Yasushi Kageyama

Enhanced Recombinant Protein Productivity by Genome Reduction in Bacillus subtilis

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.

Combined Effect of Improved Cell Yield and Increased Specific Productivity Enhances Recombinant Enzyme Production in Genome-Reduced Bacillus subtilis Strain MGB874

ABSTRACT Genome reduction strategies to create genetically improved cellular biosynthesis machineries for proteins and other products have been pursued by use of a wide range of bacteria. We reported previously that the novel Bacillus subtilis strain MGB874, which was derived from strain 168 and has a total genomic deletion of 874 kb (20.7%), exhibits enhanced production of recombinant enzymes. However, it was not clear how the genomic reduction resulted in elevated enzyme production. Here we report that deletion of the rocDEF-rocR region, which is involved in arginine degradation, contributes to enhanced enzyme production in strain MGB874. Deletion of the rocDEF-rocR region caused drastic changes in glutamate metabolism, leading to improved cell yields with maintenance of enzyme productivity. Notably, the specific enzyme productivity was higher in the reduced-genome strain, with or without the rocDEF-rocR region, than in wild-type strain 168. The high specific productivity in strain MGB874 is likely attributable to the higher expression levels of the target gene resulting from an increased promoter activity and plasmid copy number. Thus, the combined effects of the improved cell yield by deletion of the rocDEF-rocR region and the increased specific productivity by deletion of another gene(s) or the genomic reduction itself enhanced the production of recombinant enzymes in MGB874. Our findings represent a good starting point for the further improvement of B. subtilis reduced-genome strains as cell factories for the production of heterologous enzymes.

Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp0 mutant of Bacillus subtilis triggers YvyD-dependent dimerization of ribosome

To elucidate the biological functions of small (p)ppGpp synthetases YjbM and YwaC of Bacillus subtilis, we constructed RIK1059 and RIK1066 strains carrying isopropyl‐β‐D‐thiogalactopyranoside (IPTG) inducible yjbM and ywaC genes, respectively, in the ΔrelA ΔyjbM ΔywaC triple mutant background. While the uninduced and IPTG‐induced RIK1059 cells grew similarly in LB medium, the growth of RIK1066 cells was arrested following the addition of IPTG during the early exponential growth phase. Induction of YwaC expression by IPTG also severely decreased the intracellular GTP level and drastically altered the transcriptional profile in RIK1066 cells. Sucrose density gradient centrifugation analysis of the ribosomal fractions prepared from the IPTG‐induced RIK1066 cells revealed three peaks corresponding to 30S, 50S, and 70S ribosome particles, and also an extra peak. Electron microscope studies revealed that the extra peak fraction contained dimers of 70S ribosomes, which were similar to the Escherichia coli 100S ribosomes. Proteomic analysis revealed that the 70S dimer contained an extra protein, YvyD, in addition to those found in the 70S ribosome. Accordingly, strain resulting from the disruption of the yvyD gene in the RIK1066 cells was unable to form 70S dimers following IPTG induction, indicating that YvyD is required for the formation of these dimers in B. subtilis.

13C-metabolic flux analysis in heterologous cellulase production by Bacillus subtilis genome-reduced strain

The great potential of Bacillus subtilis to produce biomaterials would be further enhanced by the development of strains with deletions of non-essential genomic regions. Here, using stationary (13)C-metabolic flux analysis ((13)C-MFA), we investigated the metabolism during cellulase production by the genome-reduced B. subtilis strain MGB874. We transformed MGB874 and wild-type strains with the heterologous cellulase gene, and cultured these on a synthetic medium containing glucose as carbon source. The addition of glutamate and the genome reduction enhanced cellulase production, which led us to use (13)C-MFA to assess the effects of glutamate addition and gene deletions on metabolism. We found that there was a significant increase in the flux in the pentose phosphate (PP) pathway, whereas the fluxes of reactions from acetyl-CoA to α-ketoglutarate were repressed in the presence of glutamate. We hypothesize that the increase in the PP pathway flux was caused by the decrease of citrate synthase flux through the accumulation of glycolytic intermediates. Excess NADPH produced by the PP pathway may affect the increase in cellulase production. Furthermore, the fluxes on glycolysis and the acetate formation of the cellulase-producing wild-type strain were significantly larger than that of the cellulase-producing MGB874 strain when the strains were cultured with glucose and glutamate.

Enhanced Extracellular Production of Heterologous Proteins in Bacillus subtilis by Deleting the C-terminal Region of the SecA Secretory Machinery

In this study, we examined the effects of modifying the C-terminal region of the SecA protein on the production of heterologous proteins in Bacillus subtilis. SecA was selected as a candidate among the components of the Sec system due to its ability to interact directly with both the precursors and membrane translocases. A phylogenetic comparison demonstrated that the C-terminal region is not well conserved among eubacterial SecA proteins. The deletion of the 61 amino acids at the C-terminal region led to an 83% increase in extracellular alkaliphilic Bacillus sp. thermostable alkaline cellulase (Egl-237) activity. Moreover, the productivity of human interferon α (hIFN-α2b) was increased by 2.2-fold compared to the wild-type SecA, by deletion of these 61 amino acids. We indicated that the deletion of the C-terminal domain (CTD) of SecA enhanced the secretion of two different heterologous protein, Egl-237 and hIFN-α2b. This study provides a useful method to enhance the extracellular production of heterologous proteins in B. subtilis.

Improved production of secreted heterologous enzyme in Bacillus subtilis strain MGB874 via modification of glutamate metabolism and growth conditions

BackgroundThe Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular enzymes under batch fermentation conditions. We predicted that deletion of the gene for RocG, a bi-functional protein that acts as a glutamate dehydrogenase and an indirect repressor of glutamate synthesis, would improve glutamate metabolism, leading to further increased enzyme production. However, deletion of rocG dramatically decreased production of the alkaline cellulase Egl-237 in strain MGB874 (strain 874∆rocG).ResultsTranscriptome analysis and cultivation profiles suggest that this phenomenon is attributable to impaired secretion of alkaline cellulase Egl-237 and nitrogen starvation, caused by decreased external pH and ammonium depletion, respectively. With NH3-pH auxostat fermentation, production of alkaline cellulase Egl-237 in strain 874∆rocG was increased, exceeding that in the wild-type-background strain 168∆rocG. Notably, in strain 874∆rocG, high enzyme productivity was observed throughout cultivation, possibly due to enhancement of metabolic flux from 2-oxoglutarate to glutamate and generation of metabolic energy through activation of the tricarboxylic acid (TCA) cycle. The level of alkaline cellulase Egl-237 obtained corresponded to about 5.5 g l-1, the highest level reported so far.ConclusionsWe found the highest levels of production of alkaline cellulase Egl-237 with the reduced-genome strain 874∆rocG and using the NH3-pH auxostat. Deletion of the glutamate dehydrogenase gene rocG enhanced enzyme production via a prolonged auxostat fermentation, possibly due to improved glutamate synthesis and enhanced generation of metabolism energy.

Plant regeneration from patchouli protoplasts encapsulated in alginate beads

Mesophyll protoplasts were isolated from leaves of in vitro grown patchouli (Pogostemon cablin Benth.). The protoplasts were encapsulated in alginate beads, approximately 2–3×103 protoplasts per 25 μl bead. Successful colony formation was induced when the protoplast beads were inoculated into a liquid medium supplemented with 10-6 M NAA and 10-5 M BA. The frequency of colony formation was improved greatly by the inclusion of several beads per ml medium. To induce high colony formation for a single bead, it was essential to culture protoplasts in the presence of nurse beads containing actively-growing cells of the same species. Rapid regeneration of plants from protoplast-derived calluses was accomplished by a two-step culture procedure with liquid and then solid media. Gas-chromatographic analyses showed that regenerated plants produced an essential oil comprising a full-set of patchouli sesquiterpenes.

Intragenomic diversity of the V1 regions of 16S rRNA genes in high-alkaline protease-producing Bacillus clausii spp

Alkaliphilic Bacillus sp. strain KSM-K16, which produces high-alkaline M-protease, was characterized phenotypically, biochemically and genetically. This strain was identified as Bacillus clausii based on the results of taxonomic studies, including sequencing of the 16S rRNA gene and DNA-DNA hybridization. Seven rRNA operons in the genome were identified by pulsed-field gel electrophoresis. Sequencing of cloned 16S rRNA genes revealed two distinct types of variable region V1. Moreover, some cloned 16S rRNA genes in some of the reference strains of B. clausii had a V1 region of yet another type. The B. clausii strains could clearly be divided into at least two subgroups based on the frequencies of the types of cloned V1 sequence. Bacillus sp. strain KSM-K16 was found to be in a different phylogenetic position from other high-alkaline protease-producing strains of B. clausii.

Secretion of biologically-active human interferon-β by Bacillus subtilis.

Human interferon-β (hIFN-β) was used as a heterologous model protein to investigate the effects of the Bacillus subtilis AmyE propeptide and co-expression of PrsA in enhancing the secretion of heterologous proteins in B. subtilis. Secretion and activity of hIFN-β with AmyE propeptide increased by more than four-fold compared to that without AmyE propeptide. Moreover, under conditions of co-expressed PrsA, the secretion production and activity of hIFN-β with AmyE propeptide increased by more than 1.5-fold. AmyE propeptide and co-expression of PrsA thus have an additive effect on enhancing the production of the hIFN-β in B. subtilis.

High external pH enables more efficient secretion of alkaline α-amylase AmyK38 by Bacillus subtilis

BackgroundBacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Here, we investigated the suitability of strain MGB874 for the production of α-amylase, which was anticipated to provoke secretion stress responses involving the CssRS (Control secretion stress Regulator and Sensor) system.ResultsCompared to wild-type strain 168, the production of a novel alkaline α-amylase, AmyK38, was severely decreased in strain MGB874 and higher secretion stress responses were also induced. Genetic analyses revealed that these phenomena were attributable to the decreased pH of growth medium as a result of the lowered expression of rocG, encoding glutamate dehydrogenase, whose activity leads to NH3 production. Notably, in both the genome-reduced and wild-type strains, an up-shift of the external pH by the addition of an alkaline solution improved AmyK38 production, which was associated with alleviation of the secretion stress response. These results suggest that the optimal external pH for the secretion of AmyK38 is higher than the typical external pH of growth medium used to culture B. subtilis. Under controlled pH conditions, the highest production level (1.08 g l-1) of AmyK38 was obtained using strain MGB874.ConclusionsWe demonstrated for the first time that RocG is an important factor for secretory enzyme production in B. subtilis through its role in preventing acidification of the growth medium. As expected, a higher external pH enabled a more efficient secretion of the alkaline α-amylase AmyK38 in B. subtilis. Under controlled pH conditions, the reduced-genome strain MGB874 was demonstrated to be a beneficial host for the production of AmyK38.