Markerless tool development for chromosome engineering in Bacillus methanolicus
We here present the screening and evaluation of potential counterselection markers for Bacillus methanolicus, and the establishment of a counterselection system conducive for genome modifications. 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 in the genome of B. methanolicus. We tested substrates of each candidate to confirm their lack of toxicity to wild type B. methanolicus and then we evaluated the ability to chosen genes to convert their non-toxic substrates to toxic products when heterologously expressed in B. methanolicus. Experimental results confirmed that none of the tested substrates affected the growth of wild-type B. methanolicus at following concentrations: X-gal, 20 µg mL-1; sucrose, 10%, 5-flourooratete and 5-fluorocytosine, 100 µg mL-1. Subsequently, the selected genes were individually cloned into a low-copy plasmid pTH1mp and transformed into B. methanolicus, leading to the creation of recombinant strains, whereof there demonstrated counterselection activity. A novel conjugation method for high-efficiency transformation of B. methanolicus was developed and used for established 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, confirming the utility of the established counterselection system. The deletion strain exhibited reduced sensitivity to the toxic compound 5-fluorouracil, demonstrating the practical application of the counterselection markers in genetic engineering of B. methanolicus.