title: [Identification of the phaB genes and analysis of the PHBV precursor supplying pathway in Haloferax mediterranei].
abstract: OBJECTIVE
  Identification and characterization of the genes involved in precursor supplying for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in the haloarchaeon Haloferax mediterranei.
  
  
  METHODS
  By using BLAST (Basic Local Alignment Search Tool) search methodology, we obtained five genes (phaB1, phaB2, phaJ1, phaJ2 and phaJ3) that were possibly involved in the 3-hydroxyacyl-CoA precursor supplying for PHBV biosynthesis in H. mediterranei. Firstly, we proved that these five genes were all transcribed under the PHBV-accumulating condition in H. mediterranei. Then, we knocked out these genes individually or in combination, by double-crossover homologous recombination, resulting in the following mutants: deltaphaB1, deltaphaB2, AphaJ1, deltaphaJ2, deltaphaJ3, deltaphaB1phaB2, deltaphaJ1phaJ2 and deltaphaJ1phaJ2phaJ3. Finally, we performed the complementation analysis of the deltaphaB1phaB2 strain, with the phaB1 and phaB2 genes, respectively.
  
  
  RESULTS
  Whenever the three phaJ genes were knocked out individually or in combination, there was no obvious influence on PHBV accumulation in H. mediterranei. Knockout of phaB1 also did not affect the PHBV accumulation obviously. However, when phaB2 was knocked out, the yield of PHBV and the fraction of the 3-HV monomer decreased significantly. Notably, when the phaB1 and phaB2 were knocked out in combination, the
  
  
  CONCLUSIONS
  The PHBV-specific acetoacetyl-CoA reductases mutant deltaphaB1phaB2 no longer produced PHBV. (PhaB) involved in the precursor supplying for PHBV biosynthesis are encoded by phaB1 and phaB2 in H. mediterranei.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. feng.bo1986@163.com
authors.forename: Bo
authors.initials: B
authors.lastname: Feng
authors.forename: Shuangfeng
authors.initials: S
authors.lastname: Cai
authors.forename: Jing
authors.initials: J
authors.lastname: Han
authors.forename: Hailong
authors.initials: H
authors.lastname: Liu
authors.forename: Jian
authors.initials: J
authors.lastname: Zhou
authors.forename: Hua
authors.initials: H
authors.lastname: Xiang
mesh_terms: D020155:3-Hydroxybutyric Acid / Q000096:biosynthesis* / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D019623:Haloferax mediterranei / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D010421:Pentanoic Acids / Q000378:metabolism*; D011091:Polyesters / Q000378:metabolism; D011355:Prodrugs; D011995:Recombination, Genetic
chemical_list: D010421:Pentanoic Acids; D011091:Polyesters; D011355:Prodrugs; C013056:beta-hydroxyvaleric acid; D000429:Alcohol Oxidoreductases; C023744:acetoacetyl-CoA reductase; D020155:3-Hydroxybutyric Acid