Kazuhisa Sawada

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.

Purification and properties of a high-molecular-weight, alkaline exopolygalacturonase from a strain of Bacillus

1An exopolygalacturonase [exo-PG; poly (1,4-alpha-D-galacturonide) digalacturonohydrolase, EC 3.2.1.82] was found in a culture of Bacillus sp. strain KSM-P576. The purified exo-PG had a molecular weight of approximately 115,000 and an isoelectric point of pH 4.6. The N-terminal amino acid sequence was Thr-Glu-Val-Ser-Pro-Lys-Ser-Pro-Ala-Ser-Pro-Val. Maximum activity toward polygalacturonic acid (PGA) was observed at 55 degrees C and pH 8.0 in 100 mM Tris-HCl buffer. The exo-PG was quite stable in various pH buffers between pH 6 and 12 when incubated at 30 degrees C for 1 h. Mg(2+,) Mn(2+,) Pd(2+) and Ca(2+) ions stimulated the enzyme activity. The exo-PG released digalacturonic acid from PGA, tri-, tetra-, and penta-galacturonic acids. The apparent K(m) values for oligogalacturonic acids were almost identical, and k(cat) values increased with the chain length of the substrates.

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.

A new high-alkaline and high-molecular-weight pectate lyase from a Bacillus isolate: enzymatic properties and cloning of the gene for the enzyme.

A pectate lyase (Pel; pectate transeliminase: EC4.2.2.2.), designated Pel-15H, was found in an alkaline culture of Bacillus sp. strain KSM-P15 and purified to homogeneity by sequential column chromatographies. The molecular weight of the enzyme determined by SDS-polyacrylamide gel electrophoresis was approximately 70,000 and the pI was around pH 4.6. Pel-15H randomly trans-eliminated polygalacturonate in the presence of Ca2+ ions, and the maximum activity was observed at pH 11.5 and at 55°C in glycine-NaOH buffer. The gene for Pel-15H was cloned and sequenced, and the structural gene contained a 2,031-bp open reading frame that encoded 677 amino acids including a possible 28-amino-acid signal sequence. The mature enzyme (649 amino acids, molecular weight 69,550) showed very low similarity to Pels from Bacillus with 12.7-18.2% identity. Interestingly, part of the amino acid sequence of Pel-15H had fairly high similarity only to an N-terminal half of PelL and a C-terminal half of PelX from Erwinia chrysanthemi 3937, and a C-terminal half of PelX from E. chrysanthemi EC16 (approximately 35% identity for all).

Molecular cloning and sequencing of the gene encoding an exopolygalacturonase of a Bacillus isolate and properties of its recombinant enzyme.

An exopolygalacturonase (exo-PGase; EC 3.2.1.82) was found in the culture broth of a Bacillus isolate. The gene encoding the exo-PGase, pehK, was cloned by polymerase chain reaction using mixed primers designed from N-terminal and internal amino acid (aa) sequences of the enzyme (PehK). The determined nucleotide (nt) sequence of pehK revealed a 2940 bp open reading frame (980 aa) that encoded a putative signal sequence (27 aa) and a mature protein (953 aa; 103810 Da). The recombinant enzyme was purified to homogeneity from a culture broth of Bacillus subtilis harboring a pehK-containing plasmid. It had a molecular mass of 105 kDa and a pI value of 5.0. The maximum activity was observed at pH 8 and 55 degrees C in Tris-HCl buffer. The degradation products from polygalacturonic or oligogalacturonic acids were digalacturonic acid, like the exo-PGases, PehX of Erwinia chrysanthemi and PehB of Ralstonia solanacearum. The deduced aa sequence of PehK exhibited moderate homology to those of PehX and PehB with approx. 30% identity for both. High homology was observed in a suitably aligned internal region of the three enzymes (65% identity), and some of the conserved aa residues appeared to form the catalytic core of the enzymes.

Bifunctional pectinolytic enzyme with separate pectate lyase and pectin methylesterase domains from an alkaliphilic Bacillus

The gene for a novel enzyme having pectate lyase (Pel) and pectin methylesterase (Pme) activities found in the genome of an alkaliphilic Bacillus, KSM-P358, was sequenced. The structural gene contained a long open reading frame of 4314 bp corresponding to a 32-amino-acid signal peptide and a 1406-amino-acid mature enzyme with a molecular mass of 155,666. The mature enzyme contained two uncontiguous regions at amino acids 800–1051 and 1105–1406 exhibiting homology to a Pel from a Bacillus strain with 43.7% and a Pme from Erwinia chrysanthemi with 33.4% identity, respectively. The recombinant enzyme expressed in Bacillus subtilis cells had a molecular mass of 160 kDa and exhibited pH and temperature optima for Pel activity of 10 and 40 °C and those for the Pme activity of 8.5 and 45 °C. The genes for the domains for the Pel and Pme could be separately expressed in Escherichia coli cells, and the catalytic properties of the respective protein fragments were essentially identical to those of the intact enzyme. This novel enzyme is ‘mosaic’ in that some regions before the two domains exhibited limited but substantial similarity to some regions of carbohydrate-active enzymes. The regions contained parts of a gene for Pels from a Bacillus sp. and Pseudomonas fluorescens, a xylanase from P. fluorescens subsp. cellulosa, a 1,4-β-mannanase from a Pyromyces sp., a putative Pel from a Streptomyces coelicolor cosmid, a (1,3-1,4)-β-glucanase from Clostridium thermocellum.

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.