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  Genes encoding genetic stability, gene expression and folding proteinsUSPTO Application #: 20070037262Title: Genes encoding genetic stability, gene expression and folding proteins Abstract: The invention relates to novel nucleic acid molecules, to the use thereof for constructing genetically improved microorganisms and to methods for preparing fine chemicals, in particular amino acids, with the aid of said genetically improved microorganisms. (end of abstract) Agent: Lahive & Cockfield, LLP - Boston, MA, US Inventors: Oskar Zelder, Markus Pompejus, Hartwig Schroder, Burkhard Kroger, Corinna Klopprogge, Gregor Haberhauer USPTO Applicaton #: 20070037262 - Class: 435115000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Preparing Alpha Or Beta Amino Acid Or Substituted Amino Acid Or Salts Thereof, Lysine; Diaminopimelic Acid; Threonine; Valine The Patent Description & Claims data below is from USPTO Patent Application 20070037262. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0001] Particular products and byproducts of naturally occurring metabolic processes in cells are used in many branches of industry, including the food industry, the animal feed industry, the cosmetics industry and the pharmaceutical industry. These molecules which are collectively referred to as "fine chemicals" comprise organic acids, both proteinogenic and nonproteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins, cofactors and enzymes. They are best produced by means of cultivating, on a large scale, bacteria which have been developed to produce and secrete large amounts of the molecule desired in each particular case. An organism which is particularly suitable for this purpose is Corynebacterium glutamicum, a Gram-positive nonpathogenic bacterium. Using strain selection, a number of mutant strains have been developed which produce various desirable compounds. The selection of strains which are improved with respect to the production of a particular molecule is, however, a time-consuming and difficult process. BRIEF DESCRIPTION OF THE INVENTION [0002] The present invention provides novel nucleic acid molecules which can be used for identifying or classifying Corynebacterium glutamicum or related bacterial species. C. glutamicum is a Gram-positive, aerobic bacterium which is normally widely used in industry for the large-scale production of a number of fine chemicals and also for the degradation of hydrocarbons (for example in the case of crude oil spills) and for the oxidation of terpenoids. The nucleic acid molecules may therefore be used for identifying microorganisms which can be used for producing fine chemicals, for example by fermentation processes. Although C. glutamicum itself is nonpathogenic, it is, however, related to other Corynebacterium species such as Corynebacterium diphteriae (the diphtheria pathogen), which are major pathogens in humans. The ability to identify the presence of Corynebacterium species may therefore also be of significant clinical importance, for example in diagnostic applications. Moreover, said nucleic acid molecules may serve as reference points for mapping the C. glutamicum genome or genomes of related organisms. [0003] These novel nucleic acid molecules encode proteins which are referred to herein as proteins for gene stability, gene expression or protein secretion/protein folding (SES proteins). These SES proteins may, for example, exert a function which is involved in repair or recombination of DNA, transposition of genetic material, expression of genes (i.e. which are involved in transcription or translation), protein folding or protein secretion in C. glutamicum. Owing to the availability of cloning vectors which can be used in Corynebacterium glutamicum, as disclosed, for example in Sinskey et al., U.S. Pat. No. 4,649,119, and of techniques for the genetic manipulation of C. glutamicum and the related Brevibacterium species (e.g. lactofermentum) (Yoshihama et al., J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159: 306-311 (1984); and Santamaria et al. J. Gen. Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of the invention can be used for genetic manipulation of said organism in order to make it a more efficient producer of one or more fine chemicals. This improved production or efficiency of production of a fine chemical may be caused directly by manipulation of a gene of the invention or indirectly by such a manipulation. [0004] There is a number of mechanisms by which modification of an SES protein of the invention can directly influence the yield, production and/or efficiency of production of a fine chemical from a C. glutamicum strain containing this modified protein. For example, modulation of proteins directly involved in transcription or translation (e.g. polymerases or ribosomes) so as to increase their number or activity should overall increase cellular transcription or translation (or the rate of these processes). This increased cellular gene expression should include those proteins which are involved in the biosynthesis of fine chemicals so that the yield, production or efficiency of production of one or more compounds of interest can be increased. Modifications of the transcriptional/translational protein machinery of C. glutamicum so as to modify regulation of these proteins may also enable the increased expression of genes involved in the production of fine chemicals. Modulation of the activity of a number of proteins involved in peptide folding may increase the overall production of correctly folded molecules in the cell, thereby increasing the possibility of proteins of interest (e.g. proteins of the biosynthesis of fine chemicals) functioning correctly. Furthermore, it may be possible, by mutating proteins involved in the secretion from C. glutamicum so as to increase their number or activity, to increase secretion of a fine chemical (e.g. an enzyme) from cells in a fermentative culture from which said fine chemical can be readily obtained. [0005] Genetic modification of the SES molecules of the invention may also modulate indirectly the production of one or more fine chemicals. For example, it is possible, by increasing the number or activity of a DNA-repair or DNA-recombination protein of the invention, to increase the ability of the cell to detect and repair DNA damage. This should effectively increase the ability of the cell to keep a mutated gene in its genome and thereby increase the probability of a transgene genetically introduced into C. glutamicum (which encodes, for example, a protein which increases the biosynthesis of a fine chemical) not being lost during cultivation of the microorganism. In contrast, it may be possible, by reducing the number or activity of one or more DNA-repair or DNA-recombination proteins, to increase the genetic instability of the organism. These manipulations should improve the ability of said organism to be modified by mutagenesis, without correcting the introduced mutation. The same is true for proteins which are involved in the transposition or rearrangement of genetic elements in C. glutamicum (e.g. transposons). Mutagenesis of these proteins so as to either increase or reduce their number or activity makes it possible to increase or reduce at the same time the genetic stability of the microorganism. This crucially affects the possibility of introducing another mutation into C. glutamicum and of retaining the introduced mutation. Transposons likewise provide a suitable mechanism which makes possible the mutagenesis of C. glutamicum; duplication of genes of interest (e.g. genes of the biosynthesis of fine chemicals) as well as disruption of unwanted genes (e.g. genes involved in the degradation of fine chemicals of interest) can be readily carried out by means of transposon mutagenesis. [0006] It may be possible, by modulating one or more proteins (e.g. sigma factors) which are involved in the regulation of transcription or translation in reaction to particular environmental conditions, to prevent the cell from slowing down or stopping protein synthesis under unfavorable environmental conditions as found in a large-scale fermentative culture. This should increase gene expression, and this in turn may enable the increased biosynthesis of fine chemicals of interest under said conditions. Mutagenesis of proteins involved in secretion systems may result in modulated secretion rates. Many of these secreted proteins have functions which are important for cell viability (e.g. cell surface proteases or cell surface receptors). A change in the secretion pathway so that these proteins are transported more readily to their extracellular location may increase the overall viability of the cell and thus result in higher numbers of C. glutamicum cells able to produce fine chemicals during large-scale cultivation. It is furthermore known that the secretion apparatus (e.g. the sec system) is also involved in the insertion of integral membrane proteins (e.g. pores, channels or transporters) into the membrane. Thus, modulation of the activity of proteins involved in protein secretion from C. glutamicum may influence the ability of the cell to secrete waste products or to import necessary metabolites. If the activity of these secretory proteins is increased, the ability of the cell to produce fine chemicals may likewise be increased. If the activity of said secretory proteins is reduced, there may not be enough nutrients to support overproduction of compounds of interest or waste products may interfere with this biosynthesis. [0007] The invention provides novel nucleic acid molecules encoding proteins which are referred to herein as SES proteins and which may be involved, for example, in the repair or recombination of DNA, transposition of genetic material, expression of genes (i.e. in transcriptional or translational processes), protein folding or protein secretion in Corynebacterium glutamicum. Nucleic acid molecules encoding an SES protein are referred to herein as SES nucleic acid molecules. In a preferred embodiment, an SES protein is involved in improving or reducing the genetic stability in C. glutamicum, in the expression of genes (e.g. in transcription or translation) or in protein folding in this organism or in protein secretion from C. glutamicum. Examples of such proteins are those encoded by the genes listed in Table 1. [0008] Consequently, one aspect of the invention relates to isolated nucleic acid molecules (e.g. cDNAs) comprising a nucleotide sequence which encodes an SES protein or biologically active sections thereof and also nucleic acid fragments which are suitable as primers or hybridization probes for detecting or amplifying SES-encoding nucleic acid (e.g. DNA or mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule comprises any of the nucleotide sequences listed in Appendix A or the coding region or a complement thereof of any of these nucleotide sequences. In other preferred embodiments, the isolated nucleic acid molecule encodes any of the amino acid sequences listed in Appendix B. The preferred SES proteins of the invention likewise have preferably at least one of the SES activities described herein. [0009] Appendix A defines hereinbelow the nucleic acid sequences of the sequence listing together with the sequence modifications at the relevant position, described in Table 1. [0010] Appendix B defines hereinbelow the polypeptide sequences of the sequence listing together with the sequence modifications at the relevant position, described in Table 1. [0011] In a further embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule which comprises a nucleotide sequence of Appendix A. The isolated nucleic acid molecule preferably corresponds to a naturally occurring nucleic acid molecule. The isolated nucleic acid more preferably encodes a naturally occurring C. glutamicum SES protein or a biologically active section thereof. [0012] A further aspect of the invention relates to vectors, for example recombinant expression vectors, which contain the nucleic acid molecules of the invention and to host cells into which said vectors have been introduced. In one embodiment, an SES protein is prepared by using said host cell which is cultivated in a suitable medium. The SES protein may then be isolated from the medium or the host cell. [0013] A further aspect of the invention relates to a genetically modified microorganism into which an SES gene has been introduced or in which an HA gene has been modified. In one embodiment, the genome of said microorganism has been modified by introducing at least one inventive nucleic acid molecule which encodes the mutated SES sequence as transgene. In another embodiment, an endogenous SES gene in the genome of said microorganism has been modified, for example, functionally disrupted, by homologous recombination with a modified SES gene. In a preferred embodiment, the microorganism belongs to the genus Corynebacterium or Brevibacterium, with Corynebacterium glutamicum being particularly preferred. In a preferred embodiment, the microorganism is also used for preparing a compound of interest, such as an amino acid, lysine being particularly preferred. [0014] A further aspect of the invention relates to an isolated SES protein or a section thereof, for example a biologically active section. In a preferred embodiment, the isolated SES protein or its section may take part in the repair or recombination of DNA, transposition of genetic material, gene expression (i.e. transcriptional or translational processes), protein folding or protein secretion in Corynebacterium glutamicum. In another preferred embodiment, the isolated SES protein or a section thereof is sufficiently homologous to an amino acid sequence of Appendix B for the protein or its section to retain the ability, for example, to take part in the repair or recombination of DNA, transposition of genetic material, gene expression (i.e. transcriptional or translational processes), protein folding or protein secretion in Corynebacterium glutamicum. [0015] Another preferred embodiment are host cells having more than one of the nucleic acid molecules described in Appendix A. Such host cells can be prepared in various ways known to the skilled worker. They may be transfected, for example, by vectors carrying several of the nucleic acid molecules of the invention. However, it is also possible to use a vector for introducing in each case one nucleic acid molecule of the invention into the host cell and therefore to use a plurality of vectors either simultaneously or sequentially. Thus it is possible to construct host cells which carry numerous, up to several hundred, nucleic acid sequences of the invention. Such an accumulation can often produce superadditive effects on the host cell with respect to fine-chemical productivity. [0016] Moreover, the invention provides an isolated SES protein preparation. In preferred embodiments, the SES protein comprises an amino acid sequence of Appendix B. In a further preferred embodiment, the invention relates to an isolated full-length protein which is essentially homologous to a complete amino acid sequence of Appendix B (which is encoded by an open reading frame in Appendix A). [0017] The SES polypeptide or a biologically active section thereof may be functionally linked to a non-SES polypeptide in order to produce a fusion protein. In preferred embodiments, this fusion protein has a different activity from that of the SES protein alone. In other preferred embodiments, said fusion protein takes part in the repair or recombination of DNA, transposition of genetic material, gene expression (i.e. transcriptional or translational processes), protein folding or protein secretion in Corynebacterium glutamicum. In particularly preferred embodiments, integration of said fusion protein into a host cell modulates the production of a compound of interest by the cell. [0018] A further aspect of the invention relates to a method for preparing a fine chemical. The method provides for the cultivation of a cell containing a vector which causes expression of an SES nucleic acid molecule of the invention so that a fine chemical is produced. In a preferred embodiment, this method moreover comprises the step of obtaining a cell containing such a vector, said cell being transfected with a vector which causes expression of an SES nucleic acid. In a further preferred embodiment, said method moreover comprises the step in which the fine chemical is obtained from the culture. In a particularly preferred embodiment, the cell belongs to the genus Corynebacterium or Brevibacterium. [0019] A further aspect of the invention relates to methods for modulating the production of a molecule from a microorganism. These methods comprise contacting the cell with a substance which modulates SES-protein activity or SES nucleic-acid expression such that a cell-associated activity is modified in comparison with the same activity in the absence of said substance. In a preferred embodiment, the cell is modulated with regard to one or more C. glutamicum processes which are involved in genetic stability, gene expression, protein folding or protein secretion, so as to improve the yield, production or efficiency of production of a fine chemical of interest by this microorganism. The substance which modulates SES protein activity may be a substance which stimulates SES-protein activity or SES nucleic-acid expression. Examples of substances stimulating SES protein activity or SES nucleic-acid expression include small molecules, active. SES proteins and nucleic acids which encode SES proteins and have been introduced into the cell. Examples of substances which inhibit SES activity or SES expression include small molecules and SES antisense nucleic acid molecules. [0020] A further aspect of the invention relates to methods for modulating the yields of a compound of interest from a cell, comprising introducing an SES wild-type gene or HA-mutant gene into a cell, which gene either remains on a separate plasmid or is integrated into the genome of the host cell. Integration into the genome may take place randomly or via homologous recombination so that the native gene is replaced by the introduced copy, leading to the production of the compound of interest from the cell to be modulated. In a preferred embodiment, said yields are increased. In a further preferred embodiment, the chemical is a fine chemical which, in a particularly preferred embodiment, is an amino acid. In a particularly preferred embodiment, this amino acid is L-lysine. DETAILED DESCRIPTION OF THE INVENTION [0021] The present invention provides SES nucleic acid and SES-protein molecules which are involved in the repair or recombination of DNA, transposition of genetic material, gene expression (i.e. transcriptional or translational processes), protein folding or protein secretion in Corynebacterium glutamicum. The molecules of the invention can be used for modulating the production of fine chemicals from microorganisms such as C. glutamicum either directly (for example, if overexpression or optimization of the activity of a protein involved in the secretion of a fine chemical (e.g. an enzyme) has a direct effect on the yield, production and/or efficiency of production of a fine chemical from the modified C. glutamicum cells) or via an indirect effect which nevertheless causes an increase in the yield, production and/or efficiency of the compound of interest (for example, if modulating the activity or copy number of a C. glutamicum DNA-repair protein to changes in the ability of the microorganism to maintain the introduced mutation, and this in turn may influence the production of one or more fine chemicals from said strain). The aspects of the invention are further illustrated below. Continue reading... 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