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 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.
Highlights
Bacillus methanolicus is a thermophilic bacterium, able to grow on methanol as a sole carbon and energy source (Schendel et al, 1990; Arfman et al, 1992)
We decided to analyze a range of different replicons with regard to their applicability for gene overexpression in B. methanolicus MGA3
All rolling circle (RC) plasmids used belong to the pC194/pUB110 family, which is characterized by similarity in Rep protein and the sequences of sites involved in the replication with pNW33N and pUB110 sharing identical Rep protein sequences
Summary
Bacillus methanolicus is a thermophilic bacterium, able to grow on methanol as a sole carbon and energy source (Schendel et al, 1990; Arfman et al, 1992). Considerable progress has been made in the elucidation of the methanol utilization pathway starting from sequencing of the full genome (Heggeset et al, 2012; Irla et al, 2014), characterization of the enzymes involved in the methanol oxidation and the ribulose monophosphate (RuMP) pathway (Krog et al, 2013; Stolzenberger et al, 2013a,b; Markert et al, 2014; Ochsner et al, 2014; Wu et al, 2016), unraveling of the transcriptome by the means of microarray analysis (Heggeset et al, 2012) and RNA-seq (Irla et al, 2015), of the proteome (Müller et al, 2014), and the metabolome (Kiefer et al, 2015; Müller et al, 2015b) These findings enabled a better understanding of the metabolic processes taking place during growth on methanol, and on the limited number of alternative C-sources for this facultative methylotroph, in particular on mannitol. By contrast B. methanolicus MGA3 (pTH1xpx-amy) showed a halo on starch LB plates containing xylose as an inducer and incubated at 37◦C (Figure 9B) indicating that expression of amy from S. griseus plasmid allowed for starch degradation by recombinant B. methanolicus
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