title: Genome-based analysis and gene dosage studies provide new insight into 3-hydroxy-4-methylvalerate biosynthesis in Ralstonia eutropha.
abstract: Recombinant Ralstonia eutropha strain PHB(-)4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB(-)4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB(-)4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone.
authors: Department of Innovative and Engineered Materials, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Japan azusa-saika@aist.go.jp tsuge.t.aa@m.titech.ac.jp.
authors: Azusa
authors: A
authors: Saika
authors: Department of Innovative and Engineered Materials, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Japan.
authors: Kazunori
authors: K
authors: Ushimaru
authors: Department of Innovative and Engineered Materials, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Japan.
authors: Shoji
authors: S
authors: Mizuno
authors: Department of Innovative and Engineered Materials, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Japan azusa-saika@aist.go.jp tsuge.t.aa@m.titech.ac.jp.
authors: Takeharu
authors: T
authors: Tsuge
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020118:Cupriavidus necator / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D016680:Genome, Bacterial; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D014631:Valerates / Q000378:metabolism*; D014633:Valine / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014631:Valerates; D007930:Leucine; D014633:Valine
references: Biomacromolecules. 2010 Jun 14;11(6):1615-22
references: 20441146
references: Arch Mikrobiol. 1970;71(3):283-94
references: 4097070
references: Int J Syst Evol Microbiol. 2001 Sep;51(Pt 5):1709-13
references: 11594600
references: Biotechnol Lett. 2003 May;25(9):665-70
references: 12882163
references: Biomacromolecules. 2009 Oct 12;10(10):2866-74
references: 19681605
references: Int J Biol Macromol. 1993 Aug;15(4):253-5
references: 8373746
references: J Biol Chem. 1970 Apr 10;245(7):1828-35
references: 4314598
references: J Bacteriol. 2009 Jan;191(1):52-64
references: 18820024
references: BMC Microbiol. 2013;13:169
references: 23879744
references: Appl Microbiol Biotechnol. 2010 Jan;85(3):491-506
references: 19844702
references: Appl Microbiol Biotechnol. 2011 Mar;89(5):1527-36
references: 21279346
references: J Biosci Bioeng. 2014 Jun;117(6):670-5
references: 24484910
references: J Biol Chem. 1989 Sep 15;264(26):15298-303
references: 2670936
references: Bacteriol Rev. 1976 Mar;40(1):42-54
references: 773366
references: Arch Microbiol. 1977 Sep 28;114(3):203-10
references: 20865
references: J Mol Biol. 2001 Mar 16;307(1):465-77
references: 11243831
references: Int J Biol Macromol. 1992 Dec;14(6):321-5
references: 1476987
references: Nat Commun. 2013;4:1414
references: 23361005
references: Macromol Biosci. 2005 Feb 23;5(2):112-7
references: 15729719
references: J Biosci Bioeng. 2003;95(1):77-81
references: 16233370
references: Biochem J. 2003 Nov 15;376(Pt 1):15-33
references: 12954080
references: PLoS One. 2014;9(5):e95907
references: 24787649
references: J Bacteriol. 1997 Aug;179(15):4821-30
references: 9244271
references: Biomacromolecules. 2000 Spring;1(1):17-22
references: 11709837
references: Int J Biol Macromol. 1991 Apr;13(2):83-8
references: 1888716
references: AMB Express. 2011 May 18;1(1):6
references: 21906338
references: Appl Microbiol Biotechnol. 2015 Jan;99(2):761-74
references: 25081555
references: J Biosci Bioeng. 2012 Mar;113(3):286-92
references: 22088761
references: Int J Biol Macromol. 2002 Apr 8;30(2):97-104
references: 11911900
references: FEMS Microbiol Lett. 2004 Feb 9;231(1):77-83
references: 14769470
references: J Mol Biol. 2003 Sep 12;332(2):369-83
references: 12948488
references: Nat Biotechnol. 2006 Oct;24(10):1257-62
references: 16964242