PRODUCTION OF POLYHYDROXYALKANOATES WITH A DEFINED COMPOSITION FROM AN UNRELATED CARBON SOURCE
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.
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.