title: Characterization and functional analyses of R-specific enoyl coenzyme A hydratases in polyhydroxyalkanoate-producing Ralstonia eutropha.
abstract: A genome survey of polyhydroxyalkanoate (PHA)-producing Ralstonia eutropha H16 detected the presence of 16 orthologs of R-specific enoyl coenzyme A (enoyl-CoA) hydratase, among which three proteins shared high homologies with the enzyme specific to enoyl-CoAs of medium chain length encoded by phaJ4 from Pseudomonas aeruginosa (phaJ4(Pa)). The recombinant forms of the three proteins, termed PhaJ4a(Re) to PhaJ4c(Re), actually showed enoyl-CoA hydratase activity with R specificity, and the catalytic efficiencies were elevated as the substrate chain length increased from C(4) to C(8). PhaJ4a(Re) and PhaJ4b(Re) showed >10-fold-higher catalytic efficiency than PhaJ4c(Re). The functions of the new PhaJ4 proteins were investigated using previously engineered R. eutropha strains as host strains; these strains are capable of synthesizing poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil. Deletion of phaJ4a(Re) from the chromosome resulted in significant decrease of 3HHx composition in the accumulated copolyester, whereas no change was observed with deletion of phaJ4b(Re) or phaJ4c(Re), indicating that only PhaJ4a(Re) was one of the major enzymes supplying the (R)-3HHx-CoA monomer through β-oxidation. Introduction of phaJ4a(Re) or phaJ4b(Re) into the R. eutropha strains using a broad-host-range vector enhanced the 3HHx composition of the copolyesters, but the introduction of phaJ4c(Re) did not. The two genes were then inserted into the pha operon on chromosome 1 of the engineered R. eutropha by homologous recombination. These modifications enabled the biosynthesis of P(3HB-co-3HHx) composed of a larger 3HHx fraction without a negative impact on cell growth and PHA production on soybean oil, especially when phaJ4a(Re) or phaJ4b(Re) was tandemly introduced with phaJ(Ac) from Aeromonas caviae.
authors.affiliation: Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
authors.forename: Yui
authors.initials: Y
authors.lastname: Kawashima
authors.forename: Wen
authors.initials: W
authors.lastname: Cheng
authors.forename: Jun
authors.initials: J
authors.lastname: Mifune
authors.forename: Izumi
authors.initials: I
authors.lastname: Orita
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nakamura
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Fukui
mesh_terms: D057585:Aeromonas caviae / Q000201:enzymology / Q000235:genetics; D020118:Cupriavidus necator / Q000201:enzymology* / Q000378:metabolism*; D004746:Enoyl-CoA Hydratase / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D054813:Polyhydroxyalkanoates / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017385:Sequence Homology; D013024:Soybean Oil / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D054813:Polyhydroxyalkanoates; D011994:Recombinant Proteins; D013024:Soybean Oil; D004746:Enoyl-CoA Hydratase
references.citation: Appl Environ Microbiol. 2003 Aug;69(8):4830-6
references.pmid: 12902277
references.citation: Cell Biochem Biophys. 2000;32 Spring:73-87
references.pmid: 11330072
references.citation: Appl Microbiol Biotechnol. 1998 Mar;49(3):333-6
references.pmid: 9581296
references.citation: Int J Biol Macromol. 2003 Jan 15;31(4-5):195-205
references.pmid: 12568928
references.citation: J Biosci Bioeng. 2008 Jul;106(1):95-8
references.pmid: 18691538
references.citation: FEMS Microbiol Lett. 2000 Mar 15;184(2):193-8
references.pmid: 10713420
references.citation: Macromol Biosci. 2004 Mar 15;4(3):238-42
references.pmid: 15468213
references.citation: J Bacteriol. 1998 Feb;180(3):667-73
references.pmid: 9457873
references.citation: Macromol Biosci. 2009 Jan 9;9(1):71-8
references.pmid: 18798179
references.citation: Biochim Biophys Acta. 2006 Sep;1761(9):973-94
references.pmid: 16766224
references.citation: Mol Microbiol. 1991 Mar;5(3):535-42
references.pmid: 2046547
references.citation: Microbiol Mol Biol Rev. 1999 Mar;63(1):21-53
references.pmid: 10066830
references.citation: J Bacteriol. 1997 Aug;179(15):4821-30
references.pmid: 9244271
references.citation: Nat Biotechnol. 2006 Oct;24(10):1257-62
references.pmid: 16964242
references.citation: J Bacteriol. 2010 Oct;192(20):5319-28
references.pmid: 20729355
references.citation: Biochim Biophys Acta. 2006 Dec;1763(12):1413-26
references.pmid: 17028011
references.citation: Appl Microbiol Biotechnol. 2000 Apr;53(4):401-9
references.pmid: 10803895
references.citation: Appl Environ Microbiol. 2011 May;77(9):2847-54
references.pmid: 21398488
references.citation: Biotechnol Prog. 2004 Sep-Oct;20(5):1332-6
references.pmid: 15458314
references.citation: Curr Microbiol. 2007 Jul;55(1):20-4
references.pmid: 17534562
references.citation: FEMS Microbiol Lett. 1999 Jan 1;170(1):69-75
references.pmid: 9919653
references.citation: FEMS Microbiol Lett. 2007 Dec;277(2):217-22
references.pmid: 18031343
references.citation: Appl Environ Microbiol. 2010 Aug;76(16):5373-82
references.pmid: 20601511
references.citation: Antonie Van Leeuwenhoek. 2008 Mar;93(3):285-96
references.pmid: 17906975
references.citation: Appl Microbiol Biotechnol. 2011 Mar;89(5):1527-36
references.pmid: 21279346
references.citation: Appl Microbiol Biotechnol. 2011 May;90(3):951-9
references.pmid: 21327961
references.citation: Eur J Biochem. 1992 Oct 1;209(1):15-30
references.pmid: 1396693
references.citation: J Bacteriol. 2003 Sep;185(18):5391-7
references.pmid: 12949091
references.citation: J Biol Chem. 2003 Jan 3;278(1):617-24
references.pmid: 12409309
references.citation: J Bacteriol. 2010 Oct;192(20):5454-64
references.pmid: 20709892
references.citation: Arch Microbiol. 2002 Aug;178(2):149-60
references.pmid: 12115060
references.citation: Gene. 1994 Jul 22;145(1):69-73
references.pmid: 8045426
references.citation: Biomacromolecules. 2001 Spring;2(1):248-54
references.pmid: 11749180
references.citation: Appl Microbiol Biotechnol. 2000 Feb;53(2):209-18
references.pmid: 10709984
references.citation: Antonie Van Leeuwenhoek. 1992 Aug;62(1-2):63-78
references.pmid: 1444337