沼泽红假单胞菌phbC-hupL双突变株构建及产氢测定

利用湖底淤泥分离的沼泽红假单胞菌(Rhodopseudomonas palustris)CQU01和该菌株的吸氢酶基因hupL缺失菌株CQU012作为出发菌株,分别构建聚羟基丁酸酯合成酶基因phbC单突变株及聚羟基丁酸酯合成酶基因phbC与吸氢酶基因hupL双突变株,以提高其在光照培养条件下的产氢量.以同源重组双交换方法构建含有Em抗性基因的自杀载体,通过接合转移转化R. palustris CQU01菌株,经PCR扩增以及测序验证,成功获得了沼泽红假单胞菌phbC单突变株R. palustris CQU013及phbC-hupL双突变株R. palustris CQU014.相同条件下测定突变菌株与野生菌株的生长和产氢特性,结果显示,突变菌株生长曲线与野生菌株有明显差异,两株突变菌株的产氢量分别是原始菌株的1.31和1.76倍,达到454mL/L和604mL/L.双突变菌株产氢能力较phbC基因和hupL基因单突变菌株的产氢能力有明显提高,说明phbC和hupL基因对菌株R. palustris 的产氢代谢有着显著的影响.

Construction of Rhodopseudomonas palustris phbC-hupL double mutant and determination of H2 production

To improve the hydrogen produce ability of bacteria strain, a di-mutant bioengineering strain of Rhodopseudomonas palustris, with phbC (PHB synthase) and hupL (uptake hydrogenase) gene knocked-out, was constructed. The bacteria strains of R. palustris CQU01 isolated from silt for phbC gene knocked out, and R. palustris CQU012 with hupL knocked out which had been constructed before in our lab were used as the initial strains. The single mutant strain R. palustris CQU013 with phbC knocked out and double mutant strain R. palustris CQU014 with phbC-hupL knocked out were obtained by constructing a suicide vector containing erythromycin cassette used homologous double exchange and recombining into R. palustris CQU01 with conjugative transfer. The correct integration of the mutants was confirmed by PCR test and subsequent sequence analysis. The bacterial growth curves and hydrogen production characteristics of the mutant strains showed that the biological characters of the mutant strains had some differences with the wild strain R. palustris CQU01. The total H2 yield of R. palustris CQU013 and R. palustris CQU014 increased by 31% and 76% respectively comparing to wild strain, which up to 454mL and 604mL per liter fermentation broth in 216 hours. The hydrogen produce ability of di-mutant R. palustris CQU014 increased remarkably after the phbC gene and hupL gene be knocked out. This approved that both phbC gene and hupL gene in R. palustris affected the yield of hydrogen produce. The mutant strain R. palustris CQU014 may be a promising bacterial strain in hydrogen bio-produce.