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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

Abstract

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 11.3 to 17.5 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.

Category

Academic article

Language

English

Author(s)

Affiliation

  • University of Bielefeld
  • SINTEF Industry / Biotechnology and Nanomedicine
  • Norwegian University of Science and Technology

Year

2016

Published in

Frontiers in Microbiology

ISSN

1664-302X

Publisher

Frontiers Media S.A.

Volume

7

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