颗粒状甲烷单加氧酶异源表达方法

Heterologous expression of particulate methane monooxygenase in different host cells

摘要:

由于甲烷氧化菌只能利用甲烷作为唯一的碳源和能源, 存在生长缓慢、细胞密度低、培养困难等问题, 限制了其工业应用。解决该问题的有效途径之一是在容易实现高密度培养的异源宿主菌中表达甲烷单加氧酶(Methane monooxygenase, MMO)。本实验室前期首次在一种红球菌中成功地表达了来自于甲烷氧化菌(Methylosinus trichosporium)OB3b的pMMO(颗粒状甲烷单加氧酶), 但比酶活较原始菌低很多。本试验在该结果的基础上, 通过选用不同的启动子和宿主细胞探索表达pMMO的可能性, 结果得到了具有氧化甲烷活性的重组菌, 但是产物检测到乙醇的生成, 且该重组菌的pMMO活性不稳定, 暗示pMMO的催化特性可能发生了变化。另外, 很多重组菌检测到pMMO蛋白的表达, 但没有催化活性, 说明pMMO在宿主细胞中的正确组装是其功能表达的关键。

Abstract:

Methanotrophs uses methane as the sole carbon and energy source, which cause slow growth, low cell density and hinders its industrial applications. One promising solution is to heterologously express methane monooxygenase (MMO) in other host cells that can be easily cultivated at high cell density. We systematically exploited the possibility of functional expression of pMMO by choosing different promoters and different host cells. The results showed that the recombinants could oxidize methane to methanol. In particular, ethanol could also be detected in the oxidized products, but the enzyme activity was instable, implying that some changes of pMMO expressed in the host cells might have occurred. In addition, SDS-PAGE analysis showed that many recombinants could express the subunits of pMMO, but the enzyme activity could not be detected. In conclusion, correct fold of pMMO in the host cells is important for its functional expression.