A Novel Inducible Protein Production System and Neomycin Resistance as Selection Marker forMethanosarcina mazei

Abstract

Methanosarcina mazeiis 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 inE. coli. However, a protein production system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins inMs. 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 monitoringβ-glucuronidase production. The promoter was inactive during growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding theβ-glucuronidase fromE. coliwas fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was overproduced inMs. mazeiand was purified in an active form by affinity chromatography. (ii) Puromycin is currently the only antibiotic used as a selectable marker inMs. mazeiand its relatives. We established neomycin resistance as a second selectable marker by designing a plasmid that confers neomycin resistance inMs. mazei.