Bacillus subtilis, which is one of the most important host microorganisms for largescale industrial production of useful proteins, has a genome of 4.2 Mb with approximately 4106 protein-coding genes. Some of these genes are expected to be unnecessary for industrial production of proteins under controlled conditions and may be wasteful with regard to energy consumption. We attempted to reduce the genome size of B. subtilis by deleting unnecessary regions of the genome to allow the construction of simplified host cells as a platform for the further development of novel genetic systems with increased productivity. First, we generated the strain MGB469 with deletion of all prophage (SPβ and PBSX) and prophage-like (pro1-7 and skin) sequences, with the exception of pro7, as well as two large operons that produce secondary metabolites (pks and pps). These cells showed normal growth, but no beneficial effects were observed with regard to recombinant protein production from plasmids carrying the corresponding genes. Second, we constructed several multiple-deletion mutants containing additional deletions in the MGB469 genome, resulting in total genome size reductions of 0.78 to 0.99 Mb. In most of the multiple-deletion series, extensive deletion mutants showed no beneficial improvements in traits as host strains. The strain MG1M with a total genome size reduction of 0.99 Mb showed unstable phenotypes with regard to growth rate, cell morphology, and recombinant protein productivity after successive culture, making it inappropriate for further studies. In addition, strain MGB943 derived from another lineage with genome reduction of 0.94 Mb showed reduced recombinant cellulase productivity. Finally, we generated another multiple-deletion series including the mutant MGB874 with a total genome deletion of 0.87 Mb. In comparison to wild-type cells, the metabolic network of the mutant strain was reorganized after entry into the transition state due to the synergistic effects of multiple deletions. Moreover, the levels of production of extracellular cellulase and protease from transformed plasmids carrying the corresponding genes were markedly increased. Our results demonstrated the effectiveness of a synthetic genomic approach with reduction of genome size to generate novel and useful bacteria for industrial uses.
|ホスト出版物のタイトル||Bacterial DNA, DNA Polymerase and DNA Helicases|
|出版社||Nova Science Publishers, Inc.|
|出版ステータス||Published - 2009 12 1|
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