Development of a markerless tool for targeted chromosome modification in the thermophilic and methylotrophic bacterium Bacillus methanolicus

Bacillus methanolicus is a promising candidate to become an industrial workhorse for methanol-based bioproduction due to its methylotrophy. However, genetic toolbox for this biotechnologically relevant thermophilic bacterium is still limited. We here present the establishment of a counterselection system conducive to genome modifications in Bacillus methanolicus MGA3. We first identified four candidate genes or operons feasible to become counterselection markers: lacZ from Bacillus coagulans, sacB from Bacillus subtilis, codBA from Escherichia coli, and oroP from Lactococcus lactis, based on their absence from the genome of B. methanolicus. We tested substrates of the encoded enzymes to confirm their lack of toxicity to wild type B. methanolicus. Experimental results confirmed that none of the tested substrates affected the growth of B. methanolicus wild type at physiologically relevant concentrations. Subsequently, the selected genes were individually cloned into a low-copy plasmid pTH1mp and used to transform B. methanolicus. We evaluated the conversion of these non-toxic substrates to toxic products upon heterologous expression of the respective marker genes in B. methanolicus. The recombinant strains were demonstrated to possess the desired counterselection activity through lack of growth in the presence of their relevant substrate. A novel transconjugation method for high-efficiency plasmid-delivery of B. methanolicus was developed and used for the establishment of genome modification via non-replicating suicide vector designed for homologous recombination. Deletion of the chromosomal upp gene, crucial for uracil metabolism, was achieved using this method. In this study, we confirmed the utility of the established oroP-based counterselection system for the genome modifications through deletion of upp gene in B. methanolicus. The deletion strain exhibited reduced sensitivity to 5-fluorouracil, the toxic substrate of the upp encoded enzyme, demonstrating the practical application of the counterselection markers in genome engineering of B. methanolicus.