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  Global transcription machinery engineeringUSPTO Application #: 20070072194Title: Global transcription machinery engineering Abstract: The invention relates to global transcription machinery engineering to produce altered cells having improved phenotypes. (end of abstract) Agent: Wolf Greenfield & Sacks, PC - Boston, MA, US Inventors: Hal S. Alper, Gregory Stephanopoulos USPTO Applicaton #: 20070072194 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20070072194. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The invention relates to global transcription machinery engineering to produce altered cells having improved phenotypes. BACKGROUND OF THE INVENTION [0002] It is now generally accepted that many important cellular phenotypes, from disease states to metabolite overproduction, are affected by many genes. Yet, most cell and metabolic engineering approaches rely almost exclusively on the deletion or over-expression of single genes due to experimental limitations in vector construction and transformation efficiencies. These limitations preclude the simultaneous exploration of multiple gene modifications and confine gene modification searches to restricted sequential approaches where a single gene is modified at a time. [0003] U.S. Pat. No. 5,686,283 described the use of a sigma factor encoded by rpoS to activate the expression of other bacterial genes that are latent or expressed at low levels in bacterial cells. This patent did not, however, describe mutating the sigma factor in order to change globally the transcription of genes. [0004] U.S. Pat. No. 5,200,341 provides a mutated rpoH gene identified as a suppressor of a temperature sensitive rpoD gene by selection of temperature-resistant mutants of a bacterial strain having the temperature sensitive rpoD gene. No mutagenesis of the bacteria was undertaken, nor was the suppressor strain selected for a phenotype other than temperature resistance. When the mutant rpoH gene is added to other bacteria that are modified to express heterologous proteins, the heterologous proteins are accumulated at increased levels in the bacteria. [0005] U.S. Pat. No. 6,156,532 describes microorganisms that are modified by introduction of a gene coding for a heat shock protein and a gene coding for a sigma factor (rpoH) that specifically functions for the heat shock protein gene to enhance expression amount of the heat shock protein in cells. The modified microorganisms are useful for producing fermentative products such as amino acids. The sigma factor used in the microorganisms was not mutated. [0006] Directed evolution has been applied to microorganisms by shuffling of bacterial genomes for antibiotic (tylosin) production by Streptomyces (Zhang et al., Nature, 415, 644-646 (2002)) and acid tolerance of Lactobacillus (Patnaik et al.,A Nature Biotech. 20, 707-712 (2002)). These methods did not target mutations in any specific gene or genes, but instead non-recombinantly shuffled the genomes of strains having a desired phenotype using protoplast fusion, followed by selection of strains having improvements in the desired phenotype. SUMMARY OF THE INVENTION [0007] The invention utilizes global transcription machinery engineering to produce altered cells having improved phenotypes. In particular, the invention is demonstrated through the generation of mutated bacterial sigma factors with varying preferences for promoters on a genome-wide level. The cells resulting from introduction of the mutated sigma factors have rapid and marked improvements in phenotypes, such as tolerance of deleterious culture conditions or improved production of metabolites. [0008] The introduction of mutant transcription machinery into a cell, combined with methods and concepts of directed evolution, allows one to explore a vastly expanded search space in a high throughput manner by evaluating multiple, simultaneous gene alterations in order to improve complex cellular phenotypes. [0009] Directed evolution through iterative rounds of mutagenesis and selection has been successful in broadening properties of antibodies and enzymes (W. P. Stemmer, Nature 370, 389-91 (1994)). These concepts have been recently extended and applied to non-coding, functional regions of DNA in the search for libraries of promoter activity spanning a broad dynamic range of strength as measured by different metrics (H. Alper, C. Fischer, E. Nevoigt, G. Stephanopoulos, Proc Natl Acad Sci USA 102, 12678-12683 (2005)). However, no evolution-inspired approaches have been directed towards the systematic modification of the global transcription machinery as a means of improving cellular phenotype. Yet, detailed biochemical studies suggest that both the transcription rate and in vitro preference for a given promoter sequence can be altered by modifying key residues on bacterial sigma factors (D. A. Siegele, J. C. Hu, W. A. Walter, C. A. Gross, J Mol Biol 206, 591-603 (1989); T. Gardella, H. Moyle, M. M. Susskind, J Mol Biol 206, 579-590 (1989)). Such modified transcription machinery units offer the unique opportunity to introduce simultaneous global transcription-level alterations that have the potential to impact cellular properties in a very profound way. [0010] According to one aspect of the invention, methods for altering the phenotype of a cell are provided. The methods include 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. In some embodiments, the methods also include determining the phenotype of the altered cell or comparing the phenotype of the altered cell with the phenotype of the cell prior to alteration. In further embodiments, the methods also include repeating the mutation of the nucleic acid to produce a n.sup.th generation altered cell. In still other embodiments, the methods also include determining the phenotype of the n.sup.th generation altered cell or comparing the phenotype of the n.sup.th generation altered cell with the phenotype of any prior generation altered cell or of the cell prior to alteration. In preferred embodiments, the step of repeating the mutation of the global transcription machinery includes 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. [0011] In certain embodiments, the cell is a prokaryotic cell, preferably a bacterial cell or an archaeal cell. In such embodiments, the global transcription machinery preferably is a sigma factor or an anti-sigma factor. Nucleic acid molecules encoding the sigma factors include rpoD (.sigma..sup.70) genes, rpoF (.sigma..sup.28) genes, rpoS (.sigma..sup.38) genes, rpoH (.sigma..sup.32) genes, rpoN (.sigma..sup.54) genes, rpoE (.sigma..sup.24) genes and fecI (.sigma..sup.19) genes. The sigma factor or anti-sigma factor can be expressed from an expression vector. [0012] In other embodiments, the cell is a eukaryotic cell. Preferred eukaryotic cells include yeast cells, mammalian cells, plant cells, insect cells, stem cells and fungus cells. In certain embodiments, one or more of the eukaryotic cells are contained in, or form, a multicellular organism. In some embodiments, the nucleic acid is expressed in the cell from a tissue-specific promoter, a cell-specific promoter, or an organelle-specific promoter. [0013] In still other eukaryotic embodiments, 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. Preferred global transcription machinery includes TFIID or a subunit thereof, such as TATA-binding protein (TBP) or a TBP-associated factor (TAF). Nucleic acid molecules encoding the global transcription machinery include GAL11 genes, SIN4 genes, RGR1 genes, HRS1 genes, PAF1 genes, MED2 genes, SNF6 genes, SNF2 genes and SW11 genes. The global transcription machinery, in other embodiments, is a nucleic acid methyltransferase, a histone methyltransferase, a histone acetylase or a histone deacetylase. The global transcription machinery is expressed from an expression vector in certain embodiments. [0014] The nucleic acid in some embodiments is a nucleic acid of an organelle of the eukaryotic cell, preferably a mitochondrion or a chloroplast. The nucleic acid optionally is part of an expression vector. [0015] The nucleic acid in certain embodiments is a member of a collection (e.g., a library) of nucleic acids. Thus the methods of the invention include, in some embodiments, introducing the collection into the cell. [0016] In further embodiments, the step of expressing the nucleic acid includes integrating the nucleic acid into the genome or replacing a nucleic acid that encodes the endogenous global transcription machinery. [0017] The mutation of the nucleic acid, in certain embodiments, includes directed evolution of the nucleic acid, such as mutation by error prone PCR or mutation by gene shuffling. In other embodiments, the mutation of the nucleic acid includes synthesizing the nucleic acid with one or more mutations. [0018] Nucleic acid mutations in the invention can include one or more point mutations, and/or one or more truncations and/or deletions. [0019] In some embodiments of the invention, a promoter binding region of the global transcription machinery is not disrupted or removed by the one or more truncations or detections. In other embodiments, the mutated global transcription machinery exhibits increased transcription of genes relative to the unmutated global transcription machinery, decreased transcription of genes relative to the unmutated global transcription machinery, increased repression of gene transcription relative to the unmutated global transcription machinery, and/or decreased repression of gene transcription relative to the unmutated global transcription machinery. [0020] In still other embodiments, the methods also include selecting the altered cell for a predetermined phenotype. Preferably, the step of selecting includes culturing the altered cell under selective conditions and/or high-throughput assays of individual cells for the phenotype. [0021] A wide variety of phenotypes can be selected in accordance with the invention. In some preferred embodiments, the phenotype is increased tolerance of deleterious culture conditions. Such phenotypes include: solvent tolerance or hazardous waste tolerance, e.g., 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, and tolerance of a plurality of deleterious conditions. Continue reading... Full patent description for Global transcription machinery engineering Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Global transcription machinery engineering patent application. 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