Identification of the phaB genes and analysis of the PHBV precursor supplying pathway in Haloferax mediterranei

Abstract:

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: ΔphaB1, ΔphaB2, ΔphaJ1, ΔphaJ2, ΔphaJ3, ΔphaB1phaB2, ΔphaJ1phaJ2 and ΔphaJ1phaJ2phaJ3. Finally, we performed the complementation analysis of the ΔphaB1phaB2 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 mutant ΔphaB1phaB2 no longer produced PHBV. [Conclusions] The PHBV-specific acetoacetyl-CoA reductases (PhaB) involved in the precursor supplying for PHBV biosynthesis are encoded by phaB1 and phaB2 in H. mediterranei.