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.

Figure 1

Design and basal phenotypic analysis of MGB874. (A) Outer concentric ring: genome coordinate (bases) of the B. subtilis 168 genome. Ring 2 (green): positions of deleted sequences in MGB874, including prophages and prophage-like regions (SPβ, PBSX, skin, pro1-7) and polyketide and plipastatin synthesis operons (pks, pps). Ring 3 (dark blue): regions of single deletion. Rings 4 and 5 (light blue): protein coding regions in clockwise (Ring 4) and counterclockwise (Ring 5) orientations. Ring 6 (red): rRNA and tRNA genes. (B) Growth profiles of MGB874 (red squares) and wild-type 168 (blue diamonds) cells in LB and SMM medium. The doubling time of growth is specified. (C) Cell morphology, chromosome distribution, and mean values of cell lengths of wild-type 168 and MGB874 cells. MGB874 and 168 cells were cultured at 37°C in LB or SMM medium, and images were obtained during the exponential growth phase after staining with 4,6-diamindino-2-phenylindole (DAPI). The average cell length is indicated (∼200 cells analyzed).

Figure 2

Sequential introduction of large-scale deletions into the B. subtilis genome. A derivative of B. subtilis 168, 168 Δupp, in which the upp gene encoding uracil-phosphoribosyl-transferase is inactivated by replacement with the erythromycin-resistance gene (erm), was used as the starting strain for generation of the deletion mutant series. The entire length of the tetracycline-resistant gene (tet) cassette with its 5′-regulatory region was amplified from the pBEST307 plasmid.¹⁹ At least 500 bp of sequences flanking both sides of the region to be deleted were amplified by PCR and joined upstream and downstream of the tet cassette by ligation using overlapping sequences in primers. The B. subtilis 168 Δupp strain and its derivatives were transformed with the resultant fragment to obtain a strain in which the target sequence was replaced with the tet gene (Step 1). Next, to obtain markerless mutants, fragments upstream and downstream of the target sequence were amplified, ligated, and cloned into the pBRcat/upp plasmid harboring the upp and chloramphenicol resistance (cat) gene. The resultant plasmid was integrated into the genome of the primary transformant with selection for tetracycline and chloramphenicol resistance (Step 2). The resultant strain became 5-FU sensitive as a result of introduction of the functional upp gene, and mutants without the plasmid sequence were selected on LB plates containing 10 μM 5FU (Step 3). The crosses indicate the recombination site.

Figure 3

Productivity of extracellular enzymes by the multiple-deletion series strains. (A) Relative activities of cellulase Egl237 (black bars) and M-protease (white bars) in growth medium of the multiple-deletion series, compared with those of the wild-type 168 strain after 75 h cultivation in 2xL-Mal medium, are indicated with error bars (average of three experiments). (B) The growth profiles of wild-type 168 (open diamonds) and MGB874 (closed squares) are presented. Arrows with a–f indicate the times of cell collection for transcriptome analysis. (C) Cellulase production as a function of cell growth. Extracellular cellulase activities of wild-type 168 (white bars) and MGB874 (black bars) cultures (0.4 μL) are shown. (D) Consumption of sugars in the growth medium. Wild-type 168 (open diamonds) and MGB874 (closed squares) cultures were collected at the indicated times. The total amount of sugar was determined according to a previous report.²¹

Figure 4

Comparison of gene expression in wild-type 168 and MGB874 cells. Total RNA was extracted from both strains grown in 2xL-Mal medium for 1 (A), 7.5 (B), 13 (C), 26 (D), 40 (E), and 60 (F) hours (Fig. 3B) and used for tiling chip analysis. The average signal intensities of probes in each coding sequence were calculated after removal of the lowest and highest intensities. Scatter plots of expression in wild-type 168 and MGB874 cells are presented. Genes deleted in MGB874 were excluded from the analysis. The correlation coefficient is indicated.

Figure 5

Identification of genes with significantly disrupted expression in MGB874 cells, compared with wild-type cells. The average signal intensities of probes in each coding sequence in wild-type 168 (open diamonds) and MGB874 (closed squares) cells grown in 2xL-Mal medium for 1 (A), 7.5 (B), 13 (C), 26 (D), 40 (E), and 60 (F) h (Fig. 3B) are indicated. Regulators of expression are specified in parentheses. (A) ComA and ComK-dependent genes. (B) DegU-dependent genes. (C) Transition state regulators. (D) Sporulation genes. (E) Genes with markedly elongated expression in MGB874 cells. (F) Genes with markedly suppressed expression in MGB874 cells. (G) Genes that are specifically induced in MGB874 cells. (H) Phosphate metabolism-related genes.