Construction and Characteristics of Poly-3-hydroxybutyrate Depolymerase Encoding Gene (phbD ) Mutant in Sinorhizobium meliloti
Dai Meixue 1 Wu Bo 2 Bai Xueliang 2 Zhang Chenggang 1 Ma Qingsheng 2
(1. Shenyang Institute of Applied Ecology, Chinese Academy of Sciences , Shenyang 110015, China ; 2. Key Laboratory of Agricultural Molecular Genetics, Ministry of Agriculture, Guangxi University, Nanning 530005,China )
Abstract：Abstract: Rhizobia, as so many other bacteria, can synthesize the carbon storage compound, polyhydroxyalkanoates (PHAs), when non-carbon nutrient , such as N, P or O2, is limited to grow and excessive carbon is available. When subsequent conditions make carbon limit, the internal PHA stores may serve as sources of carbon and energy. Poly-3-hydroxybutyrate (PHB) is a common member of PHAs family.The ability to degrade PHAs depends on the production PHA depolymerases, which release dimers or monomers from the PHA polymer. Bacterial PHA depolymerases are classified as intracellular or extracellular and widely distributed among microorganisms. So far, intracellular PHA or PHB depolymerase mutants have only been described in two strains of bacteria: Pseudomonas oleovorans and Ralstonia eutropha. All Sinorhizobium meliloti PHB pathway mutants other than phbD mutant have been characterized. Creation of S. meliloti phbD mutant would be useful to study the role of phbD gene in the PHB pathway and to understand the PHB cycle in more detail. The S. meliloti phbD gene sequence was obtained from the S.meliloti 1021 genome sequencing project. A homologue of the Ralstonia eutropha phaZ gene, encoding intracellular PHB depolymerase, was identified and designated to be phbD. Using a pair of primers designed by the sequence of S. meliloti Rm1021 genome which is homologous to phaZ gene of R. eutropha , an 835 bp fragment of phbD gene was amplified by PCR from S. meliloti genome and cloned onto vector pGEM?襆-T Easy and generated the recombinant plasmid pDC45. The insert was sequenced to ensure that the cloned fragment does contain the desired sequence. A unique Kpn21 site was identified to be located within the coding region at 299 bp from the translation start codon. Interposon ΩSmSp was inserted into phbD gene at the Kpn21 site and generated the plasmid pDC48. phbD :: ΩSmSp fragment was excised from pDC48 as an Eco RⅠfragment and ligated with Eco RⅠdigested plasmid pK19mobsacB to get pDC50. Plasmid pDC50 was introduced into Rm5000 by conjugation and Rf RSmRSpR transconjugants selection. Putative double crossover homogenotes were selected on TY Sm Sp containing 5% sucrose and subsequently screened for the loss of vector-encoded NmR. A RfRSmRSpRNmS mutant was thus isolated and designated to be Rm11417. The insertion of Ω into phbD of Rm11417 was transduced into Rm1021 to generate mutant Rm11430.The putative phbD mutant Rm11430 was confirmed by Southern blotting analysis using theDIG-labelled phbD PCR product as probe. The probe hybridized to an about 1.3 kb Eco RⅠfragment of genomic DNA from the wild-type strain, while with genomic DNA from the putative mutant Rm11430, the hybribizing fragment was about 3.3 kb, consistent with the presence of a 2 kb interposon insertion within the about 1.3 kb Eco RⅠfragment. The S. meliloti phbD mutant accumulated 1~2.6 times PHB more than that of wild-type strain and showed non-mucoid colony on YMA and TY plates but mucoid colony on M9 minimum media with acetoacetate (AA) or 3-hydroxybutyrate (HB) as sole carbon source. The alkaline phosphatase in phbD mutant assay results indicated that exoF-phoA fusion introduced low activity expression in YMB but high activities in M9-AA and M9-HB.
