Thiamin production by fermentation

Abstract

The present invention provides a method 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 by such assays are provided.

1 . A microorganism selected from the group consisting of Bacillaceae, Lactobacillaceae, Streptococcaceae, Corynebacteriaceae and Brevibacteriaceae, the microorganism containing a mutation that deregulates thiamin production and causes thiamin products to be released into a culture media.  
     
     
         2 . The microorganism of  claim 1  wherein the mutation is selected from the group consisting of ΔthiL, txl, tx26 and combinations thereof.  
     
     
         3 . The microorganism of  claim 1  which is selected from the group consisting of  Bacillus, Lactobacillus, Lactococcus, Corynebacterium  and Brevibacterium.  
     
     
         4 . The microorganism of  claim 3  which is a  Bacillus subtilis  cell.  
     
     
         5 . The microorganism of  claim 4  which is  B. subtilis  TH95 (ATCC PTA-5221).  
     
     
         6 . The microorganism according to  claim 1  further comprising a DNA cassette containing a polynucleotide sequence being selected from the group consisting of: 
 (a) a polynucleotide sequence that encodes a thiA gene product, wherein one or more copies of said polynucleotide sequence are contained in said DNA cassette;    (b) a polynucleotide sequence that encodes gene products from a thiKC operon, wherein one or more copies of said polynucleotide sequence are contained in said DNA cassette; and    (c) a polynucleotide sequence that encodes gene products of a tenAl-thiOSGFD operon; which polynucleotide sequence is operatively controlled by a strong constitutive promoter.    
     
     
         7 . The microorganism according to  claim 1  further comprising (a) a DNA cassette containing a polynucleotide sequence that encodes gene products of a tenAlthiOSGFD 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.  
     
     
         8 . The microorganism according to  claim 6  which is selected from the group consisting of  B. subtilis  TH116 (ATCC PTA-5224), TH115 (ATCC PTA-5223), TH404 (DSM 16333) and TH405 (DSM 16334).  
     
     
         9 . The microorganism according to  claim 1  further comprising a first mutation that deregulates expression of a purine operon of  B. subtilis  and a second mutation that blocks conversion of 5-aminoimidazole ribotide (AIR) to carboxyaminoimidazole ribotide (CAIR).  
     
     
         10 . The microorganism according to  claim 9  wherein the first mutation comprises a mutation within the leader region of the pur operon and the second mutation comprises a mutation within the purE gene encoding phosphoribosylaminoimidazole carboxylase 1.  
     
     
         11 . The microorganism according to  claim 10  which is  B. subtilis  TH101 (ATCC PTA-5222).  
     
     
         12 . A process for producing thiamin products comprising: 
 (a) culturing, in a suitable medium, a microorganism according to  claim 1  that overproduces thiamin products into the medium; and    (b) recovering the thiamin products.    
     
     
         13 . The process according to  claim 12  further comprising culturing the microorganism in the presence of a component which is selected from the group consisting of: 
 (a) thiamin precursors,    (b) a thiamin precursor and a purine source,    (c) a precursor of a HET pathway,    (d) a precursor of a HMP pathway, and    (e) a derivative of HMP.    
     
     
         14 . The process according to  claim 13  wherein the thiamin precursors are selected from the group consisting of 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), 5-(2-hydroxyethyl)-4-methylthiazole (HET) and a combination thereof.  
     
     
         15 . The process according to  claim 13  wherein the thiamin precursor is HET and the purine source is xanthine.  
     
     
         16 . The process according to  claim 13  wherein the precursor of the HET pathway is selected from the group consisting of glycine, cysteine, isoleucine, threonine and combinations thereof.  
     
     
         17 . The process according to  claim 13  wherein the derivative of HMP is 4-amino-2-methyl-5-pyrimidinemethaneamine (Grewe Diamine).  
     
     
         18 . An isolated polynucleotide sequence comprising a txl mutation.  
     
     
         19 . The isolated polynucleotide sequence according to  claim 18  wherein the mutation results in a leucine to phenylalanine substitution at amino acid residue 116.  
     
     
         20 . The isolated polynucleotide sequence according to  claim 18  wherein the sequence is SEQ ID NO: 31 or a polynucleotide sequence that hybridizes to SEQ ID NO: 31 under stringent conditions and, when present in a microorganism, causes a deregulation of thiamin production.  
     
     
         21 . An isolated polynucleotide sequence comprising a first mutation with 70% linkage to ΔyufR::Tn917 (tx26-1) and a second mutation with 59% linkage to ΩmotA::Tn917 (tx26-2), wherein the presence of both of the mutations in a thiamin-producing microorganism causes a deregulation of thiamin production.  
     
     
         22 . The isolated polynucleotide sequence according to  claim 21  wherein the tx26-1 mutation is encoded by a polynucleotide sequence which is SEQ ID NO: 33 or a polynucleotide sequence that hybridizes to SEQ ID NO: 33 under stringent conditions and wherein the tx26-2 mutation is encoded by a polynucleotide sequence which is SEQ ID NO: 36 or a polynucleotide sequence that hybridizes to SEQ ID NO: 36 under stringent conditions and, when present in a microorganism, causes a deregulation of thiamin production.  
     
     
         23 . A DNA cassette comprising one or more polynucleotides according to  claim 18 .  
     
     
         24 . A microorganism containing the DNA cassette of  claim 23 .  
     
     
         25 . A method for detecting a pathogenic microorganism in a clinical sample from a patient comprising: 
 (a) determining whether a Gram +  microorganism is present in the sample,    (b) determining whether the microorganism contains a yloS ortholog, and    (c) determining whether the microorganism contains a thiL ortholog, wherein the presence of a yloS ortholog and the absence of a thiL ortholog in a Gram +  microorganism indicates that the microorganism is pathogenic.    
     
     
         26 . The method according to  claim 25  wherein the microorganism is selected from the group of group consisting of  Listeria, Staphylococcus, Clostridium, Enterococcus , and  Streptococcus.    
     
     
         27 . The method according to  claim 26  wherein the microorganism is selected from the group consisting of  Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Clostridium tetani, Clostridium perfringens, Enterococcus  sp.,  Streptococcus agalactiae, Streptococcus pyogenes , and  Streptococcus pneumoniae.    
     
     
         28 . An assay for identifying an antibiotic comprising: 
 (a) contacting an assay composition comprising a YloS protein with a test compound, and    (b) determining whether the test compound inhibits YloS protein activity, wherein the compound is identified as an antibiotic based on the compound's ability to inhibit the activity of the YloS protein activity.    
     
     
         29 . The assay according to  claim 28  wherein the YloS protein comprised in the assay composition is selected from the group consisting of a purified YloS protein, a partially purified YloS protein, and a crude cell extract from a cell producing YloS protein.  
     
     
         30 . The assay according to  claim 28  wherein the YloS protein is encoded by a polynucleotide derived from a pathogenic microorganism selected from the group consisting of  Listeria, Staphylococcus, Clostridium, Enterococcus , and  Streptococcus.    
     
     
         31 . The assay according to  claim 30  wherein the microorganism is selected from the group consisting of  Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Clostridium tetani, Clostridium perfringens, Enterococcus  sp.,  Streptococcus agalactiae, Streptococcus pyogenes , and  Streptococcus pneumoniae.