柴油降解基因工程菌的构建及降解特性

Construction and characterization of engineered diesel-degrading bacterium

摘要:

【目的】构建柴油降解基因工程菌，提高柴油降解速率，研究p450基因在柴油降解过程中的作用。【方法】将Alcanivorax borkumensis SK2的p450基因合成后，连接至烷烃响应表达载体pCom8中，构建该基因的表达载体p450-SK2/pCom8，并将其转入大肠杆菌DH5α中，通过SDS-PAGE检测该基因在大肠杆菌DH5α中的表达，并将重组质粒p450-SK2/pCom8转入柴油降解菌Acinetobacter sp. Y9中，构建基因工程菌p450-SK2/Y9，研究工程菌p450-SK2/Y9对柴油的降解特性及p450基因在构建的工程菌p450-SK2/Y9中的表达。【结果】PCR、酶切及测序结果表明重组质粒p450-SK2/pCom8构建正确。当柴油诱导浓度大于1%时，目的基因在大肠杆菌DH5α中的蛋白表达量较大，且随着诱导时间的延长而呈增加趋势。通过PCR检测构建的基因工程菌p450-SK2/Y9中的p450基因表明，工程菌构建正确，利用单菌株降解柴油时，宿主菌Y9与工程菌p450-SK2/Y9的柴油降解效率未见明显差异，但工程菌p450-SK2/Y9在构建的菌群中对柴油降解的促进效果明显。SDS-PAGE结果表明，p450基因在构建的工程菌p450-SK2/Y9中能得到准确表达，在混合菌中的表达量高于单菌株。【结论】柴油降解基因工程菌在混合菌群中对柴油降解具有促进作用，而在单菌株情况下未见促进作用，且p450基因的蛋白表达在混合菌中也高于单菌株，这对于提高柴油的降解速率及研究p450基因在柴油降解过程中的作用机理具有一定意义。

Abstract:

[Objective] We constructed engineered bacterium to improve diesel biodegradation and to study the effect of p450 gene on diesel biodegradation. [Methods] The p450 gene of Alcanivorax borkumensis SK2 was constructed in pCom8 and the p450-SK2/pCom8 plasmid was transformed into Escherichia coli DH5α. The expression of AlkB protein in E. coli DH5α was detected using SDS-polyacrylamide gel electrophoresis. The recombinant plasmids expressed rightly were transformed into diesel-degrading Acinetobacter sp. strain Y9. The degrading characteristics and the expression of p450 in engineered bacterium p450-SK2/Y9 were studied. [Results] The identifying results by PCR and digestion with Sal I and Nde I indicated that the recombinant plasmids and the engineered bacterium p450-SK2/Y9 were constructed rightly. When the induction concentration of diesel was above 1% (v/v), the expression level of p450 gene was higher and increased slightly with the increasing of induction time. Diesel degradation ratios of p450-SK2/Y9 were not higher than that of Y9 when it was inoculated solely, but the p450-SK2/Y9 could facilitate the biodegradation of diesel in bacterial consortium. The results of SDS-PAGE showed that p450 gene could be expressed p450-SK2/Y9, and the expression level was higher in the bacterial consortium than that of single bacterium. [Conclusion] The engineered bacterium p450-SK2/Y9 could facilitate the biodegradation of diesel in bacterial consortium, and the expression level was higher in the bacterial consortium than that of single bacterium. These results are useful to improve diesel biodegradation and study on the mechanism of p450 gene for diesel biodegradation.