Takeko Kodama

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.

The Bacillus subtilis yabG gene is transcribed by SigK RNA polymerase during sporulation, and yabG mutant spores have altered coat protein composition.

The expression of six novel genes located in the region from abrB to spoVC of the Bacillus subtilis chromosome was analyzed, and one of the genes, yabG, had a predicted promoter sequence conserved among SigK-dependent genes. Northern blot analysis revealed that yabG mRNA was first detected from 4 h after the cessation of logarithmic growth (T(4)) in wild-type cells and in a gerE36 (GerE(-)) mutant but not in spoIIAC (SigF(-)), spoIIGAB (SigE(-)), spoIIIG (SigG(-)), and spoIVCB (SigK(-)) mutants. The transcription start point was determined by primer extension analysis; the -10 and -35 regions are very similar to the consensus sequences recognized by SigK-containing RNA polymerase. Inactivation of the yabG gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yabG spores in L-alanine and in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was also the same as that of wild-type spores. On the other hand, the protein preparation from yabG spores included 15-, 18-, 21-, 23-, 31-, 45-, and 55-kDa polypeptides which were low in or not extracted from wild-type spores under the same conditions. We determined their N-terminal amino acid sequence and found that these polypeptides were CotT, YeeK, YxeE, CotF, YrbA (31 and 45 kDa), and SpoIVA, respectively. The fluorescence of YabG-green fluorescent protein fusion produced in sporulating cells was detectable in the forespores but not in the mother cell compartment under fluorescence microscopy. These results indicate that yabG encodes a sporulation-specific protein which is involved in coat protein composition in B. subtilis.

Identification and characterization of a novel polysaccharide deacetylase C (PdaC) from Bacillus subtilis

Background: Peptidoglycan modification is a very important process that bacteria use to adjust to various environmental conditions. Results: B. subtilis pdaC was associated with lysozyme sensitivity. Surprisingly PdaC is able to deacetylate N-acetylmuramic acid but not N-acetylglucosamine in peptidoglycan. But chitin oligomers were deacetylated by PdaC. Conclusion: PdaC is a unique enzyme exhibiting two different deacetylase activities. Significance: Novel deacetylase is characterized. Cell wall metabolism and cell wall modification are very important processes that bacteria use to adjust to various environmental conditions. One of the main modifications is deacetylation of peptidoglycan. The polysaccharide deacetylase homologue, Bacillus subtilis YjeA (renamed PdaC), was characterized and found to be a unique deacetylase. The pdaC deletion mutant was sensitive to lysozyme treatment, indicating that PdaC acts as a deacetylase. The purified recombinant and truncated PdaC from Escherichia coli deacetylated B. subtilis peptidoglycan and its polymer, (-GlcNAc-MurNAc[-l-Ala-d-Glu]-)n. Surprisingly, RP-HPLC and ESI-MS/MS analyses showed that the enzyme deacetylates N-acetylmuramic acid (MurNAc) not GlcNAc from the polymer. Contrary to Streptococcus pneumoniae PgdA, which shows high amino acid sequence similarity with PdaC and is a zinc-dependent GlcNAc deacetylase toward peptidoglycan, there was less dependence on zinc ion for deacetylation of peptidoglycan by PdaC than other metal ions (Mn2+, Mg2+, Ca2+). The kinetic values of the activity toward B. subtilis peptidoglycan were Km = 4.8 mm and kcat = 0.32 s−1. PdaC also deacetylated N-acetylglucosamine (GlcNAc) oligomers with a Km = 12.3 mm and kcat = 0.24 s−1 toward GlcNAc4. Therefore, PdaC has GlcNAc deacetylase activity toward GlcNAc oligomers and MurNAc deacetylase activity toward B. subtilis peptidoglycan.

A Novel Small Protein of Bacillus subtilis Involved in Spore Germination and Spore Coat Assembly

Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T5 of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine–SDS–PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.

Approaches for Improving Protein Production in Multiple Protease-Deficient Bacillus subtilis Host Strains

Bacillus subtilis is a Gram-positive, nonpathogenic organism which is widely used as a host for enzyme production, due to its ability to secrete large amounts of proteins into the growth medium (Simonen et al., 1993; Westers et al., 2004). The secretion of a target protein leads to the natural separation of the product from cell components, which simplifies downstream processing of the protein. Accordingly, there has been a great deal of research performed regarding protein production in B. subtilis (Simonen et al., 1993; Westers et al., 2004). Nevertheless, the yields of heterologous protein obtained from this organism are often insufficient (Harwood, 1992). Several bottlenecks in the B. subtilis secretion pathway have been reported, including poor targeting to the translocase, degradation of the secretory protein, and incorrect folding (Westers et al., 2004). One of the major bottlenecks involves the degradation of the produced protein by extracellular proteases; therefore, inactivation of extracellular proteases is essential for improvement of protein production with B. subtilis as the host.

Approaches for Improving Protein Production by Cell Surface Engineering

Bacillus subtilis is an attractive host organism because it has a naturally high secretory capacity and exports proteins directly into the extracellular medium. Secreted proteins emerge from translocation to the compartment between the cell wall and cytoplasmic membrane. It has been reported that the increased net negative charge of the cell wall is involved in protein folding and stability at the cytoplasmic membrane–cell wall interface of B. subtilis. In Escherichia coli, Sec translocase requires a functional interaction with the membrane acidic lipids such as phosphatidylglycerol (PG) and cardiolipin (CL). In Sect. 13.1, we describe altered compositions of anionic polymers on the cell surface and cell membrane lipid of B. subtilis, which is improved for protein secretion. Inactivation of extracellular proteases is essential for improvement of secreted proteins with B. subtilis as a host. Previously we reported that the decrease of extracellular protease amounts in B. subtilis mutants led to the stabilization of autolysins, making the cells more prone to lysis (Kodama et al., J Biosci Bioeng 103:13–21, 2007). In Sect. 13.2, we describe the improvement process for protein production from the aspect of prevention of cell lysis.

Creation of Novel Technologies for Extracellular Protein Production Toward the Development of Bacillus subtilis Genome Factories

Bacillus subtilis has been widely used for the industrial production of useful proteins because of its high protein secretion ability and safety. We focused on genome reduction as a new concept for enhancing production of recombinant enzymes in B. subtilis cells based on detailed analysis of the genome mechanism. First, we reported that a novel B. subtilis strain, MGB874, depleted 20.7 % of the genomic sequence of the wild type by rationally designed deletions to create simplified cells for protein production. When compared with wild-type cells, the productivity of cellulase and protease from transformed plasmids harboring the corresponding genes was markedly enhanced. These results indicate that a bacterial factory specializing in the production of substances can be constructed by deleting the genomic regions unimportant for growth and substance production from B. subtilis. Second, deletion of the rocDEF-rocR region, which is involved in arginine degradation, was found to contribute to the improvement of enzyme production in strain MGB874. The present study indicated that our results demonstrated the effectiveness of a synthetic genomic approach with reduction of genome size to generate novel and useful bacteria for industrial uses. Furthermore, the design of the changes in the transcriptional regulatory network of the nitrogen metabolic pathway in B. subtilis cells could facilitate the generation of improved industrial protein production.