Microorganism and Method for the Fermentative Production of an Organic-Chemical Compound

Abstract

The invention relates to a microorganism which produces and/or secretes an organic-chemical compound, wherein the microorganism has increased expression, compared to the particular starting strain, of one or more protein subunits of the ABC transporter having the activity of a trehalose importer, said microorganism being capable of taking up trehalose from the medium; and to a method for the production of an organic-chemical compound, using the microorganism according to the invention, wherein accumulation of trehalose in the fermentation broth is reduced or avoided.

What is claimed is: 
     
         1 . An isolated  Corynebacterium glutamicum  bacterium that has been modified from a starting strain and which produces an L-amino acid during fermentation, wherein:
 a) compared to the starting strain, the bacterium comprises increased expression of a polynucleotide coding for a polypeptide with an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:8 or 20, wherein:
 i) said polypeptide comprises a subunit of a protein complex having the activity of a trehalose importer; 
 ii) said polypeptide has permease activity; 
   b) said bacterium is capable of taking up trehalose from the medium.   
     
     
         2 . The  Corynebacterium glutamicum  bacterium of  claim 1 , wherein, compared to the starting strain, said bacterium further comprises increased expression of at least one additional polynucleotide selected from the group consisting of a), b), c), d) and e), wherein said additional polynucleotides are as follows:
 a) a polynucleotide coding for a polypeptide with an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:2 or 14;   b) a polynucleotide coding for a polypeptide with an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:4 or 16;   c) a polynucleotide coding for a polypeptide with an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:6 or 18;   d) a polynucleotide coding for a polypeptide with an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:10 or 22; and   e) a polynucleotide coding for a polypeptide with an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:12 or 24.   
     
     
         3 . The  Corynebacterium glutamicum  bacterium of  claim 2 , wherein, compared to the starting strain, said bacterium further comprises increased expression of at least one additional polynucleotide selected from the group consisting of polynucleotides a), b) and d). 
     
     
         4 . The  Corynebacterium glutamicum  bacterium of  claim 2 , wherein, compared to the starting strain, said bacterium further comprises increased expression of at least one additional polynucleotide selected from the group consisting of polynucleotides b) and d). 
     
     
         5 . The  Corynebacterium glutamicum  bacterium of  claim 2 , wherein, compared to the starting strain, said bacterium further comprises increased expression of additional polynucleotide d). 
     
     
         6 . The  Corynebacterium glutamicum  bacterium of  claim 2 , wherein, compared to the starting strain, said bacterium further comprises, increased expression of all of the the additional polynucleotides a), b) and d). 
     
     
         7 . The  Corynebacterium glutamicum  bacterium of  claim 2 , wherein, compared to the starting strain, said bacterium further comprises increased expression of all of the additional polynucleotides a), b), c), d) and e). 
     
     
         8 . The  Corynebacterium glutamicum  bacterium of  claim 1 , wherein the L-amino acid is selected from the group consisting of: a proteinogenic L-amino acid; L-ornithine; and L-homoserine. 
     
     
         9 . The  Corynebacterium glutamicum  bacterium of  claim 1 , wherein the L-amino acid is selected from the group consisting of: L-methionine; L-valine; L-proline; L-glutamate; and L-isoleucine. 
     
     
         10 . The  Corynebacterium glutamicum  bacterium of  claim 1 , wherein the L-amino acid is L-lysine. 
     
     
         11 . The  Corynebacterium glutamicum  bacterium of  claim 1 , wherein expression of said polynucleotide coding for an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:8 or 20 is increased by one or more measures selected from the following group consisting of:
 a) using a promoter to express the sequence at least 70% identical to the amino acid sequence of SEQ ID NO:8 or 20, wherein said promoter stronger in said bacterium than in said starting strain;   b) increasing the copy number of said polynucleotide by inserting the polynucleotide into a plasmid with increased copy number in said bacterium and/or by integrating at least one copy of said polynucleotide into the chromosome of said bacterium;   c) expressing the sequence coding for said polypeptide with a ribosome binding site which is stronger in the bacterium than in the starting strain;   d) optimizing the codon usage of said polynucleotide in said bacterium compared to the starting strain;   e) using a sequence that results in a reduction of secondary structures in the mRNA transcribed;   f) using a sequence that results in an elimination of RNA polymerase terminator sequences in transcribed mRNA;   g) using mRNA-stabilizing sequences in the mRNA transcribed from said polynucleotide.   
     
     
         12 . A method for the fermentative production of an L-amino acid, comprising the steps:
 a) culturing the bacterium of  claim 1  in a medium to produce a fermentation broth; and   b) accumulating the L-amino acid in the fermentation broth of a).   
     
     
         13 . The method of  claim 12 , wherein the accumulation of trehalose in the fermentation broth is reduced. 
     
     
         14 . The method of  claim 12 , wherein, compared to said starting strain, the bacterium used for culturing comprises increased expression of at least one additional polynucleotide selected from the group consisting of a), b), c), d) and e), wherein said additional polynucleotides are as follows:
 a) a polynucleotide coding for a polypeptide with an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:2 or 14;   b) a polynucleotide coding for a polypeptide with an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:4 or 16;   c) a polynucleotide coding for a polypeptide with an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:6 or 18;   d) a polynucleotide coding for a polypeptide with an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:10 or 22; and   e) a polynucleotide coding for a polypeptide with an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:12 or 24.   
     
     
         15 . The method of  claim 14 , wherein, compared to the starting strain, said bacterium further comprises increased expression of at least one additional polynucleotide selected from the group consisting of polynucleotides a), b) and d). 
     
     
         16 . The method of  claim 14 , wherein, compared to the starting strain, said bacterium further comprises increased expression of at least one additional polynucleotide selected from the group consisting of of polynucleotides b) and d). 
     
     
         17 . The method of  claim 14 , wherein, compared to the starting strain, said bacterium further comprises increased expression of additional polynucleotide d). 
     
     
         18 . The method of  claim 14 , wherein, compared to the starting strain, said bacterium further comprises, increased expression of all of the the additional polynucleotides a), b) and d). 
     
     
         19 . The method of  claim 14 , wherein, compared to the starting strain, said bacterium further comprises, increased expression of all of the the additional polynucleotides a), b), d) and e). 
     
     
         20 . The method of  claim 14 , wherein, compared to the starting strain, said bacterium further comprises, increased expression of all of the additional polynucleotides a), b), c), d) and e).