Production of polyhydroxyalkanoates with a defined composition from an unrelated carbon source

Abstract

Cells and methods for producing polyhydroxyalkanoates. The cells comprise one or more recombinant genes selected from an R-specific enoyl-CoA hydratase gene, a PHA polymerase gene, a thioesterase gene, and an acyl-CoA-synthetase gene. The cells further have one or more genes functionally deleted. The functionally deleted genes include such genes as an enoyl-CoA hydratase gene, a 3-hydroxyacyl-CoA dehydrogenase, and a 3-ketoacyl-CoA thiolase gene. The recombinant cells are capable of using producing polyhydroxyalkanoates with a high proportion of monomers having the same carbon length from non-lipid substrates, such as carbohydrates.

What is claimed is: 
     
         1 . A recombinant cell for producing polyhydroxyalkanoate comprising one or more recombinant genes selected from the group consisting of an R-specific enoyl-CoA hydratase gene, a PHA polymerase gene, a thioesterase gene, and an acyl-CoA-synthetase gene, wherein a gene product from a gene selected from the group consisting of an enoyl-CoA hydratase gene, a 3-hydroxyacyl-CoA dehydrogenase, and a 3-ketoacyl-CoA thiolase gene is functionally deleted, and wherein the recombinant cell is capable of producing polyhydroxyalkanoate. 
     
     
         2 . The recombinant cell of  claim 1  wherein the recombinant cell is a microbial cell. 
     
     
         3 . The recombinant cell of  claim 1  wherein the recombinant cell is a bacterial cell. 
     
     
         4 . The recombinant cell of  claim 1  wherein the enoyl-CoA hydratase gene is selected from the group consisting of fadB and fadJ. 
     
     
         5 . The recombinant cell of  claim 1  wherein the 3-hydroxyacyl-CoA dehydrogenase gene is selected from the group consisting of fadB and fadJ. 
     
     
         6 . The recombinant cell of  claim 1  wherein the 3-ketoacyl-CoA thiolase gene is selected from the group consisting of fadA and fadI. 
     
     
         7 . The recombinant cell of  claim 1  wherein gene products of fadA and fadI; fadB and fadJ; or fadA, fadI, fadB and fadJ are functionally deleted. 
     
     
         8 . The recombinant cell of  claim 1  wherein a gene product of fadR is functionally deleted. 
     
     
         9 . The recombinant cell of  claim 1  wherein gene products of fadA and fadI; fad R, fadA, and fadI; fadB and fadJ; fad R, fadB, and fadJ; fadA, fadB, fadI, and fadJ; or fad R, fadA, fadB, fadI, and fadJ are functionally deleted. 
     
     
         10 . The recombinant cell of  claim 1  wherein the enoyl-CoA hydratase gene is a phaJ gene. 
     
     
         11 . The recombinant cell of  claim 1  wherein the PHA polymerase gene is a phaC gene. 
     
     
         12 . The recombinant cell of  claim 1  wherein the enoyl-CoA hydratase gene is phaJ3 and the PHA polymerase gene is phaC2. 
     
     
         13 . The recombinant cell of  claim 1  wherein the thioesterase gene is  Umbellularia californica  thioesterase or a homolog thereof. 
     
     
         14 . The recombinant cell of  claim 1  wherein the acyl-CoA-synthetase gene is PP — 0763 from  P. putida.    
     
     
         15 . The recombinant cell of  claim 1  further comprising a recombinant phasin gene. 
     
     
         16 . The recombinant cell of  claim 1  comprising the R-specific enoyl-CoA hydratase gene, the PHA polymerase gene, the thioesterase gene, and the acyl-CoA-synthetase gene, wherein the recombinant cell is capable of producing polyhydroxyalkanoate from carbohydrate in a medium devoid of a fatty acid source. 
     
     
         17 . The recombinant cell of  claim 16  wherein gene products of fadA and fadI; fad R, fadA, and fadI; fadB and fadJ; fad R, fadB, and fadJ; fadA, fadB, fadI, and fadJ; or fad R, fadA, fadB, fadI, and fadJ are functionally deleted. 
     
     
         18 . A method of producing polyhydroxyalkanoate comprising culturing a recombinant cell as recited in  claim 1 . 
     
     
         19 . The method of  claim 18  comprising culturing the recombinant cell in aerobic conditions. 
     
     
         20 . The method of  claim 18  comprising culturing the recombinant cell in a medium comprising a carbohydrate and substantially devoid of a fatty acid source. 
     
     
         21 . The method of  claim 18  wherein the culturing produces polyhydroxyalkanoate to at least about 7.5% cell dry weight. 
     
     
         22 . The method of  claim 18  wherein the culturing produces polyhydroxyalkanoate comprised of hydroxyalkanoate monomers, wherein greater than about 50% of the hydroxyalkanoate monomers comprise hydrocarbon chains comprising same number of carbons.