenous gene encoding a polypeptide that affects Fe-S cluster biosynthesis can be selected from the group consisting of FRA2, GRX3, MSN5, and combinations thereof.
[0127] The present invention also provides a method for increasing the activity of a protein that requires Fe-S cluster in a recombinant host cell that comprises providing a recombinant host cell that comprises a protein that requires Fe-S cluster, changing the expression or activity of a polypeptide that affects Fe-S cluster biosynthesis in the host cell and cultivating the recombinant host cell with the changed expression or activity under conditions under which the activity of the protein that requires Fe-S cluster is increased. Such a method can be used to increase the activity of a protein that requires an endogenous Fe-S cluster or a protein that requires a heterologous Fe-S cluster. Such a method can be used to increase the specific activity of a DHAD described in the present invention or identified by the methods described in the present invention. The increase in protein activity that requires Fe-S cluster can be in an amount selected from more than about 10%; more than about 15%; more than about 20%; more than about 25%; more than about 30%; more than about 35%; more than about 40%; more than about 45%; more than about 50%; more than about 55%; more than about 60%>; more than about 65%; more than about 70%>; more than about 75%; more than about 80%; more than about 85%; more than about 90%; and more than about 95%. The increase in activity can be more than about 3 times, more than about 5 times, more than about 8 times, or more than about 10 times. In the embodiments, the activity of the protein that requires Fe-S cluster can be in an amount that is at least about 60% theoretical, at least about 70% theoretical, at least about 80% theoretical, or at least least about 90% theoretical.
[0128] The present invention can also be used to increase flow in the Fe-S cluster biosynthetic pathway in a host cell and to identify polypeptides that increase flow in a Fe-S cluster biosynthetic pathway in a host cell . In one embodiment, a method is provided to increase flow in a Fe-S cluster biosynthetic pathway in a host cell comprising providing a recombinant host cell comprising a protein that requires Fe-S cluster and or at least one polypeptide that affects Fe-S cluster biosynthesis, at least one deletion, mutation, and / or replacement in an endogenous gene that encodes a polypeptide that affects Fe-S cluster biosynthesis, or a combination of both and cultivating the host cell recombinant under conditions according to which the flow in the Fe-S cluster biosynthetic pathway in the host cell is increased. In another embodiment, a method is provided to identify polypeptides that increase flow in a Fe-S cluster biosynthetic pathway in a host cell comprising: (a) changing the expression or activity of a polypeptide that affects the cluster biosynthesis of Fe-S; (b) measure the activity of a protein that requires a Fe-S cluster; and (c) comparing the activity of the protein that requires Fe-S cluster measured in the presence of the change in the polypeptide of the expression or activity of step (a) to the activity of the protein requiring Fe-S cluster measured in the absence of the change in the polypeptide of change or expression of step (a), in which an increase in the activity of the protein that requires Fe-S cluster indicates an increase in the flow of said Fe-S cluster biosynthetic pathway. In such methods, the protein that requires Fe-S cluster can be endogenous or heterologous to the host cell.
[0129] The expression or activity of the polypeptide that affects Fe-S cluster biosynthesis can be changed by methods well known in the art, including, but not limited to, deletion, mutation, substitution, expression, up-regulation, down-regulation, alteration of the cellular localization, alteration of the state of the protein, and / or addition of a cofactor, and combinations thereof. The change in the state of the protein may include, but is not limited to, such changes as phosphorylation or ubiquitination. Any number of methods described in the present invention or known in the art can be used to measure the activity of the protein that requires Fe-S cluster, depending on the protein that requires chosen Fe-S cluster. For example, if DHAD is the protein that requires an Fe-S cluster, the assay described in Example 7 can be used to measure DHAD activity to determine if there is an increase in flow in the Fe-S cluster biosynthetic pathway. host cell. ISOBUTANOL AND OTHER PRODUCTS
[0130] Expression of a DHAD in a recombinant host cell, as described above, provides the recombinant host cell transformed with dihydroxy acid dehydratase activity for conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α valerate -cetomethyl A product that has α-ketoisovalerate or α-cetomethylvalerate as a pathway intermediate can be produced more effectively in a host cell disclosed in the present invention that has the heterologous DHAD described. A list of such products includes, but is not limited to, valine, isoleucine, leucine, pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol and isobutanol.
[0131] For example, yeast valine biosynthesis includes the steps of converting acetolactate to 2,3-dihydroxyisovalerate by acetohydroxy acid reductisomerase (ILV5), converting 2,3-dihydroxyisovalerate to α-ketoisovalerate (also called 2-ketoisovalerate ) by dihydroxy acid dehydratase, and conversion of α-ketoisovalerate to valine by branched chain amino acid transaminase (BAT2) and branched chain amino acid aminotransferase (BAT1). Leucine biosynthesis includes the same steps for α-ketoisovalerate, followed by conversion of α-ketoisovalerate to alpha-isopropylmalate by alpha-isopropylmalate synthase (LEU9, LEU4), conversion of alpha-isopropylmalate to beta-isopropylmalate by isopropylmalate isomerase (LEU1) , conversion of beta-isopropylmalate to alpha-ketoisocaproate by beta-IPM dehydrogenase (LEU2), and finally conversion of alpha-ketoisocaproate to leucine by branched-chain amino acid transaminase (BAT2) and branched-chain aminotransferase (BAT1). The bacterial pathway is similar, involving proteins and genes named differently. The increased conversion of 2,3-dihydroxy-isovalerate to α-ketoisovalerate will increase flow in these pathways, particularly if one or more additional enzymes in a pathway are overexpressed. Thus, it is desirable for the production of valine or leucine to use a strain disclosed in the present invention.
[0132] Pantothenic acid biosynthesis includes a step performed by DHAD, as well as steps performed by ketopantoate hydroxymethyltransferase and pantothenate synthase. The manipulation of the expression of these enzymes for the marked production of pantothenic acid biosynthesis in microorganisms is described in US Patent No. 6,177,264.
[0133] DHAD's α-ketoisovalerate product is an intermediary in the isobutanol biosynthetic pathways disclosed in Patent Application Publication No. US 20070092957 A1, which is incorporated herein by reference. A diagram of the revealed isobutanol biosynthetic pathways is provided in Figure 5. The production of isobutanol in a strain disclosed in the present invention can benefit from the increased DHAD activity. As disclosed in the present invention, increased DHAD activity is provided by the expression of a DHAD in a host cell, for example, by overexpression of DHAD, by modulating the expression or activity of a polypeptide that has Fe-S cluster regulatory activity , or a combination of both DHAD expression and modulation of the expression or activity of a polypeptide that has Fe-S cluster regulatory activity. As described in Patent Application Publication No. 20070092957 A1, which is hereby incorporated by reference, the steps in an example isobutanol biosynthetic pathway include the conversion of: - pyruvate to acetolactate (see Figure 5, step via a therein), as catalyzed, for example, by acetolactate synthase, - acetolactate into 2,3-dihydroxyisovalerate (see Figure 5, step b therein) as catalyzed, for example, by acetohydroxyacid isomeroredutase; - 2,3-dihydroxyisovalerate in α-ketoisovalerate (see Figure 5, step c in it) as catalyzed, for example, by acetohydroxy acid dehydratase, also called dihydroxy acid dehydratase (DHAD); - α-ketoisovalerate in isobutyraldehyde (see Figure 5, step d in it) as catalyzed, for example, by branched chain α-keto acid decarboxylase; and - isobutyraldehyde in isobutanol (see Figure 5, pathway and in it) as catalyzed, for example, by branched chain alcohol dehydrogenase.
[0134] The substrate for product and enzyme conversions involved in these reactions, for steps f, g, h, I, j, and k of alternative pathways are described in Patent Application Publication No. US 20070092957 A1, which is incorporated into present as a reference.
[0135] The genes that can be used for the expression of the pathway enzymes named above other than the DHADs disclosed in the present invention, as well as those for additional isobutanol pathways, are described in Patent Application Publication No. US 20070092957 A1, which is incorporated by reference. Additional genes that can be used may be by one skilled in the art through bioinformatics or using methods well known in the art, such as the various methods disclosed in Application No. US 12 / 569,636, filed September 29, 2009 that is incorporated into the present as a reference, to isolate counterparts. Suitable ketol-acid redutoisomerase (KARI) enzymes are described in Patent Application Publication No. US 20080261230 A1, 20090163376, 20100197519 and Application No. US 12/893077, all of which are incorporated herein by reference. The examples of KARIs disclosed therein are not those of Vibrio cholerae, Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens PF5. Patent Application Publication No. US 2009/0269823 and Provisional Patent Application No. US 61 / 290,636, incorporated herein by reference, describe suitable alcohol dehydrogenases.
[0136] Additionally, are described in Patent Application Publication No. US 20070092957 A1, which is incorporated herein by reference, chimeric construction genes and genetic manipulation of bacteria and yeasts for the production of isobutanol using the disclosed biosynthetic pathways. ADDITIONAL MODIFICATIONS
[0137] Examples of additional modifications that may be useful in the cells provided in the present invention include modifications to reduce glycerol-3-phosphate dehydrogenase activity and / or disruption in at least one gene encoding a polypeptide that has pyruvate activity decarboxylase or an interruption in at least one gene encoding a regulatory element that controls the expression of the pyruvate decarboxylase gene as described in US Patent Application Publication No. 20090305363 (incorporated herein by reference), modifications to a host cell that provide increased carbon flux through an Entner-Doudoroff Path or balance reduction of balance equivalents as described in Patent Application Publication No. US 20100120105 (incorporated herein by reference). Other modifications include the integration of at least one polynucleotide that encodes a polypeptide that catalyzes a step in a biosynthetic pathway using pyruvate described in Provisional Application Publication No. US 61/380563 (incorporated herein by reference). Additional modifications that may be appropriate are described in Serial Order US 12/893089. In addition, host cells that comprise a heterologous polynucleotide that encodes a polypeptide with phosphocetolase activity and host cells that comprise a heterologous polynucleotide that encodes a polypeptide with phosphotransacetylase activity are described in Provisional Patent Application No. 61/356379. CULTIVATION FOR PRODUCTION
[0138] The recombinant host cells disclosed in the present invention are cultured in a fermentation medium containing suitable carbon substrates. Suitable carbon substrates may include, but are not limited to, monosaccharides such as glucose, fructose, oligosaccharides such as lactose, maltose, galactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures of raw materials renewable sources such as whey permeate, corn maceration, beet molasses and barley malt. Other carbon substrates can include ethanol, lactate, succinate or glycerol.
[0139] In addition, the carbon substrate can also be substrates of a carbon such as carbon dioxide or methanol for which metabolic conservation in key biochemical intermediates has been demonstrated. Two-carbon substrates such as ethanol may also be suitable. In addition to one or two carbon substrates, methylotrophic organisms are known to use a variety of carbon-containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeasts are known to use methylamine carbon to form trehalose or glycerol (Bellion et ah, Microb. Growth C1 Compd., [Int. Symp.], 7a (1993), 415 to 32, Editor (s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, several species of Candida will metabolize alanine or oleic acid (Suiter et ah, Arch. Microbiol. 153: 485 to 489 (1990)). Therefore, it is contemplated that the carbon source used in the present invention can encompass a wide variety of carbon containing substrates and will only be limited by the choice of the organism.
[0140] Although it is contemplated that all the carbon substrates mentioned above and mixtures thereof are suitable in the present invention, in some embodiments, the carbon substrates are glucose, fructose and sucrose, or mixtures thereof with C5 sugars such as xylose and / or arabinose for yeast cells modified to use C5 sugars. Sucrose can be derived from renewable sources of sugar such as sugar cane, beets, cassava, sweet sorghum and mixtures thereof. Glucose and dextrose can be derived from renewable sources of grains through saccharification of starch-based raw materials including grains such as corn, wheat, rye, barley, oats and mixtures thereof. In addition, fermentable sugars can be derived from renewable cellulosic or lignocellulosic biomass through pre-treatment and saccharification processes, as described, for example, in the corresponding patent application Publication No. 20070031918 A1, which is hereby incorporated into this reference title. Biomass refers to any cellulosic or lignocellulosic material and includes materials that comprise cellulose, and optionally that further comprise hemicellulose, lignin, starch, oligosaccharides and / or monosaccharides. The biomass can also comprise additional components, such as protein and / or lipid. The biomass can be derived from a single source, or the biomass can comprise a mixture derived from more than one source; for example, biomass can comprise a mixture of ears of corn and corn straw or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural waste, solid urban waste, industrial solid waste, papermaking sludge, garden waste, wood and forest waste. Examples of biomass include, but are not limited to, corn kernels, corn cobs, crop residues such as corn husks, corn husks, grasses, wheat, wheat straw, barley, barley straw, hay, straw rice, fodder, used paper, sugarcane bagasse, sorghum, soy, components obtained from grinding grains, trees, branches, roots, leaves, wood fragments, sawdust, shrubs and bushes, vegetables, fruits, flowers , animal excrement and mixtures thereof.
[0141] In addition to an appropriate carbon source, the culture medium contains minerals, salts, cofactors, buffers and other suitable components, known to those skilled in the art, suitable for the cultivation of cultures and the promotion of an enzymatic pathway comprising an protein that requires Fe-S cluster such as, for example, DHAD. CULTURE CONDITIONS
[0142] Typically the cells are grown at a temperature in the range of about 20 ° C to about 40 ° C in an appropriate medium. Suitable culture media in the present invention are commercially prepared media such as Luria Bertani broth (LB), Sabouraud Dextrose broth (SD), Yeast Medium broth (YM), or broth that includes nitrogen and yeast base, sulfate and dextrose (as the carbon / energy source) or Medium YPD, a mixture of peptone, yeast extract and dextrose in ideal proportions to grow most Saccharomyces cerevisiae strains. Other defined or synthetic culture media can also be used and the appropriate medium for cultivating the particular microorganism will be known to one skilled in the microbiology technique or fermentation science. The use of known agents to modulate the catabolite repression directly or indirectly, for example, 2 ': 3'-cyclic adenosine monophosphate, can also be incorporated into the culture medium.
[0143] The pH ranges suitable for cultivation are between about pH 5.0 to about pH 9.0, In one embodiment, about pH 6.0 to about pH 8.0 is used for the initial condition . The pH ranges suitable for yeast fermentation are typically between about pH 3.0 to about pH 9.0. In one embodiment, about pH 5.0 to about pH 8.0 is used for the initial condition . The pH ranges suitable for fermentation of other microorganisms are between about pH 3.0 to about pH 7.5. In one embodiment, about pH 4.5 to about pH 6.5 is used for the initial condition.
[0144] Cultivation can be carried out under aerobic or anaerobic conditions. In one embodiment, aerobic or anaerobic conditions are used for cultivation. INDUSTRIAL FERMENTATIONS IN BULK AND CONTINUOUS
[0145] Isobutanol, or other products, can be produced using a batch fermentation method. A classic batch fermentation is a closed system in which the composition of the medium is defined at the beginning of the fermentation and is not subjected to artificial changes during fermentation. A variation on the standard batch system is the powered batch system. The fed batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation proceeds. Batch fed systems are useful when catabolite suppression is able to inhibit cell metabolism where it is desirable to have limited amounts of substrate in the medium. Batch and fed batch fermentations are common and well known in the art and examples can be found in Thomas D. Brock and, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA. , or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36: 227, (1992), incorporated by reference into the present invention.
[0146] Isobutanol, or other products, can also be produced using continuous fermentation methods. Continuous fermentation is an open system in which a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains cultures at a constant high density where cells are primarily in log phase cultivation. Continuous fermentation allows the modulation of one factor or any number of factors that affect cell culture or the concentration of the final product. Methods of modulating nutrients and cultivation factors for continuous fermentation processes as well as techniques to maximize the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.
[0147] It is contemplated that the production of isobutanol, or other products, can be practiced using batch, fed batch or continuous processes and that any known mode of fermentation would be adequate. Additionally, it is contemplated that the cells can be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for the production of isobutanol. METHODS FOR ISOLATION OF ISOBUTANOL FROM THE FERMENTATION MEDIUM
[0148] Bioproduced isobutanol can be isolated from the fermentation medium using methods known in the art for ABE fermentations (see, for example, Durre, Appl. Microbiol. Biotechnol. 4P: 639 to 648 (1998), Groot et al, Process Biochem 27:61 to 75 (1992), and references therein). For example, solids can be removed from the fermentation medium by centrifugation, filtration, decantation or the like. Then, isobutanol can be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas extirpation, membrane evaporation or pervaporation.
[0149] Due to the fact that isobutanol forms a low boiling azeotropic mixture, distillation can be used to separate the mixture down to its azeotropic composition. Distillation can be used in combination with another separation method to achieve separation close to the azeotrope. Methods that can be used in combination with distillation to isolate and purify butanol include, but are not limited to, decantation, liquid-liquid extraction, adsorption and membrane-based techniques. In addition, butanol can be isolated using azeotropic distillation using a charger (see, for example, Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).
[0150] The mixture of butanol and water forms a heterogeneous azotrope so that distillation can be used in combination with decantation to isolate and purify isobutanol. In this method, the fermentation broth containing isobutanol is distilled to almost the azeotropic composition. Then, the azeotropic mixture is condensed and the isobutanol is separated from the fermentation medium by decantation. The decanted aqueous phase can be returned to the first distillation column as a reflux. The decanted organic phase rich in isobutanol can be further purified by distillation on a second distillation column.
[0151] Isobutanol can also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, isobutanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The isobutanol-containing organic phase is then distilled to separate the butanol from the solvent.
[0152] Distillation in combination with adsorption can also be used to isolate isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled next to the azeotropic composition and then the remaining water is removed using an adsorbent, such as molecular sieves (Aden et al. Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co- Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover, Report NREL / TP-510-32438, National Renewable Energy Laboratory, June 2002).
[0153] Additionally, distillation in combination with pervaporation can be used to isolate and purify isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled next to the azeotropic composition and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo et al., J. Membr. Sci. 245, 199 to 210 (2004 )). ACCOMPLISHMENTS OF THE INVENTIONS
[0154] Accomplishment 1 (E1). A recombinant host cell comprising at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity in which said at least one heterologous polynucleotide comprises a high copy number plasmid or a plasmid with a regulable number of copies.
[0155] E2. A recombinant host cell comprising at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity in which said at least one heterologous polynucleotide is integrated at least once in the DNA of the recombinant host cell.
[0156] E3. A recombinant host cell comprising at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity, wherein said host cell comprises at least one deletion, mutation, and / or substitution in an endogenous gene that encodes a polypeptide that affects the Fe-S cluster biosynthesis.
[0157] E4. A recombinant host cell that comprises at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity and at least one heterologous polynucleotide that encodes a polypeptide that affects Fe-S cluster biosynthesis.
[0158] E5. The recombinant host cell of any of the E3 to E4 embodiments, wherein said heterologous polynucleotide encoding a polypeptide that affects the Fe-S cluster biosynthesis is selected from the group consisting of the genes in Tables 8 and 9.
[0159] E6. The recombinant host cell of any of the E3 to E4 embodiments, wherein said heterologous polynucleotide encoding a polypeptide that affects the Fe-S cluster biosynthesis is selected from the group consisting of the genes in Table 7.
[0160] E7. The recombinant host cell of any of the E5 to E6 embodiments, wherein said heterologous polynucleotide encoding a polypeptide that affects the Fe-S cluster biosynthesis is selected from the group consisting of AFT1, AFT2, PSE1, FRA2, GRX3, MSN5 and combinations thereof.
[0161] E8. The recombinant host cell of embodiment E7, wherein said polypeptide is encoded by a polynucleotide that is a constitutive mutant.
[0162] E9. The recombinant host cell of embodiment E8, wherein said constitutive mutant is selected from the group consisting of AFT1 L99A, AFT1 L102 A, AFT1 C291F, AFT1 C293F, and combinations thereof.
[0163] E10. The recombinant host cell of the E7 embodiment, wherein said polypeptide that affects Fe-S cluster biosynthesis is encoded by a polynucleotide comprising a high copy number plasmid or a plasmid with a regulable copy number.
[0164] E11. The recombinant host cell of the E7 embodiment, wherein said polypeptide that affects Fe-S cluster biosynthesis is encoded by a polynucleotide integrated at least once in the DNA of the recombinant host cell.
[0165] E12. The recombinant host cell of the E3 realization, in which at least one deletion, mutation, and / or substitution in an endogenous gene encoding a polypeptide that affects the Fe-S cluster biosynthesis is selected from the group consisting of FRA2, GRX3 , MSN5 and combinations thereof.
[0166] E13. The recombinant host cell of the E4 embodiment, wherein the at least one heterologous polynucleotide encoding a polypeptide that affects the Fe-S cluster biosynthesis is selected from the group consisting of AFT1, AFT2, PSE1 and combinations thereof.
[0167] E14. The recombinant host cell of any of the E3 to E13 embodiments, wherein said at least one heterologous polynucleotide encoding a polypeptide that has dihydroxy acid dehydratase activity is expressed in multiple copies.
[0168] E15. The recombinant host cell of embodiment E14, wherein said at least one heterologous polynucleotide comprises a high copy number plasmid or a plasmid with a number of copies that can be regulated.
[0169] E16. The recombinant host cell of embodiment E14, wherein said at least one heterologous polynucleotide is integrated at least once in the DNA of the recombinant host cell.
[0170] E17. The recombinant host cell of any of the E3 to E16 embodiments, wherein said Fe-S cluster biosynthesis is increased compared to a recombinant host cell that has endogenous Fe-S cluster biosynthesis.
[0171] E18. The recombinant host cell of any of the E1 to E17 embodiments, wherein said host cell is a yeast host cell.
[0172] E 19. The recombinant host cell of the E 18 embodiment, wherein said yeast host cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia and Pichia.
[0173] E20. The recombinant host cell of any of the E1 to E19 embodiments, wherein said heterologous polypeptide having dihydroxy acid dehydratase activity is expressed in the cytosol of the host cell.
[0174] E21. The recombinant host cell of any of the E1 to E20 embodiments, wherein said heterologous polypeptide having dihydroxy acid dehydratase activity has an amino acid sequence that corresponds to the HMM Profile of Table 12 with an E value of <10-5 in which the polypeptide it also comprises all three conserved cysteines, corresponding to positions 56, 129 and 201 in the amino acid sequences of the DHAD enzyme of Streptococcus mutans corresponding to SEQ ID NO: 168.
[0175] E22. The recombinant host cell of any of the E1 to E21 embodiments, wherein 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.
[0176] E23. The recombinant host cell of any of the E1 to E22 embodiments, wherein said polypeptide having dihydroxy acid dehydratase activity has a specific activity selected from the group consisting of: a. more than about 5 times with respect to the control host cell that comprises at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity; B. more than about 8 times with respect to the control host cell that comprises at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity; and c. more than about 10 times with respect to the control host cell that comprises at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity.
[0177] E24. The recombinant host cell of any of the E1 to E22 embodiments, wherein said polypeptide having dihydroxy acid dehydratase activity has a specific activity selected from the group consisting of: a. more than about 0.25 U / mg; B. more than about 0.3 U / mg; ç. more than about 0.5 U / mg; d. more than about 1.0 U / mg; and. more than about 1.5 U / mg; f. more than about 2.0 U / mg; g. more than about 3.0 U / mg; H. more than about 4.0 U / mg; i. more than about 5.0 U / mg; j. more than about 6.0 U / mg; k. more than about 7.0 U / mg; l. more than about 8.0 U / mg; m. more than about 9.0 U / mg; n. more than about 10.0 U / mg; The. more than about 20.0 U / mg; and p. more than about 50.0 U / mg.
[0178] E25. The recombinant host cell of any of the embodiments E1 to E24, wherein said recombinant host cell produces isobutanol.
[0179] E26. The recombinant host cell of embodiment E25, wherein said recombinant host cell comprises an isobutanol biosynthetic pathway.
[0180] E27. A method of producing a product that comprises: a. providing the recombinant host cell of any of the E1 to E24 embodiments; and b. placing the recombinant host cell of (a) in contact with a fermentable carbon substrate in a fermentation medium under conditions in which said product is produced; wherein the product is selected from the group consisting of branched amino acids, pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol, isobutanol and combinations thereof.
[0181] E28. A method of producing isobutanol which comprises: a. providing the recombinant host cell of any of the E1 to E24 embodiments; B. placing the recombinant host cell of (a) in contact with a fermentable carbon substrate in a fermentation medium under conditions in which isobutanol is produced.
[0182] E29. A method for converting 2,3-dihydroxyiso valerate to an α-ketoisovalerate comprising: a. providing the recombinant host of any of the E1 to E24 embodiments; B. cultivating the recombinant host cell of (a) under conditions where 2,3-dihydroxyisovalerate is converted to α-ketoisovalerate, where 2,3-dihydroxyisovalerate is converted to α-ketoisovalerate.
[0183] E30. A method for increasing the specific activity of a heterologous polypeptide that has dihydroxy acid dehydratase activity in a recombinant host cell that comprises: a. providing a recombinant host cell of any of the E1 to E24 embodiments; and b. cultivating the recombinant host cell of (a) under conditions under which the heterologous polypeptide that has dihydroxy acid dehydratase activity is expressed in functional form that has a specific activity greater than the same host cell that lacks said heterologous polypeptide.
[0184] E31. A method for increasing flow in a Fe-S cluster biosynthetic pathway in a host cell comprising: a. providing a recombinant host cell of any of the E3 to E24 embodiments; and b. culturing the recombinant host cell from (a) under conditions by which the flow in the Fe-S cluster biosynthetic pathway in the host cell is increased.
[0185] E32. A method for increasing the activity of a protein that requires a Fe-S cluster in a recombinant host cell comprising: a. providing a recombinant host cell that comprises a protein that requires Fe-S cluster; B. changing the expression or activity of a polypeptide that affects Fe-S cluster biosynthesis in said host cell; and c. cultivating the recombinant host cell of (b) under conditions by which the activity of the protein that requires Fe-S cluster is increased.
[0186] E33. The E32 realization method, in which said increase in activity is a selected quantity from the group consisting of: a. more than about 10%; B. more than about 20%; ç. more than about 30%; d. more than about 40%; and. more than about 50%; f. more than about 60%; g. more than about 70%; H. more than about 80%; i. more than about 90%; and j. more than about 95%.
[0187] E34. The E32 realization method, in which said increase in activity is a selected quantity from the group consisting of: a. more than about 5 times, b. more than about 8 times, c. more than about 10 times.
[0188] E35. A method for identifying polypeptides that increases flow in a Fe-S cluster biosynthetic pathway in a host cell comprising: (a) changing the expression or activity of a polypeptide that affects Fe-S cluster biosynthesis; (b) measure the activity of a heterologous protein that requires a Fe-S cluster; and (c) comparing the activity of the heterologous protein that requires Fe-S cluster measured in the presence of the expression or changed activity of a polypeptide from step (a) to the activity of the heterologous protein that requires Fe-S cluster measured in the absence of expression or changed activity of a polypeptide from step (a), wherein an increase in the activity of the heterologous protein that requires Fe-S cluster indicates an increase in flow in said Fe-S cluster biosynthetic pathway.
[0189] E36. A method for identifying polypeptides that increases flow in a Fe-S cluster biosynthetic pathway in a host cell comprising: a. changes the expression or activity of a polypeptide that affects Fe-S cluster biosynthesis; B. measuring the activity of a polypeptide that has dihydroxy acid dehydratase activity; and c. compare the activity of the polypeptide that has dihydroxy acid dehydratase activity measured in the presence of the change in expression or activity of a polypeptide from step (a) to the activity of the polypeptide that has dihydroxy acid dehydratase activity measured in the absence of change in the expression or activity of a polypeptide from step (a), in which an increase in the activity of the polypeptide that has dihydroxy acid dehydratase activity indicates an increase in flow in said Fe-S cluster biosynthetic pathway.
[0190] E37. The method of any of the E30 to E36 embodiments, wherein said change in the expression or activity of a polypeptide that affects the Fe-S cluster biosynthesis comprises deletion, mutation, substitution, expression, upward regulation, downward regulation, alteration of cell localization, change in protein status, and / or addition of a cofactor.
[0191] E38. The method of any of the realizations E32 to E37, in which the protein that requires Fe-S cluster has dihydroxy acid dehydratase activity and in which the said protein that requires Fe-S cluster that has dihydroxy acid dehydratase activity has an amino acid sequence that corresponding to the HMM Profile of Table 12 with an E value of 10-5 in which the polypeptide further comprises all three conserved cysteines corresponding to positions 56, 129, and 201 in the amino acid sequences of the DHAD enzyme of Streptococcus mutans corresponding to SEQ ID NO : 168.
[0192] E39. Method according to any of the realizations E32 to E38, in which said polypeptide that affects Fe-S cluster biosynthesis is selected from the group consisting of the genes in Tables 7, 8 and 9.
[0193] E40. A recombinant host cell that comprises at least one polynucleotide that encodes a polypeptide identified by the methods of any of the embodiments E35 to E37.
[0194] E41. The recombinant host cell of embodiment E40, wherein said host cell further comprises at least one heterologous polynucleotide that encodes a polypeptide that has dihydroxy acid dehydratase activity.
[0195] E42. The recombinant host cell of embodiment E41, wherein said heterologous polynucleotide encoding a polypeptide that has dihydroxy acid dehydratase activity is expressed in multiple copies.
[0196] E43. The recombinant host cell of embodiment E41, wherein said heterologous polynucleotide comprises a high copy number plasmid or a plasmid with a regulable copy number.
[0197] E44. The recombinant host cell of embodiment E41, wherein said heterologous polynucleotide is integrated at least once in the DNA of the recombinant host cell.
[0198] E45. The E35 or E36 embodiment method, wherein said host cell is a yeast host cell.
[0199] E46. The method of carrying out E45, wherein said yeast host cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia and Pichia.
[0200] E47. Method according to any of the embodiments E28 to E39, wherein said host cell is a yeast host cell.
[0201] E48. The E47 embodiment method, wherein said yeast host cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia and Pichia.
[0202] E49. The recombinant host cell of any of the E40 to E44 embodiments, wherein said recombinant host cell is a yeast host cell.
[0203] E50. The recombinant host cell of embodiment E49, wherein said yeast host cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia and Pichia.
[0204] E51. The recombinant host cell of any of the E40 to E44 or E49 to E50 embodiments, wherein said heterologous polypeptide having dihydroxy acid dehydratase activity is expressed in the cytosol of the host cell.
[0205] E52. The recombinant host cell of any of the E40 to E44 or E49 to E50 embodiments, wherein said heterologous polypeptide having dihydroxy acid dehydratase activity has an amino acid sequence that corresponds to the HMM Profile of Table 12 with an E value of <10-5em that the polypeptide further comprises all three conserved cysteines corresponding to positions 56, 129 and 201 in the amino acid sequences of the DHAD enzyme of Streptococcus mutans corresponding to SEQ ID NO: 168.
[0206] E53. The recombinant host cell of any of the E40 to E44 or E49 to E50 embodiments, wherein 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.
[0207] E54. The recombinant host cell of embodiment E53, wherein said recombinant host cell produces isobutanol.
[0208] E55. The recombinant host cell of embodiment E54, wherein said recombinant host cell comprises an isobutanol biosynthetic pathway. EXAMPLES
[0209] The meaning of the abbreviations used is as follows: "min" means minute (s), "h" means hour (s), "sec" means second (s), "μl" means microliter (s), "ml "means milliliter (s)," l "means liter (s)," nm "means nanometer (s)," mm "means millimeter (s)," cm "means centimeter (s)," μm 'means micrometer (s ), "mM" means millimolar, "M" means molar, "mmol" means millimol (es), "μmol" means micromol (s), "g" means gram (s), "μg" means microgram (s), "mg" means milligram (s), "rpm" means revolutions per minute, "w / v" means weight / volume, "OD" means optical density and "OD600" means optical density measured at a wavelength of 600 nm. GENERAL METHODS
[0210] The standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY, 1989, by TJ Silhavy, ML Bennan, and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1984, and by Ausubel, FM et ah, Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987.
[0211] Suitable materials and methods for the maintenance and cultivation of bacterial cultures are also well known in the art. Techniques suitable for use in the following Examples can be found in Manual of Methods for General Bacteriology, Phillipp Gerhardt, RGE Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds., American Society for Microbiology, Washington, DC, 1994, or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA, 1989. All reagents, enzymes from restriction and materials used for the cultivation and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, WI), BD Diagnostic Systems (Sparks, MD), Life Technologies (Rockville, MD), or Sigma Chemical Company (St. Louis, MO), unless otherwise specified. EXAMPLE 1. DHAD PROTEIN OVEREXPRESSION CODED BY THE S. MUTANS ILVD GENE WITH THE USE OF A PLASMID-BASED SYSTEM IN YEAST CYTOSOL
[0212] Overexpression of a recombinant polynucleotide can be accomplished by increasing the number of copies of a plasmid comprising the recombinant polynucleotide. To overexpress the DHAD protein in yeast, an inducible vector was constructed. The pHR81 vector contains a Ura3 marker as well as a LEU marker with a defective promoter (see Patent Application Publication No. US 2007/0092957, which is incorporated herein by reference). When the synthetic yeast abandonment culture medium (SD; also known as complete minimum medium; Teknova) is switched from SD less uracil to SD less leucine, the number of copies of the plasmid pHR81 increases, resulting in a much higher level of expression of the recombinant polynucleotide. The main structure of the pHR81 vector was derived from pLH472 JEG4y (SEQ ID NO: 921) and was prepared by digesting the pLH472 JEG4y vector with Spel and SacII.
[0213] For overexpression of a DHAD protein, the S. mutans DHAD gene ilvD (SEQ ID NO: 167) was used (see Published Patent Application No. US2009-0305363A1, which is incorporated herein as reference). This gene was cloned under the control of the FBA promoter in the vector pRS423 FBA ilvD Strep-lumio (see Published Patent Application No. US2009-0305363A1, which is hereby incorporated by reference). The region containing the FBA promoter, the ilvD gene and the FBA terminator cassette was amplified with the FBAp-F (NheI) and FBAt-R (SacII) primer set (SEQ ID NOs: 915 and 916) and cloned into the pHR81 vector. The resulting expression vector was designated as pHR81 FBA-IIvD (Sm) (SEQ ID NO: 917; Figure 1A).
[0214] To overexpress the DHAD protein of S. mutans, the pHR81 expression vector FBA-I1vD (Sm) was transformed into the wild type yeast strain BY4741. The transformants were selected on agar plates with SD less uracil. For overexpression, the yeast strains that contained the plasmid were initially cultured at 30 ° C in SD medium less uracil. A fresh overnight culture (5 ml) was then transferred to a 125 ml flask containing 75 ml of SD medium minus leucine. As a control, another 5 ml of fresh overnight culture was transferred into a flask containing 75 ml of SD less uracil. The cultures were incubated overnight before collection by centrifugation. DHAD activity was measured in raw extracts from these samples using the assay described in Example 7.
[0215] The specific activity of DHAD obtained in the raw extract in control samples grown in in SD less uracil was in the range of 0.2 to 0.3 U mg-1. The average specific activity obtained from strains grown in the SD medium less leucine, however, was 1.6 U mg-1, much higher (~ 5 to 8 times higher) than the activity of the control samples. DHAD requires Fe-S cluster for its function and it was not previously known whether the native yeast Fe-S cluster biosynthetic pathway could accommodate a protein that requires overexpressed Fe-S cluster in the yeast cytosol. In a previous screening experiment using a low copy number vector not inducible to DHAD of S. mutans it could be expressed recombinantly in yeast cytosol with a specific activity in the range of 0.1 to 0.2 U mg -1 in the raw extract (see Patent Application No. US 12 / 569,636, filed on September 29, 2009, which is incorporated herein by reference). Thus, in one embodiment, overexpression of a protein that requires a Fe-S cluster, such as DHAD, in yeast using a high copy number vector provides increased specific activity, in which the specific activity is increased by at least about 5 times to at least about 8 times. EXAMPLE 2. DHAD PROTEIN OVEREXPRESSION CODED BY THE S. MUTANS ILVD GENE THROUGH CHROMOSOME INTEGRATION
[0216] An alternative way to increase the expression of a yeast gene is to integrate multiple copies of the gene of interest into the host cell's chromosome. To integrate the S. mutans ilvD gene (SEQ ID NO: 167) into a yeast chromosome, the integration vector pZK-Delta (s) -Leu2-FBA-ilvD (Sm) -FBAt (SEQ ID NO: 918; Figure 1B) was built. The main structure of the integration vector was derived from pSuperscript (Stratagene, La Jolla, CA). The S. mutans ilvD gene (nucleotides 1306 to 3018 of the complement filament) was cloned into the integration vector under the control of the FBA promoter (nucleotides 3026 to 4023 of the complement filament) so that the ilvD gene could be flanked by a yeast delta sequence (nucleotides 118 to 267 and 5,061 to 5,760 of the complement filament). S. cerevisiae contains more than 200 delta yeast sequences (Kim J M et al. Genome Res. 1998; 8: 464 to 478). These delta strings are targets for multiple integrations. The integration vector was also manipulated to contain the defective LEU2 marker (nucleotides 4,100 to 5,191 of the complement filament) for selection of transformed strains with multiple integration events.
[0217] For integration, the vector DNA was linearized with digestion of AscI and AatII to generate filaments flanked by the vector's delta DNA sequence comprising the ilvD gene, which were then transformed to form the yeast strain BY4741. The transformants were selected using SD agar minus leucine. These transformants were then grown in liquid SD medium less leucine at 30 ° C, and the cultures were collected and analyzed by DHAD activity. The specific DHAD activity obtained in the crude extract was in the range of 0.7 to 1.2 U mg-1. This specific activity was about 3 to 6 times higher than that found in BY4741 strains transformed with a plasmid containing the ilvD gene without overexpression EXAMPLE 3. IMPROVEMENT OF DHAD'S SPECIFIC ACTIVITY IN YEAST DELETION STREETS
[0218] Although the overexpression strains described in Examples 1 and 2 had a high level of activity, not all of the expressed DHAD protein was activated. For example, the overexpressed DHAD protein accounted for approximately 5 to 10% of the total cellular protein, while yielding a specific activity of about 0.7 to 1.6 U mg-1. Given that the specific activity of the purified DHAD enzyme of S. mutans is 100 U mg-1, the expression of DHAD at 10% of total cellular protein could be expected to yield a specific activity above 5 to 10 U mg-1. While not wishing to link to a theory, the difference between the expected and observed specific activity was probably a result of insufficient Fe-S cluster loading. Thus, increasing Fe-S cluster loading by additional manipulation of overexpression strains could be used to increase specific DHAD activity.
[0219] In order to improve specific activity, yeast strains with deletions in the genes involved in iron metabolism and Fe-S cluster perception were used to investigate their effects on specific DHAD activity. These strains (antecedents of BY4741) were purchased from Open Biosystem (Huntsville, AL) and are listed in Table 10. As described in Example 1, the high pHR81 copy number plasmid FBA-IlvD (Sm) was transformed into these strains and overexpression of DHAD was induced by changing the culture medium in SD less leucine. Raw extracts from the cultures were prepared and tested by DHAD activity. The results are shown in Table 10. TABLE 10. EFFECTS OF THE DELETING OF GENES INVOLVED IN THE METABOLISM OF


[0220] Surprisingly, specific DHAD activity in the raw extract in strains with one deletion or in the FRA2 gene or in the GRX3 gene increased by 2 to 3 times, which was expected since many of the deletions tested did not increase the specific activity of DHAD. It was shown that the mechanism of assembly of cytosolic iron and sulfur (CIA) in yeast is responsible for the assembly of Fe-S clusters for cytosolic proteins such as isopropylmalata isomerase (Leua). The previous results indicate that this CIA mechanism is independent of the iron detection system that involves Aft1 and a Grx3 / Grx4-Fra2 heterodimer as the repressor (Rutherford et al, JBiol Chem. 250: 10.135 to 10.140 (2005)).
[0221] Another unexpected revelation is the effect of a Grx3 erasure on DHAD activity. It was shown that Grx3 and Grx4 are equivalent in function. Although the double mutations in both the GRX3 and GRX4 genes result in drastic activation of the Fe regulation, the mutation in Grx4 alone confers minimal phenotype (Pujol-Carrion, et al, J Cell Sci. 119: 4.554 to 4.564 (2006); Ojeda , et al, J Biol Chem. 281: 17,661 to 17,669 (2006).). As shown in Table 10 above, only the deletion of GRX3 leads to a significant improvement in specific DHAD activity.
[0222] Thus, these results demonstrate that the modulation genes involved in iron metabolism can increase the activity of a protein that requires a Fe-S cluster such as DHAD when expressed in yeast cytosol. As shown in Figure 10, the effects of deletions of the FRA2 and GRX3 genes on specific DHAD activity could result from, for example, activating the transcription of one or more of the genes in iron regulation through the global regulator Aft1p. Although not wishing to link to a theory, the activation of such genes could lead to an increase in iron absorption and an increase in cytoplasmic Fe-S cluster biosynthesis, leading to higher Fe-S cluster loading of the protein (Figure 10). The demonstration of the increased Fe-S cluster loading is described in Example 11. EXAMPLE 4. EFFECT OF THE EXPRESSION OF AFT1 P AND ITS MUTANTS ON THE SPECIFIC ACTIVITY OF DHAD.
[0223] As described in Example 3 and represented in Figure 10, Fra2, Grx3, and Grx4 are repressors that regulate the function of Aft1p (Kumanovics, et al., J. Biol. Chem. 283: 10,276 to 10,286 (2008)) . Aft1p is a global iron regulator. The activation of the genes involved in iron absorption and metabolism requires the nuclear localization of Aft1p. The expression of constitutive mutants of Aft1 or an increase in the expression of wild-type Aft1p, could lead to the activation of Fe regulation in a wild-type strain or in an AFT1 erase strain (Yamaguchi-Iwai, et al, EMBO J . 14: 1,231 to 1,239 (1995); Yamaguchi-Iwai, et al, J. Biol. Chem. 277: 18,914 to 18,918 (2002); Kaplan, et al, Chem. Rev.109: 4,536 to 4,552 (2009)) . Based on the new disclosures described in Example 3, it is possible that the expression of Aft1p protein and its constitutive mutants may enhance the active fraction of the DHAD enzyme that exhibits Fe-S clusters for its activity.
[0224] To examine this possibility, the wild type AFT1 gene and its constitutive mutants were cloned using a centromere vector pRS411 (ATCC® Number: 87,538 or SEQ ID NO: 919). This vector has an ampicillin selection marker for cultivation in E. coli and a nutritional methionine marker for selection in yeast. The wild type AFT1 gene, including its own promoter and terminator, can be cloned between the KpnI and SacI sites, resulting in the construct pRS411- Aft1 + flanking (SEQ ID NO: 920; Figure 2). A similar strategy can be used to clone genes that encode constitutive mutants of Aft1. The constitutive mutants of Aft1 with substitutions at amino acids L99 to A and C291 to F (with respect to SEQ ID NO: 703) were first analyzed. The constructs pRS411 with genes encoding the wild type AFT1 gene or constitutive mutants were transformed, together with the expression vector pHR81 FBA IlvD (Sm), into the wild yeast strain BY4741 or a yeast strain with an AFT1 erasure. , GRX3 or FRA2. The transformants were selected on agar plates with SD medium less methionine and uracil. The transformed strains were grown in SD medium less methionine and leucine to overexpress the DHAD protein in the presence of these genes or mutants. DHAD activity in the crude extract of these cultures was measured.
[0225] Results of the expression of wild type Aft1p, Aft1p (C291F) and Aft1p (L99A) are shown in Table 11. A moderate increase in specific DHAD activity was observed with Aft1p (C291F) compared to wild type Aft1p . A much greater increase in DHAD activity was seen with Aft1p (L99A). The specific activity of DHAD in the yeast that expresses Aft1p (L99A) was similar to the specific activity obtained in the GRX3 erasure strain (see Table 10). TABLE 11. EFFECTS OF THE EXPRESSION OF AFT1 PE ITS MUTANTS ON THE DHAD DE S. MUTANS ACTIVITY IN THE ΔAFTI CEPA.
EXAMPLE 5. INCREASE IN THE SPECIFIC ACTIVITY OF CYTOSOLIC DHAD IN A DECCC1 ERASING CLEAN
[0226] The exact mechanism of increasing Fe-S cluster biosynthesis capacity for cytosolic DHAD protein is unknown. Based on revelations with FRA2 and GRX3 erasure strains (Example 3) and with expression of Aft1p mutants (Example 4), increasing the availability of Fe content in the cytosol may also improve specific DHAD activity. The CCC1 erasure has been shown to increase the Fe content of the cytosol (Li L, et al, J Biol Chem. 276: 29,515 to 29,519 (2001)). To test this hypothesis, the CCC1 erase strain of BY4741 was transformed with the plasmid pHR81 FBA-IIvD (Sm) as described in Example 1. The raw extracts of the cells with the plasmid were assayed by DHAD activity. Table 13 shows the results of the experiment. When the CCC1 erase strain with plasmid DHAD was cultured in SD medium devoid of uracil, an increase in DHAD activity was found compared to wild type cells with the same plasmid. When extra Fe was added, an additional increase in DHAD was observed in the CCC1 erase strain. The addition of Fe showed no effect on the specific activity of DHAD in wild-type cells. To achieve overexpression of the DHAD protein, the strains were grown in SD medium devoid of leucine (Example 1). Under these conditions, an increase in specific DHAD activity was detected. TABLE 13. EXPRESSION OF DHAD DE S. MUTANS IN CEPA BY4741 (ΔCCEI)
EXAMPLE 6. IMPROVEMENT OF DHAD'S SPECIFIC ACTIVITY IN L. LACTIS EXPRESSED IN YEAST
[0227] Examples 1 to 5 used the DHAD enzyme from S. mutans to identify new ways to increase specific DHAD activity when expressed in yeast. In this example, the application of these methods to improve the specific activity of the L. lactis DHAD enzyme (SEQ ID NO: 958) was investigated. The L. lactis IlvD gene (SEQ ID NO: 959) was cloned to form the pHR81 vector under the control of the FBA promoter (Figure 11). The resulting pHR81 FBA-IlvD (Ll) -ADHt construct (Figure 11; SEQ ID NO: 960) was transformed into the strains with an erasure in either of the FRA2 or GRX3 genes. To study the effect of the constitutive mutant Aft1p (L99A) on L. lactis DHAD, pHR81 FBA-IlvD (Ll) -ADHt was co-transformed into the yeast host along with the vector pRS411-Aft1 (L99A) (see Example 4). To overexpress the IlvD gene, transformants were cultured in a yeast-free medium devoid of leucine or devoid of either leucine or methionine, depending on the strains. The results of the enzymatic assay are summarized in Table 14. The deletions in the FRA2 and GRX3 genes increased the specific activity of the DHAD of L. lactis when expressed in yeast. In addition, the expression of the constitutive mutant of Aft1 L99A similarly increased the specific activity of the DHAD of L. lactis. TABLE 14. L. LACTIS BACTERIAL DHAD OVEREXPRESSION IN S. CEREVISIAE. SPECIFIC SPECIFIC ACTIVITY (U / MG)
EXAMPLE 7. DETERMINATION OF DHAD'S SPECIFIC ACTIVITY. (TEST METHOD)
[0228] The quantification of the activity of proteins that require Fe-S clusters can be done in an assay format. If the protein is an enzyme, such as DHAD, the activity is typically expressed in terms of units of activity. One unit of enzyme activity was defined by the International Biochemistry Union Enzyme Commission as the amount of enzyme that will catalyze the formation of 1 micromol of the substrate per minute under standard conditions (International Biochemistry Union, Commission Report on Enzymes, Oxford: Pergamon Press, 1961). Furthermore, the term specific activity is defined as the units of activity in a given amount of enzymes. Thus, the specific activity is not directly measured, but calculated by dividing 1) the activity in units / ml of the enzyme sample by 2) the protein concentration in that sample, so the specific activity is expressed as units / mg. The specific activity of a pure, fully active enzyme sample is a characteristic of the enzyme. The specific activity of a sample of a protein mixture is a measure of the relative fraction of protein in that sample that is composed of the active enzyme of interest. DHAD activity can be measured by spectrophotometry in an end point assay using the 2,4-dinitrophenylhydrazine (2,4-DNPH) method as described in Flint, D.H. and M.H. Emptage, J. Biol. Chem. 263: 3,558 to 64 (1988). In this assay, 2,4-DNPH reacts with the keto group of 2-ketoisovaleric acid to form a hydrazone, which is detected by its absorbance at 550 nm. The assay buffer contains 50 mM Tris-HCl, 10 mM MgCl2, pH 8.0 (TM8 buffer). Sufficient 2,3-dihydroxyisovaleric acid is added to the test buffer so that its final concentration in the test mixture is 10 mM. In each test, a solution containing enzyme and buffer containing sufficient substrate are mixed so that the final volume is 1 ml. The test mixture is usually incubated at 37 ° C for 30 minutes.
[0229] The test is stopped by adding 250 μl of 10% (P / V) trichloroacetic acid. A few minutes later, 500 μl of a saturated solution of 2,4-DNPH in 1N HCl is added. The mixture is incubated at room temperature for at least 10 min to allow the hydrazone to form. Then, 1.75 ml of NaOH is added to solubilize the hydrazone and to precipitate unreacted 2,4-DNPH. A few minutes before, NaOH is added, the test tubes are placed in a sonicator bath for 10 min until degassing. The tubes are then centrifuged in a bench top centrifuge at a higher speed for 2 min to settle the precipitate.
[0230] The absorbance of the supernatant is then read at 550 nm within 1 hour. The absorbance of the sample assays minus the control assays is divided by 2,600 (determined from a standard curve of α-ketoisovaleric acid) to find the units of enzyme activity in the assay. This assay was used in the Examples described in the present invention where the specific activity of DHAD was determined. EXAMPLE 8. PURIFICATION AND CHARACTERIZATION OF DHAD OF S. MUTANS EXPRESSED IN E. COLI.
[0231] DHAD of S. mutans was purified and characterized as follows. Six liters of E. coli Turner strain culture that houses the plasmid pET28a that contains the S. mutans ilvD gene were cultured and induced with IPTG. The DHAD of S. mutans was purified by disrupting the cells with a TM8 buffer sonicator (see Example 7), centrifuging the raw extract to remove cell debris, then loading the supernatant of the raw extract onto a Q Sepharose column (GE Healthcare) and eluting DHAD with an increasing concentration of NaCl in TM8 buffer. The fractions containing DHAD were pooled, brought to (NH4) 2SO4 at 1 M, and loaded onto a Phenyl-Sepharose column (GE Healthcare) equilibrated with (NH4) 2SO4 at 1 M. DHAD was eluted with a decreasing concentration of (NH4) 2SO4. The fractions containing DHAD were pooled, concentrated to <10 ml, loaded onto a Superdex-200 column of 35 x 600 cm (577 ml of bed volume) (GE Healthcare) and eluted with TM8 buffer. As judged by SDS, the purity of the S. mutans DHAD eluted from the Superdex column was estimated to be> 90%.
[0232] The UV visible spectrum of the purified S. mutans DHAD is shown in Figure 3. The number of peaks above 300 nm is typical of proteins with [2Fe-2S] clusters. The DHAD of S. mutans was reduced with sodium dithionite and its EPR spectrum was measured at various temperatures. Figure 4 shows the EPR spectra measured at temperatures between -253.15 ° C (20K) and -203.15 ° C (70K). The EPR spectrum of the S. mutans DHAD is measurable up to -203.15 ° C (70K), which indicates that it contains a [2Fe-2S] cluster and not a [4Fe-4S] cluster due to the fact that that the EPR spectra of proteins containing [4Fe-4S] clusters are not observable at temperatures well above -263.15 ° C (10K).
[0233] The exact protein content of the purified S. mutans DHAD batch with the highest specific activity using Bradford protein assay was determined by quantitative amino acid analysis. A for that batch. The iron content of this batch determined by ICP-MS using the methodology known in the art was 2 iron molecules per DHAD molecule. This is consistent with this DHAD batch of S. mutans that contains a complete complement of [2Fe-2S] clusters. EXAMPLE 9. SEPARATION OF THE FORMS OF DHAD IN RAW YEAST EXTRACT FROM OTHER PROTEINS IN THE CELL AND BETWEEN YOU TO MEASURE THE AMOUNT OF DHAD PRESENT.
[0234] The DHAD protein in yeast cells exists in the form of dimers with two Fe-S clusters / dimers, one Fe-S cluster / dimer and zero Fe-S cluster / dimer. A method to measure the concentration of these three forms of the DHAD protein in raw yeast extracts was developed using a Mono Q column and a Source 15 PHE PE 4.6 / 100 column (both columns obtained from GE Healthcare) and is described below.
[0235] The frozen yeast cells were thawed, suspended in 50 mM Tris-HCl, 10 mM MgCl2, pH 8.0 (TM8), then disrupted by tapping microspheres. The disrupted cells are centrifuged to remove cell debris and generate the raw yeast extract.
[0236] The crude extract was loaded onto a 4 ml Mono Q column fixed to an AKTA chromatographic system (GE Healthcare) with buffer A being TM8 and buffer B being TM8 containing 0.5 M NaCl. The column was balanced with buffer A before the sample is loaded. The DHAD of S. mutans linked to the Mono Q column under these conditions. After the sample was loaded onto the column, the column was washed with 10 ml of TM8 buffer, then the concentration of NaCl in the eluent was increased to NaCl at 0.22 M. This was followed by a linear gradient of 30 ml of NaCl at 0 , 22 M to 0.35 M. During chromatography, the A215 of the column eluate was monitored and 1 ml fractions were collected. The fractions were tested for DHAD activity. The sum of DHAD activity in fractions outside the Mono Q column was close to that in the raw extract. Good separations using this column were obtained with up to 5 ml of crude extract that represents up to 1 g of yeast cell paste. Fractions containing DHAD were pooled and made 1.35 M into (NH4) 2SO4 in preparation for chromatography on the PHE column.
[0237] The Source 15 PHE PE 4.6 / 100 column was also fixed to an AKTA chromatographic system with buffer A being TM8 containing (NH4) 2SO4 at 1.5 M and buffer B being TM8. Before each cycle, the column was equilibrated with 90% A. The grouped fractions of the Mono Q column made 1.35 M in (NH4) 2SO4 were loaded onto the PHE column and at that concentration of (NH4) 2SO4, the DHAD bound to the column. During chromatography, the A215 of the column eluate was monitored and 1 ml fractions were collected. The DHAD eluted from the column in three peaks when the column was developed with a decreasing linear gradient of 30 ml of (NH4) 2SO4 at 1.35 M to 0 M. The area of each of the DHAD peaks was determined by integration. This elution scheme was revealed to be ideal for separating DHAD from S. mutans from other yeast proteins that coeluted with them outside the Mono Q column. SDS gels run in fractions where the eluted peaks showed that well over 90% of the proteins present in these peaks were DHAD when it was expressed in 1% of the soluble protein in yeast cells. The fractions containing each of the three DHAD peaks were grouped separately. Based on the UV-visible absorbent spectrum and the iron and sulfide levels of DHAD in these peaks, it was determined that the first peak contained DHAD with two clusters / dimers of [2Fe-2S], the second peak contained DHAD with a cluster / dimer of [2Fe-2S] and the third peak contained DHAD with zero cluster / dimer of [2Fe-2S]. Thus, without its native state, the DHAD enzyme of S. mutans appears to exist as a dimer of two monomeric DHAD proteins.
[0238] A standard curve that reports the amount of DHAD present in a sample to the sum of the area of the three DHAD peaks outside the PHE column was obtained as follows. The crude yeast cell extract does not contain S. mutans DHAD was boosted with various amounts of purified S. mutans DHAD. These extracts were subjected to chromatography in Mono Q and a column of PHEs as described above. The area under each of the three DHAD peaks has been integrated. The sum of these areas was plotted against the amount of pure DHAD reinforced in the raw yeast extracts. The plot was used to derive the following equation: # μg of DHAD in the sample of raw extract = 0.507 x (counting the area added of the three peaks of DHAD)
[0239] The activity of DHAD in a raw yeast extract can be readily determined by the method described in Example 7. The amount of DHAD protein in the raw yeast extracts can be determined by the procedure outlined in that Example. With these data, a person can calculate the specific activity of the S. mutans DHAD protein per se in the raw extracts according to the procedure in Example 10. EXAMPLE 10. METHODS FOR DETERMINING DHAD FRACTION IN RAW YEAST EXTRACT LOADED WITH FE-S CLUSTERS.
[0240] When a purified Fe-S cluster that needs protein contains a full complement of clusters, it will have a specific characteristic activity. As previously mentioned, for the DHAD of S. mutans this specific activity is 100 units / mg when it has a total complement of clusters.
[0241] A DHAD sample that averages one Fe-S / cluster per dimer could contain some dimers with two clusters, some dimers with one cluster, and some dimers with no cluster. Alternatively, if the cluster addition to the dimer is total or none and on average there is a Fe-S / dimer cluster in a sample, half of the DHAD dimers would have a full complement of clusters and half would be without clusters. From the results in Example 9, it is known that all or no behavior is not followed by S. mutans DHAD because a species with a cluster per dimer can be isolated. It was found that S. mutansque DHAD dimers have a Fe-S cluster have% of the dimer activity with two Fe-S / dimer clusters, that is, the specific DHAD activity of S. mutans with% of one total complement of Fe-S clusters is 50 units / mg. This means the absence of a Fe-S cluster in one of the monomers of one dimer does not influence the activity of the other monomer and it may contain a cluster of Fe-S.
[0242] With the information obtained from the procedures described in Example 9 and the information described up to that point in that Example, a person can determine the degree of cluster loading of Fe-S in a DHAD sample in two different ways.
[0243] First, a person can compare the ratio of the quantities of the three peaks of DHAD to determine the relative quantity that has two clusters per dimer, one cluster per dimer, and zero clusters per dimer. This provides the degree of cluster loading. For example, if peak area 1 outside the PHE column was 25%, peak 2 was 50%, and peak 3 was 25% of the sum of peak 1, peak 2, and peak 3 areas, the percentage of monomers loaded with clusters can be calculated to be 50% according to the following equation: 100 * [2 * (peak area 1) + 1 * (peak area 2) + 0 * (peak area 3 )] / [2 * (total peak area)] =% of DHAD monomers with Fe-S clusters.
[0244] Second, a person can use the specific DHAD activity present to calculate the degree of cluster loading. The specific activity is determined by dividing the determined activity as described in Example 7 with the amount of DHAD protein determined as described in Example 9. The specific activity is then divided by 100 U / mg to determine the fraction of monomers loaded with the clusters. This fraction is multiplied by 100 to determine the percentage of DHAD monomers with Fe-S clusters.
[0245] For example, if the specific activity is found to be 50 U / mg, the fraction loaded with clusters is 0.5 and the percentage of DHAD monomers with Fe-S clusters is 50%.
[0246] To perform such a calculation, the specific activity needs to be based on the concentration of the DHAD protein in the raw extract (not the total protein). Determining the DHAD concentration of S. mutans in the presence of other proteins can be performed using the methods described in Example 9. EXAMPLE 11. SPECIFIC ACTIVITIES AND INFERRED FRACTION OF PROTEINS LOADED WITH DHAD.
[0247] To determine the fraction of DHAD monomers loaded with Fe-S clusters in different yeast strains grown under different conditions, the methods described above were used. The results are shown in Table 15. TABLE 15. SPECIFIC ACTIVITIES AND INFERRED FRACTION OF PROTEINS LOADED WITH DHAD.


[0248] These results indicate that in these culture conditions, the level of Fe-S cluster loading in DHAD in strains that lack FRA2 and GRX3 is higher than in strains that contain functional copies of these genes. Thus, a higher fraction of the DHAD protein is in the active form in the deletion strains because of the content of Fe-S clusters (which are necessary for activity) which is greater. EXAMPLE 12. CONSTRUCTION OF SACCHAROMYCES CEREVISIAEPNY1505, PNY1541, AND PNY1542.
[0249] The purpose of this Example was to build strains of Saccharomyces cerevisiae PNY1505, PNY1541, and PNY1542. These strains were derived from PNY1503 (BP1064). PNY1503 was derived from CEN.PK 113-7D (CBS 8340; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversiry Center, Netherlands). The construction of PNY1503 (BP1064) is described in order in US 61 / 368,436, incorporated by reference in this document and in Example 13 below. PNY1505 contains a deletion of the FRA2 gene. PNY1541 and PNY1542 contain an integration of the AFT1 gene with the L99A mutation (AFT1-L99A) at the YPRCΔ15 locus.
[0250] Deletions / integrations were created by homologous recombination with PCR fragments that contain regions of homology upstream and downstream of the target gene and the URA3 gene for the selection of transformants. The URA3 gene was removed by homologous recombination to create a deletion / integration without scarring.
[0251] The deletion / integration procedure without scars was adapted from Akada et al., Yeast, 2J (5): 399 to 405 (2006). The PCR cassette for each deletion / integration was made by combining four fragments, ABUC, or by overlapping PCR or by cloning the individual fragments and the gene to be integrated, in a plasmid before amplification and the cassette as a whole by PCR for the deletion / integration procedure. The PCR cassette contained a selectable / counter-selectable marker, URA3 (Fragment U), which consists of the native URA3 gene CEN.PK 113-7D together with the promoter regions (250 bp upstream of the URA3 gene) and a terminator (150 bp downstream of the URA3 gene). Fragments A (150 bp to 500 bp in length) and C (250 bp in length) corresponded to the sequence immediately upstream of the target gene (Fragment A) and the 3 'sequence of the target gene (Fragment C). Fragments A and C were used to integrate the cassette into the chromosome by homologous recombination. Fragment B (500 bp in length) corresponded to 500 bp immediately downstream of the target gene and was used for excision of the URA3 marker and Fragment C from the chromosome by homologous recombination, as a direct repetition of the sequence corresponding to the Fragment B was created by integrating the cassette into the chromosome.
[0252] Using the ABUC cassette of PCR product, the URA3 marker was first integrated into and then excised from the chromosome by homologous recombination. The initial integration deleted the gene, excluding the 3 'sequence. Through excision, the 3 'region of the gene was also deleted. For the integration of genes using this method, the gene to be integrated was included in the cassette between fragments A and B. DELETION OF FRA2
[0253] The FRA2 deletion (also described in the application in US 61 / 380.563, incorporated by reference in this document) was designed to delete 250 nucleotides from the 3 'end of the coding sequence, which leaves the first 113 nucleotides of the sequence coding codes intact. A frame stop codon was present in 7 nucleotides downstream of the deletion. The four fragments for the PCR cassette for the scarless deletion of FRA2 were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, MA) and CEN.PK 113-7D genomic DNA as a model, prepared with a Gentra Puregene Yeast / Bact kit (Qiagen; Valencia, CA). FRA2 Fragment A was amplified with the oBP594 primer (SEQ ID NO: 961) and the oBP595 primer (SEQ ID NO: 962), which contains a 5 'end with homology to the 5' end of FRA2 Fragment B. Fragment B of FRA2 was amplified with the primer oBP596 (SEQ ID NO: 963), which contains a 5 'end with homology to the 3' end of Fragment A of FRA2, and the primer oBP597 (SEQ ID NO: 964) , which contains a 5 'end with homology to the 5' end of Fragment U of FRA2. Fragment U of FRA2 was amplified with the primer oBP598 (SEQ ID NO: 965), which contains a 5 'end with homology to the 3' end of Fragment B of FRA2, and the primer oBP599 (SEQ ID NO: 966) , which contains a 5 'end with homology to the 5' end of FRA2 Fragment C. FRA2 Fragment C was amplified with primer 0BP6OO (SEQ ID NO: 967), which contains a 5 'end with homology to the end 3 'of FRA2 U Fragment, and the 0BP6OI primer (SEQ ID NO: 968). PCR products were purified with a PCR purification kit (Qiagen). Fragment AB of FRA2 was created by overlapping PCR by mixing Fragment A of FRA2 and Fragment B of FRA2 and amplification with primers oBP594 (SEQ ID NO: 961) and oBP597 (SEQ ID NO: 964). The FRA2 UC Fragment was created by overlay PCR by mixing FRA2 Fragment U and FRA2 Fragment C and amplification with the primers oBP598 (SEQ ID NO: 965) and 0BP6OI (SEQ ID NO: 968). The resulting PCR products were purified on an agarose gel followed by a gel extraction kit (Qiagen). The ABRA FRA2 cassette was created using the overlapping PCR by mixing the FRA2 AB fragment and the FRA2 UC fragment and amplification with the oBP594 (SEQ ID NO: 961) and 0BP6OI (SEQ ID NO: 968) primers. The PCR product was purified with a PCR purification kit (Qiagen).
[0254] The PNY1503 competent cells were made and transformed with the ABUC FRA2 PCR cassette using a frozen EZ yeast transformation kit II (Zymo Research; Orange, CA). Transformation mixtures were plated in a complete synthetic medium that lacks uracil supplemented with 1% ethanol at 30 ° C. Transformants with an FRA2 knockout were screened by PCR with the primers oBP602 (SEQ ID NO: 969) and oBP603 (SEQ ID NO: 970) using genomic DNA prepared with a Gentra Puregene Yeast / Bact kit (Qiagen). A correct transformant was cultured in YPE (yeast extract, peptone, 1% ethanol) and plated in a complete synthetic medium supplemented with 1% ethanol and containing 5-fluoro-optical acid (0.1%) at 30 ° C to select for isolates that have lost the URA3 marker. The deletion and marker removal was confirmed by PCR with the primers oBP602 (SEQ ID NO: 969) and oBP603 (SEQ ID NO: 970) using genomic DNA prepared with a Gentra Puregene Yeast / Bact kit (Qiagen). The absence of the isolate's FRA2 gene was demonstrated by a negative PCR result using specific primers for the deleted FRA2 coding sequence, oPP605 (SEQ ID NO: 971) and 0BP6O6 (SEQ ID NO: 972). The correct isolate was selected as strain CEN.PK 113-7D MATa ura3Δ :: loxP his3Δ pdc6Δ pdclΔ :: P [PDC1] - DHAD | ilvD_Sm-PDClt pdc5Δ :: P [PDC5] -ADH | sadB_Ax-PDC5t gpd2Δ :: | ox2 fra2A and designed as PNY1505 (BP1135). TABLE16. INITIATORS USED IN DELETING FRA2
DELETION OF YPRCΔ15 AND INTEGRATION OF AFT1-L99A
[0255] The YPRCΔ15 locus has been deleted and replaced with AFT1- L99A along with the native promoter region (800 bp) and terminator region (800 bp) of AFT1. The scarless cassette for the Integration of YPRCΔ15 deletion AFT1L99A was first cloned into plasmid pUC19- URA3MCS (described in order in US 61 / 356,379, incorporated by reference in this document). The vector is based on pUC19 and contains the sequence of the URA3 gene of S. cerevisiae CEN.PK 113-7D located within a multiple cloning site (MCS). pUC19 (American Type Culture Collection, Manassas, VA; ATCC # 37254) contains the pMBl replicon and a gene encoding beta-lactamase for replication and selection in Escherichia coli. In addition to the coding sequence for URA3, the upstream (250 bp) and downstream (150 bp) sequences of that gene are present for the expression of the URA3 gene in yeast. The vector can be used for cloning purposes and can be used as a yeast integration vector.
[0256] The DNA covering the URA3 coding region along with 250 bp upstream and 150 bp downstream of the URA3 coding region of Saccaromyces cerevisiae CEN.PK 113-7D (CBS 8340; Centraalbureau voor Schimmelcultures ( CBS) Fungal Biodiversity Center, Netherlands) Genomic DNA was amplified with primers osBP438 (SEQ ID NO: 1033), which contains restriction sites for BamHl, Ascl, Pmel, and Fsel, and oBP439 (SEQ ID NO: 1034) , which contains XbaI, Pad, and Notl restriction sites. Genomic DNA was prepared using Yeast Gentra Puregene / Bact kit (Qiagen). The PCR product and pUC19 were linked to T4 DNA ligase after digestion with BamHl and XbaI to create the vector pUC19- URA3MCS. The vector was confirmed by PCR and sequencing with the primers oBP264 (SEQ ID NO: 1031) and oBP265 (SEQ ID NO: 1032).
[0257] Fragment A of YPRCΔ15 was amplified from genomic DNA, prepared as above, with the oBP622 primer (SEQ ID NO: 973), which contains a Kpnl restriction site, and the oBP623 primer (SEQ ID NO: 974) , which contains a 5 'end with homology to the 5' end of Fragment B of YPRCΔ15. Fragment B of YPRCΔ15 was amplified from genomic DNA with the primer oBP624 (SEQ ID NO: 975), which contains a 5 'end with homology to the 3' end of Fragment A of YPRCΔ15, and the primer oBP625 (SEQ ID NO : 976), which contains a Fsel restriction site. PCR products were purified with a PCR purification kit (Qiagen). YPRCΔ15 Fragment A - YPRCΔ15 Fragment B was created by overlapping PCR by mixing the PCR and YPRCΔ15 Fragment A amplification products and YPRCΔ15 Fragment B with the primers oBP622 (SEQ ID NO: 973) and oBP625 (SEQ ID NO: 973) and oBP625 (SEQ ID NO: 973) ID NO: 976). The resulting PCR product was digested with Kpnl and Fsel and ligated with T4 DNA ligase at the corresponding pUC19-URA3MCS sites after digestion with the appropriate enzymes. Fragment C of YPRCΔ15 was amplified from genomic DNA with the primer oBP626 (SEQ ID NO: 977), which contains a Notl restriction site, and the oBP627 primer (SEQ ID NO: 978), which contains a restriction site Pad. The YPRCΔ15 Fragment C PCR Product was digested with Notl and Pad and ligated with T4 DNA ligase at the corresponding sites on the plasmid containing AB YPRCΔ15 Fragments. AFT1-L99A, together with the native promoter region (800 bp) and the terminator region (800 bp) of AFT1, was amplified using pRS41 1-AFT1 (L99A) (described in Example 4 above) as a model with oBP744 primer (SEQ ID NO: 979), which contains an AscI restriction site, and oBP745 primer (SEQ ID NO: 980), which contains a Pmel restriction site. The PCR product was digested with AscI and Pmel and ligated with T4 DNA ligase at the corresponding sites on the plasmid containing YPRCΔ15C AB Fragments. The entire integration cassette was amplified from the resulting plasmid with the primers oBP622 (SEQ ID NO: 973) and oBP627 (SEQ ID NO: 978).
[0258] The competent PNY1503 cells were made and transformed with the YPRCΔ15 integration cassette PCR product using a frozen EZ yeast transformation kit II (Zymo Research). The transformation mixtures were plated in a complete synthetic medium that lacks uracil supplemented with 1% ethanol at 30 ° C. The transformants were grown in YPE (1% ethanol) and plated in a complete synthetic medium supplemented with 1% EtOH and containing 5-fluoro-optical acid (0.1%) at 30 ° C to select the isolates that lost their URA3 marker. The deletion of YPRCΔ15 and the integration of AFT1L99A were confirmed by PCR with external primers oBP636 (SEQ ID NO: 981) and oBP637 (SEQ ID NO: 982) and with specific primer HY840 from AFT1-L99A (SEQ ID NO: 983) and external primer oBP637 (SEQ ID NO: 982) using genomic DNA prepared with Yeast Gentra Puregene / Bact kit (Qiagen) and by colony PCR. Correct independent isolates of CEN.PK 113-7D MATa ura3Δ :: loxP his3Δ pdc6Δ pdclΔ :: P [PDC1] - DHAD | ilvD_Sm-PDClt pdc5Δ :: P [PDC5] -ADH | sadB_Ax-PDC5t gpd2Δ :: loxP yprcΔ15 AFTlL99A were designed as strains PNY1541 and PNY1542. TABLE 17. INITIATORS USED IN THE DELETION OF YPRCΔ15 AND IN THE INTEGRATION OF AFT1-L99A
EXAMPLE 13. CONSTRUCTION OF SACCHAROMYCES CEREVISIAE CEPA BP1064 (PNY1503).
[0259] The BP1064 strain was derived from CEN.PK 113-7D (CBS 8340; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Center, Netherlands) and contains deletions of the following genes: URA3, HIS3, PDC1, PDC5, PDC6, and GPD2 .
[0260] Deletions, which completely removed the entire coding sequence, were created by homologous recombination with the PCR fragments containing regions of homology upstream and downstream of the target gene and or with a G418 resistance marker or gene URA3 for the selection of transformants. The G418 resistance marker, flanked by loxP sites, was removed using Cre recombinase. The URA3 gene was removed by homologous recombination to create a scarless deletion, or if flanked by loxP sites it was removed using Cre recombinase.
[0261] The scarless deletion procedure was adapted from Akada et al. 2006 Yeast v23 p399. In general, the PCR cassette for each scarless deletion was made by combining four fragments, A-B-U-C, using the overlapping PCR. The PCR cassette contained a selectable / counter-selectable marker, URA3 (Fragment U), which consists of the native URA3 gene CEN.PK 113-7D together with the promoter (250 bp upstream of the URA3 gene) and the terminator (150 bp downstream of the URA3 gene). Fragments A and C, each 500 bp in length, corresponded to 500 bp immediately upstream of the target gene (Fragment A) and 3 'of 500 bp of the target gene (Fragment C). Fragments A and C were used to integrate the cassette into the chromosome through homologous recombination. Fragment B (500 bp in length) corresponded to 500 bp immediately downstream of the target gene and was used to excise the URA3 marker and Fragment C from the chromosome by homologous recombination, as a direct repetition of the sequence corresponding to Fragment B was created by integrating the cassette into the chromosome. Using the ABUC cassette of PCR product, the URA3 marker was first integrated into it and then excised from the chromosome by homologous recombination. The initial integration deleted the gene, excluding the 3 '500 bp. Upon excision, the 3 '500 bp region of the gene was also deleted. For the integration of genes using this method, the gene to be integrated was included in the PCR cassette between fragments A and B. DELETION OF URA3
[0262] To delete the URA3endogenous coding region, a ura3 :: loxP-kanMX-loxP cassette was amplified by PCR of the pLA54 model DNA (SEQ ID NO: 986). pLA54 contains the K. lactis TEF1 promoter and kanMX marker, and is flanked by loxP sites to allow for recombination with Cre recombinase and removal of the marker. PCR was performed using Phusion DNA polymerase and BK505 and BK506 primers (SEQ ID NOs: 987 and 988, respectively). The URA3 portion of each primer was derived from the 5 'region upstream of the URA3 promoter and from the 3' region downstream of the coding region so that integration of the loxP-kanMX-loxP marker resulted in the replacement of the coding region of URA3. The PCR product was transformed into CEN.PK 113-7D using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pages 201 to 202) and the transformants were selected in YPD containing G418 (100 μg / ml) at 30 C. The transformants were screened to verify the correct integration by PCR using the LA468 and LA492 primers (SEQ ID NOs: 989 and 990, respectively) and named CEN. PK 113-7D Δura3 :: kanMX. DEHIS3 DELETION
[0263] The four fragments for the PCR cassette for the HIS3 deletion without scars were amplified using the Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, MA) and the CEN.PK 113-7D genomic DNA as a model, prepared with a Gentra Puregene Yeast / Bact kit (Qiagen; Valencia, CA). The HIS3 A Fragments were amplified with the oBP452 primer (SEQ ID NO: 991) and the oBP453 Primer (SEQ ID NO: 992), which contains a 5 'end with homology to the 5' end of the HIS3 B Fragment. The HIS3 B fragment was amplified with the oBP454 primer (SEQ ID NO: 993), which contains a 5 'end end with homology to the 3' end of the HIS3 A fragment, and the oBP455 primer (SEQ ID NO: 994) , which contains a 5 'end with homology to the 5' end of HIS3 U Fragment. The HIS3 U Fragment was amplified with the oBP456 primer (SEQ ID NO: 995), which contains a 5 'end with homology to the 3 'end of the HIS3 B Fragment, and the oBP457 primer (SEQ ID NO: 996), which contains a 5' end with homology to a 5 'end of the HIS3 C Fragment. The HIS3 C Fragment was amplified with the oBP458 primer (SEQ ID NO: 997), which contains a 5 'end end with homology to a 3' end of HIS3 U, and an oBP459 primer (SEQ ID NO: 998). PCR products were purified with a PCR purification kit (Qiagen). The HIS3 AB Fragment was created using the overlapping PCR by mixing the HIS3 A Fragment and the HIS3 B Fragment and amplification with the primers oBP452 (SEQ ID NO: 991) and oBP455 (SEQ ID NO: 994). The HIS3 UC fragment was created using the overlapping PCR by mixing the HIS3 U fragment and the HIS3 C fragment and amplification with the oBP456 (SEQ ID NO: 995) and oBP459 (SEQ ID NO: 998) primers. The resulting PCR products were purified on an agarose gel followed by a gel extraction kit (Qiagen). The HUC3 ABUC cassette was created using the overlapping PCR by mixing the HIS3 AB fragment and the HIS3 UC fragment and amplification with the oBP452 (SEQ ID NO: 991) and oBP459 primers (SEQ ID NO: 998). The PCR product was purified with a PCR purification kit (Qiagen).
[0264] CEN.PK 113-7DAura3 :: kanMX competent cells were made and transformed with the ABUC HIS3 PCR cassette using the frozen EZ yeast transformation kit II (Zymo Research; Orange, CA). The transformation mixtures were plated in the complete synthetic medium that lacks uracil supplemented with 2% glucose at 30 ° C. Transformants with a his3 knockout were screened by PCR with primers oBP460 (SEQ ID NO: 999) and oBP461 (SEQ ID NO: 1000) using genomic DNA prepared with a Genteg Puregene Yeast / Bact kit (Qiagen). A correct transformant was selected as the CEN.PK 113-7D Δura3 :: kanMX Ahis3 :: URA3 strain. KANMX MARKER REMOVAL FROM THE ΔURA3 AND URA3 SITE MARKER REMOVAL FROM THE AHIS3 SITE
[0265] The KanMX marker was removed by transforming CEN.PK 113-7D Δura3 :: kanMX Ahis3 :: URA3 with pRS423 :: PGAL 1 -ere (SEQ ID NO: 1011, described in provisional application US 61 / 290.639 ) using the frozen EZ yeast transformation kit II (Zymo Research) and plated in a complete synthetic medium that lacks histidine and uracil supplemented with 2% glucose at 30 ° C. Transformants were cultured in YP supplemented with 1% galactose at 30 ° C for ~ 6 hours to induce excision of Cre recombinase and KanMX marker and plated on YPD (2% glucose) plates at 30 ° C for recovery. An isolate was grown overnight in YPD and plated in a complete synthetic medium containing 5-fluoro-optical acid (0.1%) at 30 ° C to select the isolates that lost the URA3 marker. The 5-FOA resistant isolates were cultured on and plated in YPD to remove plasmid from pRS423 :: PoALi-cre. The isolates were checked for loss of the KanMX marker, URA3 marker, and pRS423 :: Pgali-cre plasmid by testing the culture on YPD + G418 plates, plates of complete synthetic medium lacking uracil, and plates of complete medium synthetic that lacks histidine. A correct isolate that was sensitive to G418 and auxotrophic for uracil and histidine was selected as the strain CEN.PK 113- 7D Δura3 :: loxP Ahis3 and designed as BP857. Deletions and a removal marker were confirmed by PCR and sequencing with primers oBP450 (SEQ ID NO: 1001) and oBP451 (SEQ ID NO: 1002) for Δura3 and primers oBP460 (SEQ ID NO: 999) and oBP461 (SEQ ID NO: 1000) for Ahis3 using genomic DNA prepared with a Gentra Puregene Yeast / Bact kit (Qiagen). DEPTION DEPDC6
[0266] The four fragments for the PCR cassette for PDC6 deletion without scars were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs) and CEN.PK 113-7D genomic DNA as a template, prepared with Gentra Puregene Yeast / Bact kit (Qiagen). PDC6 Fragment A was amplified with the oBP440 primer (SEQ ID NO: 1003) and the oBP441 primer (SEQ ID NO: 1004), which contains a 5 'end with homology to the 5' end of PDC6 Fragment B. PDC6 Fragment B was amplified with oBP442 primer (SEQ ID NO: 1005), which contains a 5 'end end with homology to a 3' end of PDC6 Fragment A, and the oBP443 primer (SEQ ID NO: 1006), containing a 5 'end with homology to the 5' end of the PDC6 U Fragment. The PDC6 U Fragment was amplified with the oBP444 primer (SEQ ID NO: 1007), which contains a 5 'end end with homology to a 3' end of the PDC6 Fragment B, and the oBP445 primer (SEQ ID NO: 1008) , which contains a 5 'end with homology to a 5' end of Fragment C of PDC6. PDC6 Fragment C was amplified with the oBP446 primer (SEQ ID NO: 1009), which contains a 5 'end end with homology to a 3' end of the PDC6 U Fragment, and the oBP447 primer (SEQ ID NO: 1010) . PCR products were purified with a PCR purification kit (Qiagen). PDC6B Fragment A was created by overlapping PCR by mixing PDC6 Fragment A and PDC6 Fragment B and amplification with the primers oBP440 (SEQ ID NO: 1003) and oBP443 (SEQ ID NO: 1006). The PDC6C U Fragment was created by overlapping PCR by mixing PDC6 U Fragment and PDC6 Fragment C and amplification with the oBP444 (SEQ ID NO: 1007) and oBP447 primers (SEQ ID NO: 1010). The resulting PCR products were purified on an agarose gel followed by a gel extraction kit (Qiagen). The ABUC PDC6 cassette was created using the overlapping PCR by mixing PDC6B Fragment A and PDC6C Fragment U and amplification with the primers oBP440 (SEQ ID NO: 1003) and oBP447 (SEQ ID NO: 1010). The PCR product was purified with a PCR purification kit (Qiagen).
[0267] The competent cells of CEN.PK 113-7D Δura3 :: loxP Ahis3 were made and transformed with the ABUC PDC6 PCR cassette using the frozen EZ yeast transformation kit II (Zymo Research). The transformation mixtures were plated in the complete synthetic medium that lacks uracil supplemented with 2% glucose at 30 ° C. Transformants with a pdc6 knockout were screened by PCR with primers oBP448 (SEQ ID NO: 1012) and oBP449 (SEQ ID NO: 1013) using genomic DNA prepared with a Gentle Yeast Puregene / Bact kit (Qiagen). A correct transformant was selected as the CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 :: URA3 strain.
[0268] CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 :: URA3 was grown overnight in YPD and plated in a complete synthetic medium containing 5-fluoro-optical acid (0.1%) at 30 ° C to select the isolates that have lost the URA3 marker. The deletion and marker removal were confirmed by PCR and sequencing with the primers oBP448 (SEQ ID NO: 1012) and oBP449 (SEQ ID NO: 1013) using genomic DNA prepared with a Puregene Yeast Gentle / Bact kit (Qiagen ). The absence of the isolate's PDC6 gene was demonstrated by a negative PCR result using primers specific for the PDC6 coding sequence, oBP554 (SEQ ID NO: 1014) and oBP555 (SEQ ID NO: 1015). The correct isolate was selected as the strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 and designed as BP891. INTEGRATION OF DEPDC1 DELETION ILVDSM
[0269] The PDC1 gene has been deleted and replaced with the ilvD coding region of Streptococcus mutans ATCC # 700610. The A fragment followed by the Streptococcus mutans ilvD coding region for the PCR cassette for PDC1 deletion ilvDSm integration was amplified using the Phusion High Fidelity PCR Master Mix (New England BioLabs) and (described in the provisional application in US 61/246709) NYLA83 genomic DNA as a template, prepared with a Yeast Gentra Puregene / Bact kit (Qiagen). The PDC1 fragment A-ilvDSm (SEQ ID NO: 1053) was amplified with oBP513 primer (SEQ ID NO: 1016) and the oBP515 primer (SEQ ID NO: 1017), which contains a 5 'end with homology to one end 5 'of PDC1 Fragment B. Fragments B, U, and C for the PCR cassette for the integration of PDC1 deletion ilvDSm were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs) and CEN.PK 113-7D genomic DNA as a template, prepared with a Gentra Puregene Yeast / Bact kit (Qiagen). Fragment B of PDC1 was amplified with the primer oBP516 (SEQ ID NO: 1018) which contains a 5 'end with homology to a 3' end of Fragment A-ilvDSm of PDC1, and the primer oBP517 (SEQ ID NO: 1019 ), which contains a 5 'end end with homology to a 5' end of PDC1 U Fragment. PDC1 U Fragment was amplified with the oBP518 primer (SEQ ID NO: 1020), which contains a 5 'end end with homology to a PDC1 Fragment B 3' end, and the oBP519 primer (SEQ ID NO: 1021) , which contains a 5 'end with homology to a 5' end of Fragment C of PDC1. Fragment C of PDC1 was amplified with the primer oBP520 (SEQ ID NO: 1022), which contains a 5 'end with homology to the 3' end of Fragment U of PDC1, and the primer oBP521 (SEQ ID NO: 1023) . PCR products were purified with a PCR purification kit (Qiagen). PDC1 Fragment A-ilvDSm-B was created by overlapping PCR by mixing PDC1 Fragment A-ilvDSm and PDC1 Fragment B and amplification with the primers oBP513 (SEQ ID NO: 1016) and oBP517 ( SEQ ID NO: 1019). The PDC1 UC Fragment was created by overlapping PCR by mixing PDC1 Fragment U and PDC1 Fragment C and amplification with the primers oBP518 (SEQ ID NO: 1020) and oBP521 (SEQ ID NO: 1023). The resulting PCR products were purified on an agarose gel followed by a gel extraction kit (Qiagen). The PDC1 A-ilvDSm-BUC cassette (SEQ ID NO: 1054) was created by overlapping PCR by mixing the PDC1 A-ilvDSm-B Fragment and the PDC1 UC Fragment and amplification with the oBP513 primers ( SEQ ID NO: 1016) and oPP521 (SEQ ID NO: 1023). The PCR product was purified with a PCR purification kit (Qiagen).
[0270] CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 competent cells were made and transformed with the PDC1 PCR A-ilvDSm-BUC cassette using the frozen EZ yeast transformation kit II (Zymo Research). The transformation mixtures were plated in the complete synthetic medium that lacks uracil supplemented with 2% glucose at 30 ° C. Transformants with a pdc1 knockout ilvDSm integration were screened by PCR with primers oBP511 (SEQ ID NO: 1024) and oBP512 (SEQ ID NO: 1025) using genomic DNA prepared with a Gentra Puregene Yeast / Bact kit (Qiagen). The absence of the isolate's PDC1 gene was demonstrated by a negative PCR result using specific primers for the PDC1 coding sequence, oBP550 (SEQ ID NO: 1026) and oBP551 (SEQ ID NO: 1027). A correct transformant was selected as the CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm-URA3 strain.
[0271] CEN.PK 113-7D Aura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm- URA3 was grown overnight in YPD and plated in a completely synthetic medium containing 5-fluoro-orotic acid (0.1%) at 30 ° C to select isolates that lost the URA3 marker. PDCl deletion, ilvDSm integration and marker removal were confirmed by PCR and sequencing with oBP511 (SEQ ID NO: 1024) and oBP512 (SEQ ID NO: 1025) primers using genomic DNA prepared with a Gentra Puregene yeast kit / Bact (Qiagen). The correct isolate was selected as the CEN.PK 113-7D Aura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm strain and designated as BP907. DEPDC5 DELETION AND SADB INTEGRATION
[0272] The PDC5 gene was deleted and replaced by the sadB coding region from Achromobacter xylosoxidans. A segment of the PCR cassette for PDB5 deletion sadB Integration was first cloned into plasmid pUC19-URA3MCS.
[0273] pUC19-URA3MCS is based on pUC19 and contains the URA3 gene sequence from Saccaromyces cerevisiae located in a multiple cloning site (MCS). pUC19 contains the pMBl replicon and a gene encoding beta-lactamase for replication and selection in Escherichia coli. In addition to the coding sequence for URA3, the upstream and downstream sequences of that gene were included for expression of the URA3 gene in yeast. The vector can be used for cloning purposes and can be used as a yeast integration vector.
[0274] The DNA covering the URA3 coding region along with 250 bp upstream and 150 bp downstream of the URA3 coding region of Saccaromyces cerevisiae genomic DNA CEN.PK 113-7D has been expanded with oBP438 primers (SEQ ID NO: 1033 ), containing restriction sites BamHI, Ascl, Pmel, and Fsel, and oBP439 (SEQ ID NO: 1034), containing the restriction sites Xbal, Pad, and Notl, using Phusion High-Fidelity PCR Master Mix (New England BioLabs). Genomic DNA was prepared using a Gentra Puregene Yeast / Bact kit (Qiagen). The PCR and pUC19 product (SEQ ID NO: 1056) was ligated with T4 DNA ligase after digestion with BamHI and Xbal to create the vector pUC19-URA3MCS. The vector was confirmed by PCR and sequencing with the primers oBP264 (SEQ ID NO: 1031) and oBP265 (SEQ ID NO: 1032).
[0275] The PDB5 sadB and B-fragment coding sequence was cloned into pUC19-URA3MCS to create the sadB-BU portion of the PDC5 PCR A-sadB-BUC cassette. The sadB coding sequence was expanded with the use of pLH468-sadB (SEQ ID NO: 1051) as a model with the oBP530 primer (SEQ ID NO: 1035), containing an Ascl restriction site, and oBP531 primer (SEQ ID NO: 1036), containing the 5 'tail homologous to the 5' end of the PDC5 B-fragment. The PDC5 B-fragment was amplified with the primer oBP532 (SEQ ID NO: 1037), containing the 5 'tail with homology to the 3' end of sadB, and primer oBP533 (SEQ ID NO: 1038), containing a restriction site Pmel. PCR products were purified with a PCR purification kit (Qiagen). SadB-PDC5 B-fragment was created by overlapping PCR by mixing the sadB and PDC5 B fragment PCR products and by expanding with primers oBP530 (SEQ ID NO: 1035) and oBP533 (SEQ ID NO: 1038) . The resulting PCR product was digested with Ascl and Pmel and ligated with T4 DNA ligase at the corresponding pUC19-URA3MCS sites after digestion with the appropriate enzymes. The resulting plasmid was used as a model for amplification of sadB-fragment, B-fragment U with the use of primers oBP536 (SEQ ID NO: 1039) and oBP546 (SEQ ID NO: 1040), containing the 5 'tail with homology to 5 'end of the PDC5 C fragment. PDC5 fragment C was amplified with the primer oBP547 (SEQ ID NO: 1041) containing a 5 'tail homology to the 3' end of sadB-fragment, B-fragment U of PDC5, and primer oBP539 (SEQ ID NO: 1042 ). PCR products were purified with a PCR purification kit (Qiagen). The sadB-fragment, B-fragment, U-fragment C of PDC5 were created by overlapping the PCR by mixing the sadB-fragment, B-fragment U of PDC5 and fragment C of PDC5 and by amplifying with oBP536 primers ( SEQ ID NO: 1039) and oPP539 (SEQ ID NO: 1042). The resulting PCR product was purified on an agarose gel followed by a gel extraction kit (Qiagen). The PDC5 A-sadB-BUC cassette (SEQ ID NO: 1055) was created by amplifying the PDB5 sadB-fragment, B-fragment, U-Fragment C with oBP542 primers (SEQ ID NO: 1043), containing a tail 5 'with homology to 50 nucleotides immediately upstream of the native PDC5 coding sequence, and oPP539 (SEQ ID NO: 1042). The PCR product was purified with a PCR purification kit (Qiagen).
[0276] CEN.PK 113-7D competent cells Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm were made and transformed with the PDC5 A-sadB-BUC PCR cassette using an Frozen-EZ yeast Transformation II kit (Zymo Research). Transformation mixtures were plated in a completely synthetic medium without uracil supplemented with 1% ethanol (no glucose) at 30 ° C. Transformants with PDC5 knockout sadB integration were screened using PCR with oBP540 (SEQ ID NO: 1044) and oBP541 (SEQ ID NO: 1045) primers using genomic DNA prepared with a Gentra Puregene Yeast / Bact kit ( Qiagen). The absence of the isolate's PDC5 gene was demonstrated by a negative PCR result using primers specific for the PDC5 coding sequence, oBP552 (SEQ ID NO: 1046) and oBP553 (SEQ ID NO: 1047). A correct transformant was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB-URA3.
[0277] CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB-URA3 was grown overnight in YPE (1% ethanol) and plated in completely synthetic medium supplemented with ethanol (no glucose) and containing 5-fluoro-orotic acid (0.1%) at 30 ° C to select isolates that have lost the URA3 marker. PDC5 deletion, sadB integration and marker removal were confirmed by PCR with oBP540 (SEQ ID NO: 1044) and oBP541 (SEQ ID NO: 1045) primers using genomic DNA prepared with a Gentra Puregene Yeast / kit Bact (Qiagen). The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB and designated as BP913. GPD2 DELETION
[0278] To delete the endogenous GPD2 coding region, a gpd2 :: loxP-URA3-loxP cassette (SEQ ID NO: 1057) was expanded by PCR using loxP-URA3-oxP PCR (SEQ ID NO: 1052) as model DNA. loxP- URA3-loxP contains the URA3 marker from (ATCC # 77107) flanked by loxP recombinase sites. PCR was performed using Phusion DNA polymerase and LA512 and LA513 primers (SEQ ID NOs: 1029 and 1030, respectively). The GPD2 portion of each primer was derived from the 5 'region upstream of the GPD2 coding region and 3' region downstream from the coding region so that q integration of the loxP-URA3-loxP marker results in the replacement of the GPD2 coding region . The PCR product was transformed into BP913 and transformants were selected in a completely synthetic medium without uracil supplemented with 1% ethanol (no glucose). Transformants were screened to verify correct integration through PCR using oBP582 and AA270 primers (SEQ ID NOs: 1048 and 1049, respectively).
[0279] The URA3 marker was recycled through transformation with pRS423 :: PoALi-cre (SEQ ID NO: 1011) and plated in completely synthetic medium without histidine supplemented with 1% ethanol at 30 ° C. Transformants were tinted in a completely synthetic medium supplemented with 1% ethanol and containing 5-fluoro-orotic acid (0.1%) and incubated at 30 ° C to select isolates that lost the URA3 marker. The 5-FOA resistant isolates were grown in YPE (1% ethanol) to remove plasmid pRS423 :: PoALi-cre. The deletion and removal of the marker were confirmed by PCR with primers oBP582 (SEQ ID NO: 1048) and oBP591 (SEQ ID NO: 1050). The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Apdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB Agpd2 :: loxP and designated as BP 1064. EXAMPLE 14. SHAKE BOTTLE EXPERIMENT TO MEASURE THE ACCUMULATION OF 2,3-DIIDROXYISOVALERATE AND ISOBUTANOL PRODUCTION
[0280] The purpose of this Example was to show the effect on accumulation of the 2,3-hydroxyisovalerate intermediate isobutanol (DHIV) and to show isobutanol production in isobutanologen strains with an integrated copy of the AFT1-L99A gene or an FRA2 deletion compared to the parental strain. Strains were transformed with plasmids from the pYZ090 isobutanol pathway (SEQ ID NO: 984; described in Application No. US 61 / 368,436, incorporated by reference in this document) and pLH468 (SEQ ID NO: 985; described in Application for US No. 61 / 246,844, incorporated by reference in this document). These plasmids are also briefly described, as follows.
[0281] pYZ090 (SEQ ID NO: 984) was constructed to contain a chimeric gene that has the coding region for the alsS gene from Bacillus subtilis (nt position 457-2172) expressed from the yeast CUP1 promoter (nt 2 -449) and followed by the terminator CYC1 (nt 2181-2430) for ALS expression, and a chimeric gene that has the ilvC gene coding region from Lactococcus lactis (nt 3634-4656) expressed from the yeast ILV5 promoter (2433-3626) and followed by the ILV5 terminator (nt 4682-5304) for KARI expression.
[0282] pLH468 (SEQ ID NO: 985) was constructed to contain: a chimeric gene that has the ilvD gene coding region from Streptococcus mutans (nt position 3313-4849) expressed from the S. cerevisiae (nt FBA1 promoter) 2109-3105) followed by the FBA1 terminator (nt 4858-5757) for expression of DHAD; a chimeric gene that has the codon-optimized horse liver alcohol dehydrogenase coding region (nt 6286-7413) expressed from the S. cerevisiae GPM1 promoter (nt 7425-8181) followed by the ADH1 terminator (nt 5962-6277 ) for expression of ADH; and a chimeric gene that has the codon-optimized kivD gene coding region from Lactococcus lactis (nt 9249-10895) expressed from the TDH3 promoter (nt 10896-1 1918) followed by the TDH3 terminator (nt 8237-9235 ) for KivD expression.
[0283] A PNY1503 transformant (parental strain) was designated PNY1504. A PNY1505 transformant (fra2 deletion strain) was designated PNY1506. Transformers from PNY1541 and PNY1542 (integration strains AFT1-L99A) were designated PNY1543 and PNY1544, for PNY1541, and PNY1545 and PNY1546, for PNY1542.
[0284] Strains were grown in synthetic medium (Yeast Nitrogen Base without Amino Acids (Sigma-Aldrich, St. Louis, MO) and Yeast Synthetic Drop- Out Media Supplement without uracil and histidine (Clontech, Mountain View, CA)) supplemented with 100 mM MES and pH 5.5, 0.2% glucose, and 0.2% ethanol. Overnight, cultures were grown in 15 ml medium in ventilated 125 ml Erlenmeyer flasks at 30 ° C, 225 RPM in a New Brunswick Scientific 124 shaker. 18 ml of medium in tightly capped 125 Erlenmeyer flasks ml were inoculated with overnight culture in a 0.5 OD600 and cultured for six hours at 30 ° C, 225 RPM on a New Brunswick Scientific 124 shaker. After six hours, glucose was added to 2.5% , the yeast extract was added at 5 g / l, and peptone was added at 10 g / l (0 hour time). After 24 and 48 hours, culture supernatants (collected using Costar No. 8169 Spin-X centrifuge tube filter units) were analyzed by HPLC (method described in Patent Publication Application No. US 2007/0092957, incorporated by reference in this document) and LC / MS. Glucose and isobutanol concentrations were determined by HPLC. DHIV was separated and quantified by LC / MS in a Waters AcquityTQD system (Milford, MA), using an Atlantis T3 column (part # 186003539). The column was maintained at 30 ° C and the flow rate was 0.5 ml / min. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. Each occurrence consisted of 1 min in 99% A, a linear gradient over 1 min in 25% B, followed by 1 min in 99% A. The column effluent was monitored for peaks at m / z = 133 (ESI negative), with 32.5V cone voltage, by Waters ACQ_TQD mass spectrometry detector (s / n QBA688). DHIV typically emerged in 1.2 min. Baseline separation was obtained and peak areas for DHIV were converted to μM DHIV concentrations by reference to analyzes of standard solutions made from a 1 M aqueous stock.
[0285] Table 18 shows the molar yield of DHIV (moles of DHIV per mole of glucose consumed) and isobutanol titration of strains AFT1-L99A (PNY1543, PNY1544, PNY1545, and PNY1546) and the FRA2 deletion strain (PNY1506) compared to the previous parental strain (PNY1504) in 24 and 48 hours. The expression of AFT1 -L99A or the deletion of FRA2 led to an approximately 50% decrease in DHIV accumulation. TABLE 18. MOLAR YIELD OF DHIV AND TITRATION OF ISOBUTANOL

[0286] The data are the average of two independent vials, for PNY1504 and PNY1506, and two independent transformants for the strains AFT1-L99A (PNY1543-PNY1544 and PNY1545-PNY1546).
[0287] The aforementioned description of the specific achievements will then fully reveal the general nature of the invention, since individuals can, by applying the wisdom of elements versed in the technique, promptly modify and / or adapt the specific achievements in various applications , without undue experimentation and without departing from the general concept of the present invention. Thus, such adaptations and modifications are intended to be within the meaning and scope of equivalents of the revealed achievements, based on the teaching and guidance presented in this document. It should be understood that the expression or terminology of this document is intended to describe, and not to limit, so that the terminology or expression of this descriptive report is interpreted by the elements versed in the light of the teachings and guidelines. TABLE 12
[0288] HMMER2.0 (2.2g) Name and version of the program.
[0289] NAME dhad_for_hmm Name of the input sequence alignment file.
[0290] LENGTH 564 The alignment length includes indels.
[0291] ALPH Amino Type of waste.
[0292] MAP yes Map of the compatibility status for the alignment columns.
[0293] COM / app / public / hmmer / current / bin / hmmbuild- F_dhad-exp_hmm_dhad_form_hmm.aln Commands used to generate the file. This means that hmmbuild (default parameters) has been applied to the alignment file.
[0294] COM / app / public / hmmer / current / bin / hmmcalibrate dhad-exp_hmm Commands used to generate the file. This means that hmmcalibrate (default parameters) has been applied to the hmm profile.
[0295] NSEQ 8 Number of sequences in the alignment file.
[0296] DATE Tuesday, June 03, 2008, 10:48:24 AM When the file was generated.
[0297] XT-8455-4-1000-1000-8455-4-8455-4
[0298] NULT-4-8455 The transition probability distribution for the null model (single state G).
[0299] NULL 595-1568 35 338-294 453-1158 197 249 902- 1685-142-21-313 45 531 201 364-199 The emission probability distribution symbol for the null model (state G) consists of numbers K (for example, 4 or 20). The null probability used to convert this back to model probabilities is 1 / K.
[0300] EVD-498.650970 0.086142 The extreme value distribution parameters μ and lambda, both respectively being floating point values. Lambda is positive, not zero. These values are defined when the model is calibrated with hmmcalibrate.

权利要求:
Claims (14)
[0001]
1. Yeast recombinant host cell, characterized by comprising: (a) at least one heterologous ilvD gene that encodes a polypeptide that has dihydroxy acid dehydratase activity, in which the polypeptide is presented by SEQ ID NO: 168 or SEQ ID NO: 232; and (b) (i) at least one deletion in an endogenous gene that encodes a polypeptide that affects the Fe-S cluster biosynthesis in which the gene is selected from the group consisting of FRA2, CCC1, GRX3 and their combinations , and where FRA2 is presented by the nucleic acid sequence of SEQ ID NO: 773 or the amino acid sequence of SEQ ID NO: 706, CCC1 is presented by the nucleic acid sequence of SEQ ID NO: 811 or the amino acid sequence of SEQ ID NO: 744, and GRX3 is presented by the nucleic acid sequence of SEQ ID NO: 774 or amino acid sequence of SEQ ID NO: 707; and / or (c)) at least one heterologous polynucleotide that encodes a polypeptide that affects Fe-S cluster biosynthesis, wherein said polynucleotide is selected from the group consisting of AFT1, AFT2 and combinations thereof, and where AFT1 is presented by the nucleic acid sequence of SEQ ID NO: 770 or amino acid sequence of SEQ ID NO: 703, and AFT2 is presented by the nucleic acid sequence of SEQ ID NO: 771 or amino acid sequence of SEQ ID NO : 704; and wherein the Fe-S cluster biosynthesis in said host cell having (i) and / or (ii) is increased compared to a recombinant host cell having endogenous Fe-S cluster biosynthesis.
[0002]
2. CELL according to claim 1, characterized by said at least one heterologous ilvD gene: (a) comprising a high copy number plasmid or a plasmid with a copy number that can be regulated; or (b) be integrated at least once into the recombinant host cell DNA.
[0003]
3. CELL according to any one of claims 1 to 2, characterized in that said polypeptide that affects Fe-S cluster biosynthesis is encoded by a polynucleotide that is a constitutive mutant, wherein said constitutive mutant is selected from of the group consisting of AFT1 L99A, AFT1 L102A, AFT1 C291F, AFT1 C293F and combinations thereof.
[0004]
4. CELL according to any one of claims 1 to 2, characterized in that said polypeptide that affects the Fe-S cluster biosynthesis is FRA2 or CCC1.
[0005]
CELL according to any one of claims 1 to 4, characterized in that said host cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia and Pichia.
[0006]
CELL according to any one of claims 1 to 5, characterized in that said heterologous polypeptide which has dihydroxy acid dehydratase activity is expressed in the cytosol of said host cell.
[0007]
A CELL according to any one of claims 1 to 6, characterized in that 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.
[0008]
CELL according to any one of claims 1 to 7, characterized in that said recombinant host cell comprises an isobutanol biosynthetic pathway.
[0009]
9. METHOD FOR MANUFACTURING A PRODUCT, characterized by comprising: (a) supplying the recombinant yeast host cell, as defined in any one of claims 1 to 7; (b) contacting the recombinant yeast host cell of (a) with a fermentable carbon substrate in a fermentation medium under conditions in which said product is produced; the product being selected from the group consisting of branched chain amino acids, pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol, isobutanol and combinations thereof, preferably isobutanol.
[0010]
10. METHOD FOR CONVERTING 2,3-DIIDROXYISOVALERATE TO α-CETOISOVALERATE, characterized by comprising: (a) providing the recombinant yeast host, as defined in any one of claims 1 to 8; and (b) culturing the recombinant yeast host cell from (a) under conditions where 2,3-dihydroxyisovalerate is converted to α-ketoisovalerate
[0011]
11. METHOD FOR INCREASING THE SPECIFIC ACTIVITY OF A HETEROLOGIC POLYPEPTIDE, which has dihydroxy acid dehydratase activity in a recombinant host cell, characterized by comprising: (a) providing a recombinant yeast host cell, as defined in any of claims 1 to 8; and (b) culturing the recombinant host cell of (a) under conditions in which the heterologous polypeptide which has dihydroxy acid dehydratase activity is expressed in functional form, having a specific activity greater than the same host cell without said heterologous polypeptide.
[0012]
12. METHOD FOR INCREASING FLOW IN A BIOSYNTHETIC PATH OF Fe-S cluster in a host cell, characterized by comprising: (a) providing a recombinant yeast host cell, as defined in any of claims 1 to 8; and (b) culturing the recombinant yeast host cell from (a) under conditions where the flow in the Fe-S cluster biosynthetic pathway in the host cell is increased.
[0013]
13. METHOD FOR INCREASING A PROTEIN ACTIVITY, which requires a cluster of Fe-S in a recombinant host cell, characterized by comprising: (a) providing a recombinant host cell, as defined in any one of claims 1 to 8, which comprises a protein that requires Fe-S cluster, in which protein that requires Fe-S cluster has dihydroxy acid dehydratase activity, shown by SEQ ID NO: 168 or SEQ ID NO: 232; (b) alter the expression or activity of a polypeptide that affects the Fe-S cluster biosynthesis in said host cell, wherein said polypeptide that affects the Fe-S cluster biosynthesis is encoded by a gene selected from the group which consists of FRA2, CCC1, GRX3, AFT1, AFT2 and combinations thereof; and (c) cultivating the recombinant host cell of (b) under conditions where the activity of the protein requiring Fe-S cluster is increased.
[0014]
14. METHOD, according to claim 13, characterized in that said host cell is a yeast host cell, preferably in which said yeast host cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia, and Pichia.

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法律状态:
2018-03-06| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-06-23| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-12-01| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-29| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 17/02/2011, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-05-18| B16C| Correction of notification of the grant|Free format text: REF. RPI 2608 DE 29/12/2020 QUANTO AO TITULAR E AO TITULO. |

优先权:

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申请号 | 申请日 | 专利标题

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US30533310P| true| 2010-02-17|2010-02-17|

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US61/305,333|2010-02-17|

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PCT/US2011/025258|WO2011103300A2|2010-02-17|2011-02-17|Improving activity of fe-s cluster requiring proteins|

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