Efficient and accurate production of de novo designed large-size gene clusters by a novel bacillus subtilis-based system

Recombinant cells harnessing a set of genes designed to produce useful materials or to respond to environmental changes have been constructed. Thanks to the recent burst of genome sequence information, target genes are not limited to the existing genes; the candidates extend to those from unculturable microbes and de novo designed genes. As synthesis processes become more complex, the number of relevant enzymes can be expected to increase. The increase in the number of genes delivered to industrial microbes has also raised certain technological barriers not encountered previously. Conventional molecular cloning systems rely on Escherichia coli strain K-12, which is poorly suited for multiple gene assembly processes, the primary topic of this chapter. DNA cloning in the E. coli plasmid requires a plasmid in circular form before transformation. However, reactions to circularize long DNA and to connect multiple DNA fragments are trade-offs in solution and are thus rarely conducted for a single ligation. In contrast, our novel DNA cloning system using Bacillus subtilis strain 168 as a final host is offered to solve all plausible stumbling blocks in gene assembly technologies. We call the new system BGM (Bacillus subtilis-based gene/genome manipulation). In this chapter, we focus on the development of the BGM system and certain related achievements, highlighting the system's clear conceptual differences from E. coli systems.