Global transcription machinery engineering

Abstract

The invention relates to global transcription machinery engineering to produce altered cells having improved phenotypes.

1 . A method for altering the phenotype of a cell comprising: 
 mutating a nucleic acid encoding global transcription machinery and, optionally, its promoter, expressing the nucleic acid in a cell to provide an altered cell that includes mutated global transcription machinery, and culturing the altered cell;    optionally determining the phenotype of the altered cell, and/or    optionally repeating the mutation of the nucleic acid to produce a n th  generation altered cell, and preferably determining the phenotype of the n th  generation altered cell and/or preferably wherein the step of repeating the mutation of the global transcription machinery comprises isolating a nucleic acid encoding the mutated global transcription machinery and optionally, its promoter, from the altered cell, mutating the nucleic acid, and introducing the mutated nucleic acid into another cell.    
     
     
         2 - 5 . (canceled)  
     
     
         6 . The method of  claim 1 , wherein the cell is a prokaryotic cell, preferably a bacterial cell or an archaeal cell.  
     
     
         7 . (canceled)  
     
     
         8 . The method of  claim 6 , wherein the global transcription machinery is a sigma factor or an anti-sigma factor, preferably wherein a nucleic acid encoding the sigma factor is a rpoD (σ 70 ) gene, a rpoF (σ 28 ) gene, a rpoS (σ 38 ) gene, a rpoH (σ 32 ) gene, a rpoN (σ 54 ) gene, a rpoE (σ 24 ) gene or a fecI (σ 19 ) gene and/or wherein the sigma factor or anti-sigma factor is expressed from an expression vector.  
     
     
         9 - 10 . (canceled)  
     
     
         11 . The method of  claim 1 , wherein the cell is a eukaryotic cell. preferably a yeast cell, a mammalian cell, a plant cell, an insect cell, a stem cell. or a fungus cell; and preferably wherein the eukaryotic cell is contained in a multicellular organism; or wherein the nucleic acid is expressed in the cell from a tissue-specific promoter, a cell-specific promoter, or an organelle-specific promoter; or wherein the nucleic acid is a nucleic acid of an organelle of the eukaryotic cell, preferably wherein the organelle is a mitochondrion or a chloroplast.  
     
     
         12 - 19 . (canceled)  
     
     
         20 . The method of  claim 11 , wherein the global transcription machinery binds to an RNA polymerase I, an RNA polymerase II or an RNA polymerase III, or a promoter of an RNA polymerase I, an RNA polymerase II or an RNA polymerase III, preferably wherein the global transcription machinery is a nucleic acid methyltransferase, a histone methyltransferase, a histone acetylase or a histone deacetylase, TFIID or a subunit thereof, preferably wherein the subunit is TATA-binding protein (TBP) or a TBP-associated factor (TAF), or preferably wherein a nucleic acid encoding the global transcription machinery is a GAL11 gene, a SIN4 gene, a RGR1 gene, a HRS1 gene, a PAF1 gene, a MED2 gene, a SNF6 gene, a SNF2 gene, or a SW11 gene; and/or wherein the global transcription machinery is expressed from an expression vector.  
     
     
         20 - 27 . (canceled)  
     
     
         28 . The method of  claim 1 , wherein the nucleic acid is part of an expression vector and/or is a member of a collection of nucleic acids, preferably wherein the method further comprises introducing the collection into the cell.  
     
     
         29 - 30 . (canceled)  
     
     
         31 . The method of  claim 1 , wherein the step of expressing the nucleic acid comprises integrating the nucleic acid into the genome or replacing a nucleic acid that encodes the endogenous global transcription machinery.  
     
     
         32 . The method of  claim 1 , wherein the mutation of the nucleic acid comprises directed evolution of the nucleic acid, preferably mutation by error prone PCR or mutation by gene shuffling, or wherein the mutation of the nucleic acid comprises synthesizing the nucleic acid with one or more mutations.  
     
     
         33 - 35 . (canceled)  
     
     
         36 . The method of  claim 1 , wherein the nucleic acid mutations is/are one or more point mutations and/or one or more truncations or deletions, preferably wherein a promoter binding region of the global transcription machinery is not disrupted or removed by the one or more truncations or detections.  
     
     
         37 - 38 . (canceled)  
     
     
         39 . The method of  claim 1 , wherein the mutated global transcription machinery exhibits increased or decreased transcription of genes relative to the unmutated global transcription machinery, and/or wherein the mutated global transcription machinery exhibits increased or decreased repression of gene transcription relative to the unmutated global transcription machinery.  
     
     
         40 - 42 . (canceled)  
     
     
         43 . The method of  claim 1 , further comprising selecting the altered cell for a predetermined phenotype, preferably wherein the step of selecting comprises culturing the altered cell under selective conditions, and/or wherein the step of selecting comprises high-throughput assays of individual cells for the phenotype, and/or wherein the cell is contained in a multicellular organism.  
     
     
         44 - 45 . (canceled)  
     
     
         46 . The method of  claim 43 , wherein the phenotype is increased tolerance of deleterious culture conditions, preferably solvent tolerance or hazardous waste tolerance, such as wherein the solvent is ethanol, hexane or cyclohexane; tolerance of industrial media; tolerance of high sugar concentration; tolerance of high salt concentration; tolerance of high temperatures; tolerance of extreme pH; tolerance of surfactants, or tolerance of a plurality of deleterious conditions; increased metabolite production, preferably lycopene; polyhydroxybutyrate (PHB); or a therapeutic protein, such as an antibody or an antibody fragment, tolerance to a toxic substrate, metabolic intermediate or product, preferably an organic solvent, acetate or para-hydroxybenzoic acid (pHBA), or an overexpressed protein; antibiotic resistance; increased resistance to apoptosis; one or more growth characteristics; generation time; resistance to one or more pests or diseases, production of fruit or other parts of a plant; one or more developmental changes; one or more lifespan alterations; gain or loss of function; and/or increased robustness.  
     
     
         47 - 68 . (canceled)  
     
     
         69 . The method of  claim 1 , wherein the cell used in the method is optimized for the phenotype prior to mutating the global transcription machinery.  
     
     
         70 . The method of  claim 1 , further comprising identifying the changes in gene expression in the altered cell, preferably wherein the changes in gene expression are determined using a nucleic acid microarray.  
     
     
         71 . (canceled)  
     
     
         72 . A method for altering the phenotype of a cell comprising altering the expression of one or more gene products in a first cell that are identified by detecting changes in gene expression in a second cell, wherein the changes in gene expression in the second cell are produced by mutating global transcription machinery of the second cell.  
     
     
         73 . The method of  claim 72 , wherein altering the expression of the one or more gene products in the first cell comprises increasing expression of one or more gene products that were increased in the second cell, optionally wherein the expression of the one or more gene products is increased by introducing into the first cell one or more expression vectors that express the one or more gene products and/or by increasing the transcription of one or more endogenous genes that encode the one or more gene products, preferably wherein increasing the transcription of the one or more endogenous genes comprises mutating a transcriptional control sequence of the one or more genes.  
     
     
         74 - 76 . (canceled)  
     
     
         77 . The method of  claim 72 , wherein altering the expression of the one or more gene products in the first cell comprises decreasing expression of one or more gene products that were decreased in the altered cell, optionally wherein the expression of the one or more gene products is decreased by introducing into the first cell nucleic acid molecules that reduce the expression of the one or more gene products, preferably wherein the nucleic acid molecules are, or express, siRNA molecules, or wherein the expression of the one or more gene products is decreased by mutating one or more genes that encode the one or more gene products or a transcriptional control sequence of the one or more genes.  
     
     
         78 - 80 . (canceled)  
     
     
         81 . The method of  claim 72 , wherein the changes in gene expression in the second cell are determined using a nucleic acid microarray: and/or wherein the changes in gene expression in the second cell are used to construct a model of a gene or protein network, and wherein the model is used to select which of the one or more gene products in the network to alter.  
     
     
         82 . (canceled)  
     
     
         83 . The method of  claim 1 , wherein the global transcription machinery comprises more than one nucleic acid and/or polypeptide or is encoded by more than one nucleic acid.  
     
     
         84 . A cell produced by the method of  claim 1 .  
     
     
         85 . A method for altering the production of a metabolite, comprising 
 mutating, according to  claim 1 , global transcription machinery of a cell that produces a selected metabolite to produce an altered cell, and    isolating altered cells that produce increased or decreased amounts of the selected metabolite, preferably wherein the method further comprises culturing the isolated cells, and recovering the metabolite from the cells or the cell culture.    
     
     
         86 . (canceled)  
     
     
         87 . The method of  claim 85 , wherein the metabolite is lycopene; polyhydroxybutyrate (PHB); or a therapeutic protein, preferably a recombinant protein, an antibody or an antibody fragment.  
     
     
         88 - 91 . (canceled)  
     
     
         92 . The method of  claim 85 , wherein the cells are prokaryotic cells, preferably bacterial cells or archaeal cells; or eukaryotic cells, preferably yeast cells, mammalian cells, plant cells, insect cells, stem cells or fungus cells.  
     
     
         93 - 100 . (canceled)  
     
     
         101 . The method of  claim 85 , wherein the global transcription machinery is encoded by nucleic acid of an organelle of the eukaryotic cell, preferably a mitochondrion or a chloroplast.  
     
     
         102 . (canceled)  
     
     
         103 . A collection comprising a plurality of different nucleic acid molecule species, wherein each nucleic acid molecule species encodes global transcription machinery comprising different mutation(s), optionally wherein the global transcription machinery is a sigma factor or an anti-sigma factor, preferably wherein a nucleic acid encoding the sigma factor is a rpoD (σ 70 ) gene, a rpoF (σ 28 ) gene, a rpoS (σ 38 ) gene, a rpoH (σ 32 ) gene, a rpoN (σ 54 ) gene, a rpoE (σ 24 ) gene or a fecI (σ 19 ) gene.  
     
     
         104 - 105 . (canceled)  
     
     
         106 . The collection of  claim 103 , wherein the global transcription machinery binds to an RNA polymerase I, an RNA polymerase II or an RNA polymerase III, or a promoter of an RNA polymerase I, an RNA polymerase II or an RNA polymerase III; optionally wherein the global transcription machinery is a nucleic acid methyltransferase, a histone methyltransferase, a histone acetylase, a histone deacetylase, or TFIID or a subunit thereof, preferably wherein the subunit is TATA-binding protein (TBP) or a TBP-associated factor (TAF).  
     
     
         107 - 109 . (canceled)  
     
     
         110 . The collection of  claim 103 , wherein the nucleic acid molecule species are contained in expression vectors, preferably wherein the nucleic acid is expressed from a tissue-specific promoter, a cell-specific promoter, or an organelle-specific promoter, and/or wherein the expression vectors contain a plurality of different nucleic acid molecule species, wherein each nucleic acid molecule species encodes different global transcription machinery.  
     
     
         111 - 112 . (canceled)  
     
     
         113 . The collection of  claim 103 , wherein the global transcription machinery is mutated by directed evolution, preferably wherein the directed evolution is performed using error prone PCR or gene shuffling.  
     
     
         114 - 115 . (canceled)  
     
     
         116 . The collection of  claim 103 , wherein the mutation(s) in the global transcription machinery is/are one or more point mutations and/or one or more truncations or deletions, preferably wherein the truncation does not include the promoter binding region of the global transcription machinery.  
     
     
         117 - 118 . (canceled)  
     
     
         119 . The collection of  claim 103 , wherein the global transcription machinery of a cell is mutated according to  claim 1 .  
     
     
         120 . A collection of cells comprising the collection of nucleic acid molecules of  claim 103 , preferably comprising a plurality of cells, each of the plurality of cells comprising one or more of the nucleic acid molecules.  
     
     
         121 . (canceled)  
     
     
         122 . The collection of  claim 120 , wherein the cells are prokaryotic cells, preferably bacterial cells or archaeal cells, or eukaryotic cells, preferably yeast cells, mammalian cells, plant cells, insect cells, stem cells or fungus cells.  
     
     
         123 - 130 . (canceled)  
     
     
         131 . The collection of  claim 120 , wherein the nucleic acid molecules are integrated into the genome of the cells or replace nucleic acids that encode the endogenous global transcription machinery.  
     
     
         132 . A nucleic acid encoding global transcription machinery produced by a plurality of rounds of mutation, optionally wherein the plurality of rounds of mutation comprise directed evolution, preferably wherein the directed evolution comprises mutation by error prone PCR or mutation by gene shuffling.  
     
     
         133 - 135 . (canceled)  
     
     
         136 . The nucleic acid of  claim 132 , wherein the nucleic acid encodes a plurality of different global transcription machinery species or a plurality of different versions of the same type of global transcription machinery species.  
     
     
         137 . (canceled)  
     
     
         138 . Global transcription machinery encoded by the nucleic acid of  claim 132 .  
     
     
         139 . A truncated sigma factor protein comprising (carboxy-terminal) region 4.  
     
     
         140 . A method for bioremediation of a selected waste product, comprising 
 mutating, according to  claim 1 , global transcription machinery of a cell to produce an altered cell,    isolating altered cells that metabolize an increased amount of the selected waste product relative to unaltered cells,    culturing the isolated cells, and    exposing the altered cells to the selected waste product, thereby providing bioremediation of the selected waste product.