Enhanced Recombinant Protein Productivity by Genome Reduction in Bacillus subtilis

Abstract

The emerging field of synthetic genomics is expected to facilitate the generation of microorganisms with the potential to achieve a sustainable society. One approach towards this goal is the reduction of microbial genomes by rationally designed deletions to create simplified cells with predictable behavior that act as a platform to build in various genetic systems for specific purposes. We report a novel Bacillus subtilis strain, MBG874, depleted of 874 kb (20%) of the genomic sequence. When compared with wild-type cells, the regulatory network of gene expression of the mutant strain is reorganized after entry into the transition state due to the synergistic effect of multiple deletions, and productivity of extracellular cellulase and protease from transformed plasmids harboring the corresponding genes is remarkably enhanced. To our knowledge, this is the first report demonstrating that genome reduction actually contributes to the creation of bacterial cells with a practical application in industry. Further systematic analysis of changes in the transcriptional regulatory network of MGB874 cells in relation to protein productivity should facilitate the generation of improved B. subtilis cells as hosts of industrial protein production.

Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering

“…subtilis and E. coli genome reductions often led to beneficial traits (Manabe et al, 2011(Manabe et al, , 2013Mizoguchi et al, 2008). B. subtilis strain MGB874 lacking 20.7 % of the genome produced significantly higher levels of the heterologous enzymes alkaline cellulase and protease than the parent strain (Morimoto et al, 2008). The production of extracellular enzymes by B. subtilis MGB874 was further enhanced by deleting genes from the arginine degradation pathway (Manabe et al, 2011(Manabe et al, , 2013.…”

Building the blueprint of life

“…Efforts are under way in many labs throughout the world to produce a living strain with a minimal genome [1,7,8,14,[49][50][51]. These efforts are applying various approaches, depending on the organisms being used as a starting point and the specific scientific or industrial objectives motivating the effort.All the approaches can be classified as either top down or bottom up [4].…”

“…These efforts are applying various approaches, depending on the organisms being used as a starting point and the specific scientific or industrial objectives motivating the effort.All the approaches can be classified as either top down or bottom up [4]. Top-down approaches involve starting with the genome of an existing (often far from minimal) organism and combining deletions to produce progressively smaller genomes [7,8,14,[49][50][51]. Bottom-up approaches involve starting with a very small genome and engineering a reduced version of the entire genome for implantation and viability testing [1].…”

“…It is precisely this need for a minimal catalogue of the essential biological functions required for life that drives the pursuit for the design and creation of a minimal organism [5][6][7][8][9]. Here, we define a minimal organism as a living, dividing cell where every gene in the genome is indispensable for viability in a defined chemical environment.…”

“…These efforts are already helping to improve genome annotations [5,6,11], correct models ( [12,13] and Tanaka et al, unpublished results), and gain insights into global cell regulation [8,14].…”