10.3389/fmicb.2016.01481.s001 Marta Irla Marta Irla Tonje M. B. Heggeset Tonje M. B. Heggeset Ingemar Nærdal Ingemar Nærdal Lidia Paul Lidia Paul Tone Haugen Tone Haugen Simone B. Le Simone B. Le Trygve Brautaset Trygve Brautaset Volker F. Wendisch Volker F. Wendisch Data_Sheet_1_Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production.PDF Frontiers 2019 Bacillus methanolicus thermophile methylotroph genetic tool box theta-replicating plasmids gene expression 2019-03-12 12:01:06 Dataset https://frontiersin.figshare.com/articles/dataset/Data_Sheet_1_Genome-Based_Genetic_Tool_Development_for_Bacillus_methanolicus_Theta-_and_Rolling_Circle-Replicating_Plasmids_for_Inducible_Gene_Expression_and_Application_to_Methanol-Based_Cadaverine_Production_PDF/7831262 <p>Bacillus methanolicus is 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 gene mtlR were 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 the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was 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 6.5 to 10.2 g/L when a heterologous lysine decarboxylase gene cadA was 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 developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.</p>