Institute for Microbiology and Biotechnology, University of Bonn, 53115 Bonn, Germany.
Archaea. 2012;2012:973743. doi: 10.1155/2012/973743. Epub 2012 Jul 19.
Methanosarcina mazei is one of the model organisms for the methanogenic order Methanosarcinales whose metabolism has been studied in detail. However, the genetic toolbox is still limited. This study was aimed at widening the scope of utilizable methods in this group of organisms. (i) Proteins specific to methanogens are oftentimes difficult to produce in E. coli. However, a protein production system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins in Ms. mazei. The promoter p1687, which directs the transcription of methyl transferases that demethylate methylamines, was cloned into plasmid pWM321 and its activity was determined by monitoring β-glucuronidase production. The promoter was inactive during growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding the β-glucuronidase from E. coli was fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was overproduced in Ms. mazei and was purified in an active form by affinity chromatography. (ii) Puromycin is currently the only antibiotic used as a selectable marker in Ms. mazei and its relatives. We established neomycin resistance as a second selectable marker by designing a plasmid that confers neomycin resistance in Ms. mazei.
产甲烷八叠球菌是产甲烷目（Methanosarcinales）的模式生物之一，其代谢已被详细研究。然而，遗传工具包仍然有限。本研究旨在拓宽该组生物中可利用方法的范围。（i）产甲烷菌特有的蛋白质通常难以在大肠杆菌中生产。然而，产甲烷菌没有蛋白质生产系统。在这里，我们提出了一种在产甲烷八叠球菌中生产 Strep 标记蛋白的诱导系统。克隆了指导脱甲基甲胺甲基转移酶转录的启动子 p1687 到质粒 pWM321 中，并通过监测β-葡糖苷酸酶的产生来确定其活性。启动子在甲醇生长期间不活跃，但当三甲基胺添加到培养基中时，它会迅速激活。来自大肠杆菌的β-葡糖苷酸酶基因与 Strep 标签融合，并克隆到 p1687 启动子的下游。该蛋白在产甲烷八叠球菌中过表达，并通过亲和层析以活性形式纯化。（ii）目前，嘌呤霉素是唯一可用于产甲烷八叠球菌及其亲缘种的选择标记抗生素。我们通过设计一种在产甲烷八叠球菌中赋予新霉素抗性的质粒，将新霉素抗性确立为第二个选择标记。
