Extracellular polyhydroxyalkanoates produced by genetically engineered microorganisms

Abstract

The present invention is in the field of biosynthesis of polyhydroxyalkanoates (PHA). The invention relates to a genetically engineered microorganism having at least one gene involved in the metabolism, preferably in the production, of polyhydroxyalkanoates (PHA). This microorganism is useful in commercial production of polyhydroxyalkanoates. The present invention further relates to a method for the production of polyhydroxyalkanoates (PHA).

1 . A genetically engineered microorganism having at least one modification in at least one gene encoding a protein involved in the metabolism of polyhydroxyalkanoate(s) (PHA), or in at least one gene encoding a protein, which interferes in the metabolism of the microorganism with the production of PHA, wherein the at least one modification causes deposition and/or overproduction of medium or long chain polyhydroxyalkanoate(s) PHA. 
     
     
         2 . The genetically engineered microorganism of  claim 1 , wherein the at least one gene encodes a protein, which interferes in the metabolism of the microorganism with the production of PHA, wherein the protein is an enzyme, which competes with enzymes of the PHA synthesis pathway for intermediates of the PHA synthesis pathway. 
     
     
         3 . The genetically engineered microorganism of  claim 2 , wherein the enzyme is a thioesterase. 
     
     
         4 . The genetically engineered microorganism of  claim 2 , wherein the thioesterase acts on (R)-3-OH-acyl-CoA as a substrate. 
     
     
         5 . The microorganism of  claim 2 , wherein the thioesterase is acyl-CoA thioesterase tesB-like. 
     
     
         6 . The genetically engineered microorganism of  claim 2 , wherein the enzyme is encoded by a nucleic acid sequence comprising a (modified) nucleic acid sequence as shown in any of  FIGS. 11 and 12 , or a homolog thereof. 
     
     
         7 . The genetically engineered microorganism of  claim 2 , wherein the enzyme is encoded by a nucleic acid sequence comprising a (native) nucleic acid sequence as shown in any of  FIGS. 14 ,  16  and  18  to  25 , or a homolog thereof, wherein the native nucleic acid sequence is modified by the at least one modification. 
     
     
         8 . The genetically engineered microorganism of  claim 1 , wherein the at least one modification is causes a complete or partial inactivation of the modified gene. 
     
     
         9 . The genetically engineered microorganism of  claim 1 , wherein the at least one modification is effected by a transposon insertion selected from the group consisting of Tn5 and Tn10 transposons, preferably by a Tn5 insertion, more preferably on the gene downstream to the modified gene. 
     
     
         10 . The genetically engineered microorganism of  claim 1 , wherein the at least one modification is effected by transposon mutagenesis, preferably based on miniTn5 Km element, more preferably based on miniTn5 Str/Sp element. 
     
     
         11 . The genetically engineered microorganism of  claim 1 , wherein the gene is integrated into the chromosome of the microorganism. 
     
     
         12 . The genetically engineered microorganism of  claim 1 , wherein the at least one gene encodes a protein selected from the group consisting of PHA synthase, PHB synthase, acyl-CoA transferase, enyol-CoA hydratase or reductase. 
     
     
         13 . The genetically engineered microorganism of  claim 1 , wherein the at least one modification causes an extracellular deposition or an overproduction of polyhydroxyalkanoate(s) PHA, preferably medium or long chain polyhydroxyalkanoates (PHA), produced by the microorganism. 
     
     
         14 . The genetically engineered microorganism of  claim 13 , wherein the extracellular deposition or overproduction of polyhydroxyalkanoate(s) PHA, results in a PHA production which is at least 5 times, preferably at least 10 times, more preferably at least 15 times, more preferably at least 25 times, even more preferably at least 40 times, even more preferably at least 50 times, most preferably at least 60 times, most preferably at least 80 times, even most preferably at least 100 times higher than the PHA production of the corresponding wild type microorganism. 
     
     
         15 . The genetically engineered microorganism of  claim 1 , wherein the microorganism is selected from the group consisting of  Alcanivorax borkumensis, Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas fluorescens, Pseudomonas acitophila, Pseudomonas oleovarans, Idiomarina loihiensis, Acinetobacter sp., Caulobacter crescentus, Alcaligenes eutrophus, Alcaligenes latus, Azotobacter vinlandii, Rhodococcus eutropha, Chromobacterium violaceum , and  Chromatium vinosum.    
     
     
         16 . The genetically engineered microorganism of  claim 1 , wherein the microorganism is selected from the group consisting of  Alcanivorax , preferably  Alcanivorax borkumensis , more preferably  Alcanivorax borkumensis  SK2. 
     
     
         17 . A nucleic acid sequence which constitutes at least one gene encoding a protein involved in the metabolism of polyhydroxyalkanoate(s) (PHA), or in at least one gene encoding a protein, which interferes in the metabolism of the microorganism with the production of PHA, wherein the at least one modification causes deposition and/or overproduction of medium or long chain polyhydroxyalkanoate(s) PHA. 
     
     
         18 . The nucleic acid sequence of  claim 17 , wherein the gene is selected from the group consisting of PHA synthase, PHB synthase, acyl-CoA transferase, and enyol-CoA. 
     
     
         19 . The nucleic acid sequence of  claim 17  wherein the gene is acyl-CoA thioesterase tesB-like, preferably acyl-CoA thioesterase tesB-like of  Alcanivorax borkumensis, Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas fluorescens, Pseudomonas acitophila, Pseudomonas oleovarans, Idiomarina loihiensis, Acinetobacter sp., Caulobacter crescentus, Alcaligenes eutrophus, Alcaligenes latus, Azotobacter vinlandii, Rhodococcus eutropha, Chromobacterium violaceum , or  Chromatium vinosum.    
     
     
         20 . The nucleic acid sequence of  claim 17  wherein the gene is acyl-CoA thioesterase tesB-like, preferably acyl-CoA thioesterase tesB-like of  Alcanivorax borkumensis , more preferably acyl-CoA thioesterase tesB-like of  Alcanivorax borkumensis  SK2. 
     
     
         21 . The nucleic acid sequence of  claim 17 , wherein the at least one modification is effected by a transposon insertion, preferably Tn-5 insertion, on the gene downstream to the modified gene. 
     
     
         22 . The nucleic acid sequence of  claim 17 , wherein the at least one modification is achieved by transposon mutagenesis, preferably based on miniTn5 Km element, more preferably based on miniTn5 Str/Sp element. 
     
     
         23 . The nucleic acid sequence of  claim 17 , wherein the nucleic acid comprises a (modified) nucleic acid sequence as shown in any of  FIGS. 11 and 12 , or a homolog thereof. 
     
     
         24 . The nucleic acid sequence of  claim 17 , wherein the nucleic acid comprises a (native) nucleic acid sequence as shown in any of  FIGS. 14 ,  16  and  18  to  25 , or a homolog thereof, wherein the native nucleic acid sequence is modified by the at least one modification. 
     
     
         25 . Polypeptide encoded by a nucleic acid sequence of  claim 17 . 
     
     
         26 . Polypeptide of  claim 25 , wherein the polypeptide comprises one of the amino acid sequences shown in  FIG. 11  or  12 . 
     
     
         27 . A vector comprising the nucleic acid sequence of  claim 17 . 
     
     
         28 . A vector comprising a PHA producing gene cluster comprising a gene having at least one modification in at least one gene encoding for a protein involved in the metabolism of polyhydroxyalkanoate(s) (PHA), or in at least one gene encoding for a protein, which interferes in the metabolism of the genetically engineered microorganism of  claim 1  with the production of PHA, wherein the at least one modification causes extracellular deposition of medium or long chain polyhydroxyalkanoate(s) (PHA). 
     
     
         29 . A cell comprising the vector of  claim 27  and/or the nucleic acid sequence of  claim 17 . 
     
     
         30 . A method for producing polyhydroxyalkanoates (PHA) comprising the following steps:
 a. cultivating a microorganism of  claim 1  or a cell of  claim 29  and   b. recovering polyhydroxyalkanoates (PHA) from the culture medium.   
     
     
         31 . (canceled) 
     
     
         32 . (canceled)