title: Genome-Based Genetic Tool Development forBacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production.
abstract: Bacillus methanolicusis a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator genemtlRwere identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of themtlRregulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system fromB. megateriumwas employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase genecadAwas expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developedB. methanolicusgenetic toolbox, valuable for genetic engineering and further exploration of this bacterium.
authors.affiliation: Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University Bielefeld, Germany.
authors.forename: Marta
authors.initials: M
authors.lastname: Irla
authors.affiliation: SINTEF Materials and Chemistry, Department of Biotechnology and Nanomedicine Trondheim, Norway.
authors.forename: Tonje M B
authors.initials: TM
authors.lastname: Heggeset
authors.affiliation: SINTEF Materials and Chemistry, Department of Biotechnology and Nanomedicine Trondheim, Norway.
authors.forename: Ingemar
authors.initials: I
authors.lastname: Nærdal
authors.affiliation: Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University Bielefeld, Germany.
authors.forename: Lidia
authors.initials: L
authors.lastname: Paul
authors.affiliation: SINTEF Materials and Chemistry, Department of Biotechnology and Nanomedicine Trondheim, Norway.
authors.forename: Tone
authors.initials: T
authors.lastname: Haugen
authors.affiliation: SINTEF Materials and Chemistry, Department of Biotechnology and Nanomedicine Trondheim, Norway.
authors.forename: Simone B
authors.initials: SB
authors.lastname: Le
authors.affiliation: SINTEF Materials and Chemistry, Department of Biotechnology and NanomedicineTrondheim, Norway; Department of Biotechnology, Norwegian University of Science and TechnologyTrondheim, Norway.
authors.forename: Trygve
authors.initials: T
authors.lastname: Brautaset
authors.affiliation: Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University Bielefeld, Germany.
authors.forename: Volker F
authors.initials: VF
authors.lastname: Wendisch
keywords: Bacillus methanolicus; gene expression; genetic tool box; methylotroph; thermophile; theta-replicating plasmids