DNA sequence useful for the production of polyhydroxyalkanoates

Abstract

A genomic fragment harboring the gene phaG was cloned by phenotype complementation of Pseudomonas putida KT2440 mutants, defective in the polyhydroxyalkanoic acid (PHA) synthesis via de novo fatty acid biosynthesis but not affected in PHA biosynthesis via fatty acid beta-oxidation. The 885-bp phaG gene encodes a protein of 295 amino acids with a molecular weight of 33.876 Da. The transcriptional induction of phaG could be observed under conditions of PHA synthesis via de novo fatty acid biosynthesis whereas no induction was detected under conditions which favor PHA synthesis via fatty acid degradation by beta-oxidation. The phaG gene is useful for the production of PHAs in bacteria and plants.

What is claimed is:  
     
       1. An isolated DNA molecule, comprising a nucleotide sequence selected from the group consisting of: 
       (a) the nucleotide sequence of the coding strand shown in SEQ ID NO:1, positions 911 through 1795, or the complement thereof;  
       (b) a nucleotide sequence that hybridizes to said nucleotide sequence of (a) under a wash stringency equivalent to 0.5X SSC to 2X SSC, 0.1% SDS, at 55-65° C., and which encodes a PHA synthase  CoA- ACP acyltransferase  protein;  
       (d) a Pseudomonas putida nucleotide sequence having at least 40% identity with the nucleotide sequence of (a) and which encodes a PHA synthase  CoA- ACP acyltransferase  protein;  
       (d) a nucleotide sequence encoding the same amino acid sequence as said nucleotide sequence of (a); and  
       (e) a nucleotide sequence encoding the same amino acid sequence as said nucleotide sequence of (b).  
     
     
       2. The DNA molecule of  claim 1  having the DNA sequence of SEQ ID NO: 1, positions 911 through 1795. 
     
     
       3. A recombinant DNA comprising a DNA molecule of  claim 1  and a promoter region, operatively linked such that the promoter enhances transcription of said DNA molecule in a host cell. 
     
     
       4. The recombinant DNA of  claim 3  wherein said DNA molecule has the DNA sequence of SEQ ID NO:1, positions 911 through 1795. 
     
     
       5. A recombinant vector comprising a DNA of any one of claims  1 - 4 . 
     
     
       6. A recombinant host cell comprising a DNA of claims  3  or  4 , wherein said DNA molecule is heterologous to said promoter region. 
     
     
       7. The recombinant cell of  claim 6  which is a microbe or plant cell. 
     
     
       8. The recombinant cell of  claim 6  which is a Pseudomonas fluorescens rRNA homology group I bacterium. 
     
     
       9. A method of transforming a host cell capable of producing polyhydroxyalkanoates comprising: 
       providing a host cell having a functional PHA synthase gene; preparing a recombinant vector comprising a DNA molecule of  claim 1  and a promoter region, operatively linked such that the promoter causes transcription of said DNA molecule in said host cell, and wherein said DNA molecule is heterologous to said promoter region;  
       and transforming the host cell with said vector.  
     
     
       10. The method of  claim 9  wherein the recombinant vector comprises a DNA molecule having the nucleotide sequence of SEQ ID NO:1, positions 911 through 1795. 
     
     
       11. The method of  claim 9  wherein the host cell is a microbe or a plant cell. 
     
     
       12. The method of  claim 9  wherein the host cell is selected from bacteria of the Pseudomonas fluorescens rRNA homology group I. 
     
     
       13. A method for preparing polyhydroxyalkanoates composed of repeating units having 3-14 carbon atoms (PHA) comprising: 
       culturing transformed cells produced according to any one of claims  9 - 12  to allow expression of the pha genes; and recovering the PHA from the cells.  
     
     
       14. The method of  claim 13  wherein the transformed cells are cultured in a media containing a simple carbohydrate substrate. 
     
     
       15. The method of  claim 14  wherein the media comprises gluconate, glucose, sucrose, fructose or lactose. 
     
     
       16. The method of  claim 13  wherein the transformed cells are cultured in a media comprising corn syrup or molasses. 
     
     
       17. A substantially purified protein having the amino acid sequence of SEQ ID NO:2. 
     
     
       18. A method of producing a genetically transformed plant which expresses PHaG comprising the steps of: 
       inserting into the genome of a host plant cell a recombinant, double-stranded DNA molecule comprising:  
       (i) a promoter which functions in plant cells to enhance transcription of an adjacent DNA coding sequence;  
       (ii) a DNA molecule of  claim 1  operatively linked to the promoter; and  
       regenerating a genetically transformed plant from said host plant cell.  
     
     
       19. The method of  claim 18  wherein the DNA molecule is SEQ ID NO:1, positions 911 through 1795. 
     
     
       20. The method of  claim 18  wherein the genome of the host plant cell also includes a functional PHA synthase gene. 
     
     
       21. The method of  claim 18  further comprising harvesting the transformed plant or plant parts. 
     
     
       22. The method of  claim 21  wherein the plant parts comprise leaves, roots, seeds or tubers. 
     
     
       23. The method of  claim 18  wherein the plant is maize, potato, sugar beet, tobacco, wheat, or Arabidonsis; or a high oil seed selected from the group consisting of soybean, canola, rape seed, sunflower, flax and peanut. 
     
     
       24. A plant produced by the method of any one of claims  18 - 20  or  23 . 
     
     
       25. A mutant Pseudomonas putida microorganism unable to synthesize PHA from gluconate but able to synthesize PHA from octanoic acid. 
     
     
       26. The mutant of  claim 25  wherein the phenotypic ability to synthesize PHA from gluconate is restored by complementation with a DNA sequence of SEQ ID NO: 1.