Transgenic microbial polyhydroxyalkanoate producers

Abstract

Transgenic microbial strains are provided which contain the genes required for PHA formation integrated on the chromosome. The strains are advantageous in PHA production processes, because (1) no plasmids need to be maintained, generally obviating the required use of antibiotics or other stabilizing pressures, and (2) no plasmid loss occurs, thereby stabilizing the number of gene copies per cell throughout the fermentation process, resulting in homogeneous PHA product formation throughout the production process. Genes are integrated using standard techniques, preferably transposon mutagenesis. In a preferred embodiment wherein mutiple genes are incorporated, these are incorporated as an operon. Sequences are used to stabilize mRNA, to induce expression as a function of culture conditions (such as phosphate concentration), temperature, and stress, and to aid in selection, through the incorporation of selection markers such as markers conferring antibiotic resistance.

1 . A genetically engineered microorganism having at least one gene involved in synthesis of polyhydroxyalkanoates selected from the group consisting of thiolase, reductase, PHB synthase, PHA synthase, acyl-CoA transferase, enoyl-CoA hydratase, integrated into the chromosome, which produce polyhydroxyalkanoate.  
     
     
         2 . The microorganism of  claim 1  selected from the group consisting of  E. coli, Alcaligenes latus, Alcaligenese eutrophus, Azotobacter, Pseudomonas putida,  and  Ralstonia eutropha.    
     
     
         3 . The microorganism of  claim 1  wherein the gene is inserted using transposan mutagenesis.  
     
     
         4 . The microorganism of  claim 1  comprising multiple genes involved in synthesis of polyhydroxyalkanoate wherein the genes are integrated operably linked as an operon.  
     
     
         5 . The microorganism of  claim 1  wherein the gene is integrated operably linked under the control of a promoter.  
     
     
         6 . The microorganism of  claim 1  wherein the gene is integrated operably linked with upstream activating sequences  
     
     
         7 . The microorganism of  claim 1  wherein the gene is integrated operably linked with mRNA stabilizing sequences.  
     
     
         8 . The microorganism of  claim 5  wherein the gene is operably linked with promoter including a consensus  E. coli  pho box and −35 promoter region that is regulated by the phosphate concentration in the medium.  
     
     
         9 . The microorganism of  claim 5  wherein the promoter is selected from the group consisting of promoter that induces expression under general stress conditions such as nutrient limitation, pH or heat shock, and administration of toxic chemicals.  
     
     
         10 . The microorganism of  claim 1  wherein the gene is integrated operably linked with a selection marker.  
     
     
         11 . The microorganism of  claim 1  wherein the gene is isolated or derived from a microorganism selected from the group consisting of  A. eutrophus, Aeromonas caviae, Zoogloea ramigera, Nocardia, Rhodococcus, Pseudomonas  Sp. 61-3,  Pseudomonas acidophila, Pseudomonas oleovarans, Chromobacterium violaceum,  and  Alcaligenes latus.    
     
     
         12 . The microorganism of  claim 1  wherein the gene is selected from the group consisting of PHB polymerase from  R. eutropha  (C1), PHA polymerase from  P. oleovorans  (C3), PHB polymerase from  A. caviae  (C12), ACP::CoA transacylase from  P. putida  (G3), (R)-specific enouyl-CoA hydratase from  A. caviae  (J12), a broad substrate specific 3-ketoacyl-CoA thiolase from  R. eutropha  (A1-II), and phasins from  R. eutropha  (P1-I and P1-II).  
     
     
         13 . The microorganism of  claim 1  wherein the gene is integrated as a single copy on the chromosome of the microorganism.  
     
     
         14 . A method for screening for a gene involved in synthesis of polyhydroxyalkanoates that enhances production comprising 
 mutating a genetically engineered microorganism having at least one gene involved in synthesis of polyhydroxyalkanoates selected from the group consisting of thiolase, reductase, PHB synthase, PHA synthase, acyl-CoA transferase, enoyl-CoA hydratase, integrated into the chromosome, which produces polyhydroxyalkanoate, and    screening for enhanced production of polyhydroxyalkanoates.    
     
     
         15 . The method of  claim 14  wherein the gene is isolated or derived from a microorganism selected from the group consisting of  A. eutrophus, Aeromonas caviae, Zoogloea ramigera, Nocardia, Rhodococcus, Pseudomonas  Sp. 61-3,  Pseudomonas acidophila, Pseudomonas oleovarans, Chromobacterium violaceum,  and  Alcaligenes latus.    
     
     
         16 . The method of  claim 14  wherein the microorganisms are missing one or more genes required for production of polyhydroxyalkanoates.  
     
     
         17 . The method of  claim 14  wherein the microorganims produce polyhydroxyalkanoates, further comprising selecting genes which result in increased polyhydroxyalkanoate production.  
     
     
         18 . A method for producing polyhydroxyalkanoates comprising culturing genetically engineered microorganisms having at least one gene involved in synthesis of polyhydroxyalkanoates selected from the group consisting of thiolase, reductase, PHB synthase, PHA synthase, acyl-CoA transferase, enoyl-CoA hydratase, integrated into the chromosome, with appropriate substrate under conditions wherein the microorganisms produce polyhydroxyalkanoate.  
     
     
         19 . The method of  claim 18  wherein the microorganisms comprise multiple genes involved in synthesis of polyhydroxyalkanoate wherein the genes are integrated operably linked as an operon.  
     
     
         20 . The method of  claim 18  wherein the microorganisms comprise genes selected from the group consisting of genes integrated operably linked under the control of a promoter, genes integrated operably linked with upstream activating sequences, genes integrated operably linked with mRNA stabilizing sequences, genes operably linked with a promoter including a consensus  E. coli  pho box and −35 promoter region that is regulated by the phosphate concentration in the medium, and genes integrated operably linked with a selection marker.