沼泽红假单胞菌高效产氢hupL缺失突变株的构建

利用湖底淤泥分离的沼泽红假单胞菌(Rhodopseudomonas palustris)CQU01作为出发菌株,构建吸氢酶大亚基基因hupL缺失突变株,以提高光合细菌菌株的产氢效率.以PCR扩增的hupL两侧hupS 和 hupC基因为同源重组双交换臂,连入pMD18-T载体;再将hupS, hupC 和Kmr基因与经SalⅠ和HindⅢ双酶切的pSUP202,构建靶向自杀载体pBPZ.经接合转移转化R. palustris CQU01菌株,成功获得沼泽红假单胞菌吸氢酶活性缺失突变株R. palustris CQU012.测定突变株的吸氢酶活性及生长和产氢特性,结果表明,突变株的产氢量比野生菌株提高了约50%,而生长特性与野生菌株没有显著差异.R. palustris CQU012 吸氢酶缺失突变株可望为工业废水的生物治理提供高效产氢工程菌株.

Construction of Rhodopseudomonas palustris hupL gene deficient mutant with higher efficient of H2 production.

Application of Rhodopseudomonas palustris CQU01 isolated from silt as initial strain to construct a hupL gene deficient mutant for promoting its H2-production. The amplified hupS and hupC in bilaterally of hupL of R. palustris were used as double-exchange arms in homologous recombination. They were inserted into pMD18-T for sequencing. The confirmed hupS, hupC and Kmr cassette were recombined into the pSUP202, constructing a suicide vector pBPZ for knocking-out the structure gene hupL of uptake hydrogenase. The pBPZ was identified based on enzyme digestion, amplification, sequencing and then transformed into R. palustris CQU01 via conjunction. A mutant strain was obtained by screened on Km culture plate and confirmed by PCR assay. The assay for total volume of H2 produced and growth patterns showed that the volume of H2 produced by the mutant strain was 50% higher than that of the wild strain of R. palustris and had similar growth patterns with the wild strain. It indicated that the hupL deficient mutant of R. palustris CQU012 may have a promising capability in the treatment of waste water.