ppGpp synthetase and expression systems for improved production of protein of interest

Abstract

The present invention relates to a novel ppGpp synthetase and expression systems for improved production of proteins of interest in Gram-positive bacteria, involving use of the novel ppGpp synthetase.

What is claimed is:  
     
         1 . An isolated polynucleotide molecule selected from the group consisting of: 
 (a) polynucleotide molecules encoding a polypeptide having ppGpp synthetase activity and comprising a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 21 to nucleotide 2225;    (b) species homologs of (a);    (c) polynucleotide molecules that encode a polypeptide having ppGpp synthetase activity that is at least 70% identical to the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to amino acid residue 734;    (d) molecules complementary to (a), (b) or (c); and    (e) degenerate nucleotide sequences of (a), (b), (c) or (d).    
     
     
         2 . The isolated polynucleotide molecule of  claim 1 , wherein the polynucleotide molecule encodes a polypeptide having ppGpp synthetase activity and comprises a sequence of nucleotides as shown in SEQ ID NO:3 from nucleotide 21 to nucleotide 2225.  
     
     
         3 . An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of: 
 (a) polynucleotide molecules encoding a polypeptide having ppGpp synthetase activity and comprising a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 21 to nucleotide 2225;    (b) species homologs of (a);    (c) polynucleotide molecules that encode a polypeptide having ppGpp synthetase activity that is at least 70% identical to the amino acid sequence of SEQ ID NO:2 from amino acid residue 1 to amino acid residue 734; and    (d) degenerate nucleotide sequences of (a), (b), or (c); and a transcription terminator.    
     
     
         4 . A cultured cell into which has been introduced an expression vector of  claim 3 , wherein the cell expresses the polypeptide encoded by the DNA segment.  
     
     
         5 . An isolated polypeptide having ppGpp synthetase activity selected from the group consisting of: 
 (a) polypeptide molecules comprising an amino acid sequence as shown in SEQ ID NO:2 from residue 1 to residue 734; and    (b) polypeptide molecules that are at least 70% identical to the amino acids of SEQ ID NO:2 from amino acid residue 1 to amino acid residue 734.    
     
     
         6 . A composition comprising a purified polypeptide of  claim 5 .  
     
     
         7 . A method of producing a polypeptide having ppGpp synthetase activity comprising: 
 (a) culturing a cell into which has been introduced an expression vector according to  claim 3 , whereby the cell expresses a polypeptide encoded by the DNA segment; and    (b) recovering the polypeptide.    
     
     
         8 . An expression system for improved production of at least one protein of interest in gram-positive bacteria, which expression system comprises a gram-positive bacterium expressing smaller than wild-type amounts of a polypeptide of  claim 5 .  
     
     
         9 . The expression system of  claim 8 , wherein said species of Bacillus is a  Bacillus subtilis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus thuringiensis, Bacillus clausii,  or  Bacillus licheniformis.    
     
     
         10 . The expression system of  claim 8 , wherein the protein of interest is recombinantly produced by use of an expression vector.  
     
     
         11 . The expression system of  claim 8 , wherein the protein of interest is a homologous protein.  
     
     
         12 . The expression system of  claim 8 , wherein the protein of interest is a heterologous protein.  
     
     
         13 . The expression system of  claim 10 , wherein the protein is an exoprotein and is expressed by use of an expression vector.  
     
     
         14 . The expression system of  claim 8 , wherein the protein of interest is an amylase, bacterial protein toxin, cellulase, CGTase (cyclodextrin gluconotransferase), galactosidase, glucose isomerase, glucose oxidase, glucosyl transferase, laccase, lipase, microbial surface protein, pharmaceutical, phytase, protease, protein disulphide isomerase, pullanase, viral protein, or xylanase.  
     
     
         15 . The expression system of  claim 8 , wherein the gram-positive bacteria is a strain of Bacillus.  
     
     
         16 . The expression system of  claim 15 , wherein the strain is a  Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis,  or  Bacillus thuringiensis  strain.  
     
     
         17 . A method for improved production of at least one protein of interest in a gram-positive bacterium, which method comprises: 
 (a) culturing an expression system of claim  8 ; and    (b) purifying the protein of interest from the resulting culture broth or expression system.    
     
     
         18 . The method of  claim 17 , wherein the culturing is done as a fed-batch fermentation.