| Thiamin production by fermentation -> Monitor Keywords |
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Thiamin production by fermentationRelated 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 Heterocyclic Carbon Compound Having Only O, N, S, Se, Or Te As Ring Hetero Atoms, Nitrogen As Only Ring Hetero Atom, Containing Six-membered Hetero RingThiamin production by fermentation description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060127993, Thiamin production by fermentation. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The present invention relates to methods for producing thiamin products. More particularly, the present invention relates to methods for producing thiamin products using a microorganism containing a mutation that causes it to overproduce and release thiamin products into the medium. Biologically pure cultures of the microorganisms and isolated polynucleotides containing the mutations are also provided. In addition, methods for detecting a pathogenic microorganism in a clinical sample, assays for identifying an antibiotic, as well as, antibiotics identified using such assays are provided. [0002] Thiamin, also known as vitamin B1, is a member of the water-soluble B-complex of vitamins and is a nutritional requirement for mammals. The pyrophosphate form of thiamin acts in vivo as the coenzyme in many carbohydrate and amino acid metabolic pathways, like for example those catabolized by pyruvate dehydrogenase, pyruvate oxidase or transketolase. It is important to note that unlike other vitamin biosynthetic pathways (e.g. riboflavin and biotin), thiamin is not part of the de novo pathway, but is actually part of the salvage pathway. [0003] Most enzymatic steps and intermediates in thiamin biosynthesis have been studied in E. coli and to a lesser extent in Salmonella typhimurium and Rhizobium (for reviews, see Brown and Williamson (1987) pp. 528-532, In F. C. Neidhardt et al. (ed.) Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol. 1. American Society for Microbiology, Washington, D.C.; White and Spenser (1996) pp. 680-686, In P. C. Neidhardt et al. (ed.) Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol. 2. American Society for Microbiology, Washington, D.C.; Begley et al. (1999) Arch. Microbiol. 171: 293-300). The E. coli genes encoding the steps in the thiamin pathway are located at four distinct sites on the chromosome: a thiCEFSGH operon at 90''; a thiMD operon at 46'', individual thiJ and thiL genes are clustered in the 9.5'' vicinity and thiK at 25''. All of these genes have been cloned and sequenced and many of the enzymes encoded by these genes have been overproduced in E. coli and their enzymatic activities determined. [0004] The pyrimidine moiety, 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P), is derived from 5-aminoimidazole ribotide (AIR), an intermediate in the de novo purine biosynthetic pathway. In Gram-negative bacteria, conversion of AIR to HMP-P is catalyzed by the thiC gene product. HMP-P is then phosphorylated to HMP-PP by ThiD kinase prior to coupling with the thiazole unit. [0005] The thiazole moiety, 5-(2-hydroxyethyl)-4-methylthiazole phosphate (HET-P), is derived from L-tyrosine and 1-deoxy-D-xylulose phosphate (DXP); the sulfur atom is most likely derived from L-cysteine. This reaction requires expression of at least five genes thiF, thiS, thiG, thiH and thiI. [0006] Coupling of HMP-PP and HET-P is catalyzed by thiamin phosphate pyrophosphorylase encoded by thiE, resulting in thiamin monophosphate (TMP). TMP is then phosphorylated to form thiamin pyrophosphate (TPP) by the action of thiamin monophosphate kinase, encoded by thiL. Because thiamin is not part of the de novo pathway, E. coli requires a salvage enzyme, thiamin kinase, encoded by thiK to convert exogenous thiamin into TMP. [0007] Synthesis of thiamin in B. subtilis appears to utilize the same enzymes and intermediates as found in E. coli (see, e.g., Perkins and Pero (2001) pp. 271-286, In Sonenshein et al, (ed.) Bacillus subtilis and its relatives: from genes to cells, American Society for Microbiology, Washington, D.C.). However there are important differences. The traditional gene names are different in E. coli and B. subtilis. First, the HMP biosynthesis enzyme ThiC, thiamin-phosphate pyrophosphate ThiE, and hydroxyethylthiazole kinase ThiM from E. coli have their counterparts named ThiA, ThiC, and ThiK, respectively. Second, the known B. subtilis thiamin biosynthetic genes are organized differently, as three clusters: the thiA locus consisting of only the thiA gene, the thiB locus consisting of genes thiOSFGD1, and the thiC locus consisting of thiK and thiC genes. Third, at least one enzymatic step in thiazole biosynthesis is different. The B. subtilis genome does not contain a thiH ortholog. Instead thiO (yjbR in the thiB locus) is predicted to encode an oxidase activity involved in thiazole biosynthesis. This gene is not present in the E. coli genome, nor does it show amino acid homology to ThiH. It is homologous to one of the genes (thiO) associated with thi genes from Rhizobium etli. Fourth, two orthologs of E. coli thiD have been found in B. subtilis, yjbV (thiD1) and ywdB (thiD2), which could encode the biosynthetic and salvage HMP kinases. Finally, the thiC locus contains an unknown gene, ywbI that displays strong similarity to the lysR family of transcriptional regulators. [0008] The present invention provides a microorganism selected from the group consisting of Bacillaceae, Lactobacillaceae, Streptococcaceae, Corynebacteriaceae and Brevibacteriaceae, wherein the microorganism contains a mutation that deregulates thiamin production and causes thiamin products to be released from the cell. [0009] "Thiamin products" means thiamin, thiamin monophosphate (TMP) and/or thiamin pyrophosphate (TPP), either alone or in any combination. [0010] It is understood that a microorganism as used for the present invention means a "biologically pure culture" of said microorganism, i.e., a microorganism that is separated from constituents, cellular and otherwise, in which the microorganism is normally associated with in nature. [0011] The following materials have been deposited with the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108 USA on May 12, 2003, and with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg 1B, D-38124 Braunschweig, Germany on Apr. 5, 2004, respectively, with the corresponding accession numbers as indicated below, in accordance with the stipulations of the Budapest Treaty: Bacillus subtilis TH95 (ATCC PTA-5221), Bacillus subtilis TH101 (ATCC PTA-5222), Bacillus subtilis TH115 (ATCC PTA-5223), Bacillus subtilis TH116 (ATCC PTA-5224), Bacillus subtilis TH404 (DSM 16333), and Bacillus subtilis TH405 (DSM 16334). [0012] "Mutation" is used interchangeably herein with modification to mean a change in the wild-type DNA sequence of a microorganism, such as a bacterium, that conveys a phenotypic change to the microorganism compared to the wild type microorganism, e.g. that allows an increase or decrease of thiamin or a thiamin product either in the cell or out of the cell by any mechanism. The mutation may be caused in a variety of ways including one or more frame shifts, substitutions, insertions and/or deletions, including nonsense mutations (amber (UAG), ocher (T/UAA) and opal (T/UGA)). The deletion may be of a single nucleotide or more, including deletion of the entire gene. [0013] "Amino acid substitution" means a one-for-one amino acid replacement. Such substitutions are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements include substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Non-conservative substitutions within the scope of the present invention include replacement of amino acids having aliphatic side chains with those that have aromatic side chains, such as replacement of leucine with phenylalanine. [0014] Amino acid "insertions" or "deletions" mean changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. The variation allowed in a particular amino acid sequence may be experimentally determined by producing the peptide synthetically or by systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques. [0015] "Deregulates" or "deregulation" means an alteration or modification of the expression of a gene that encodes an enzyme/protein in a biosynthetic pathway, such that the level or activity of said enzyme/protein is altered or modified, which results in, but is not limited to, an increase in the production of a thiamin product or the release of thiamin products out of the cell, by e.g., secretion, efflux, and the like. Alterations or modifications of gene expression can occur by changes in the DNA sequence of the gene itself or regions outside of the gene, including non-protein encoding DNA regions. "Deregulates" or "deregulation" can also mean any perturbation of the intracellular levels of a metabolite that alters the expression of a biosynthetic gene of the cell, such that an increase in the production or the release of thiamin products occurs. [0016] In one embodiment, the mutation that deregulates thiamin production in a microorganism as defined above is selected from the group consisting of .DELTA.thiL, txl, tx26 and combinations thereof. Such a mutation includes .DELTA.thiL combined with txl, .DELTA.thiL combined with tx26, txl combined with tx26, and .DELTA.thiL combined with both txl and tx26. Preferred is a microorganism comprising all three mutations .DELTA.thiL, txl and tx26. [0017] In a preferred embodiment, the microorganism is selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Gorynebacterium, and Brevibacterium. More preferably, the microorganism is selected from the genus Bacillus, most preferably it is a B. subtilis cell. [0018] In one embodiment, the microorganism containing the mutation as defined above is B. subtilis TH95. [0019] In one embodiment, the present invention provides a microorganism as defined above containing a mutation which is selected from the group consisting of .DELTA.thiL, txl, tx26 and combinations thereof further comprising a DNA cassette containing at least one copy of a polynucleotide sequence that encodes a thiA gene product, which polynucleotide sequence is operatively controlled by a strong constitutive promoter. A preferred microorganism is B. subtilis TH116. [0020] In a further embodiment, the present invention provides a microorganism as defined above containing a mutation which is selected from the group consisting of .DELTA.thiL, txl, tx26 and combinations thereof further comprising a DNA cassette containing at least one copy of a polynucleotide sequence that encodes gene products from a thiKC operon, which polynucleotide sequence is operatively controlled by a strong constitutive promoter. A preferred microorganism is B. subtilis TH115. [0021] In a further embodiment, the present invention provides a microorganism as defined above containing a mutation which is selected from the group consisting of .DELTA.thiL, txl, tx26 and combinations thereof further comprising a DNA cassette containing a polynucleotide sequence that encodes gene products of a tenAl-thiOSGFD operon, which polynucleotide sequence is operatively controlled by a strong constitutive promoter. A preferred microorganism is B. subtilis TH404. [0022] In a further embodiment, the present invention provides a microorganism as defined above containing a mutation which is selected from the group consisting of .DELTA.thiL, txl, tx26 and combinations thereof further comprising (a) a DNA cassette containing a polynucleotide sequence that encodes gene products of a tenAl-thiOSGFD operon and (b) a DNA cassette containing at least one copy of a polynucleotide sequence that encodes a thiA gene product, which polynucleotide sequences are operatively controlled by a strong constitutive promoter. A preferred microorganism is B. subtilis TH405. [0023] In the present invention, "DNA cassette" means a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. DNA fragments can also be inserted into the vector DNA without restriction enzymes by the use of topoisomerase bound at the ends of linearized vector DNA; this is especially useful for direct cloning of PCR-prepared DNA fragments. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a "DNA construct". A common type of vector is a "plasmid", which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can be readily introduced into a suitable host cell. A plasmid often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. "Coding DNA" is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. The DNA cassette may, in addition to the specific nucleotide sequence, contain additional transcription control elements including enhancers and promoters for controlling transcription of the specific nucleotide sequence. "Promoter DNA" is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include those specifically described in the Examples, as well as, pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), pCR2.1Topo (Invitrogen, San Diego, Calif.), pXLTopo (Invitrogen, San Diego, Calif.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes. [0024] The DNA cassette may contain one or more copies of the coding DNA such as for example from 1-50 copies of the sequence, preferably from 1-25 copies, such as from 1-5,1-10, 1-15 and 1-20 copies. The sequences may be arranged in any order, including for example, tandemly, i.e., in a head-to-tail arrangement. Continue reading about Thiamin production by fermentation... Full patent description for Thiamin production by fermentation Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Thiamin production by fermentation patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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