Production and molecular weight variation of poly-γ-glutamic acid using a recombinant Bacillus subtilis with various pgs-component ratios.

Poly-γ-glutamic acid (PGA) has been of interest as a sustainable biopolymer in industrial applications. PGA biosynthesis in Bacillus subtilis is catalyzed by a transmembrane protein complex comprising PgsB, PgsC, and PgsA. To determine the Pgs component responsible for PGA overproduction, we constructed recombinants in which the promoter of the host-derived pgs gene was replaced with another host-derived gene promoter. These recombinants were then transformed using high-copy-number plasmids with various pgs-gene combinations to enhance Pgs component in different ratios. Subsequently, PGA production was investigated in batch cultures with l-glutamate supplemented medium. The recombinant strain enhanced with pgsB alone significantly overproduced PGA (maximum production 35.8 gL-1) than either the pgsC- or pgsA-enhanced strain. The molecular weight of the PGA produced with pgsB-enhanced strain was also greater than the pgsC- or pgsA-enhanced strain (approximately 10-fold). Hence, PgsB enhancement alone contributes to PGA overproduction with increased molecular weight.

Prognostic impact of polypharmacy and discharge medications in octogenarians and nonagenarian patients with acute heart failure.

With the increasing frequency of heart failure (HF) in elderly patients, polypharmacy has become a major concern owing to its adverse outcomes. However, reports on the clinical impact of polypharmacy and discharge medications in hospitalized super-aged patients with acute HF are rare. Data from 682 patients aged 80 years or older, hospitalized for treating acute HF, were analyzed. We recorded the number of medications at discharge and classified them into three groups: HF, non-HF cardiovascular, and non-cardiovascular medications. We investigated the correlation of polypharmacy, defined as daily administration of 10 or more medications at discharge, and the use of discharge medications with post-discharge prognosis. Polypharmacy was recorded in 24.3% of enrolled patients. Polypharmacy was not an independent predictor of all-cause mortality, the incidence of cardiac-related death, or HF-associated rehospitalization; however, the number of non-cardiovascular medications, multiple usage of potentially inappropriate medications, use of mineralocorticoid receptor antagonists, and doses of loop diuretics were associated with poor prognosis. Polypharmacy was significantly associated with higher mortality in patients with Barthel index ≥ 60 at discharge; hence, physical function at discharge was useful for the stratification of prognostic impacts of polypharmacy. The current study demonstrated that polypharmacy was not essentially associated with poor prognosis in super-aged patients with acute HF. Appropriate medications that consider the patient's physical function, rather than polypharmacy itself, are important for the management of HF.

Poly-L-gamma-glutamic acid production by recombinant Bacillus subtilis without pgsA gene.

Poly-gamma-glutamic acid (PGA) is a promising bio-based polymer that shares many functions with poly (acrylic acid) and its derivatives. Thus, technologies for efficient production and molecular size control of PGA are required to expand the application of this useful biopolymer. In Bacillus strains, PGA is synthesized by the PgsBCA protein complex, which is encoded by the pgsBCA gene operon, otherwise is known as ywsC and ywtAB operons and/or capBCA operon. Hence, we investigated responsible components of the PgsBCA complex in B. subtilis for over-production of PGA. In particular, we constructed genomic pgsBCA gene-deletion mutants of B. subtilis. And also, we assembled high copy-number plasmids harboring σA-dependent promoter, leading to high-level expression of all combinations of pgsBCA, pgsBC, pgsBA, pgsCA, pgsB, pgsC, and/or pgsA genes. Subsequently, PGA production of the transformed B. subtilis mutant was determined in batch fermentation using medium supplemented with L-glutamate. PGA production by the transformants introduced with pgsBC genes (lacking the genomic pgsBCA genes) was 26.0 ± 3.0 g L-1, and the enantiomeric ratio of D- and L-glutamic acid (D/L-ratio) in the produced PGA was 5/95. In contrast, D/L-ratio of produced PGA by the transformants introduced with pgsBCA genes (control strains) was 75/25. In conclusion, B. subtilis without pgsA gene could over-produce PGA with an L-rich enantiomeric ratio.

Combined effect of improved cell yield and increased specific productivity enhances recombinant enzyme production in genome-reduced Bacillus subtilis strain MGB874.

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.

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.

Bacillus subtilis AprX involved in degradation of a heterologous protein during the late stationary growth phase.

In Bacillus subtilis, extracellular protease-deficient mutants have been used in attempts to increase the productivity of heterologous proteins. We detected protease activity of AprX using protease zymography in the culture medium at the late stationary growth phase. An alpha-amylase-A522-PreS2 hybrid protein, in which the PreS2 antigen of human hepatitis B virus (HBV) is fused with the N-terminal 522-amino-acid polypeptide of B. subtilis alpha-amylase, has been produced in multiple-protease-deficient mutants. The B. subtilis KA8AX strain, which is deficient in eight extracellular proteases and AprX, did not show the proteolysis of alpha-amylase-A522-PreS2 in the late stationary growth phase. Moreover, the production of alpha-amylase-A522-PreS2 was about 80 mg/l, which was eight times higher than that by the KA8AX strain previously reported. In addition, we showed the degradation of the heterologous protein by AprX that leaked to the culture medium (probably caused by cell lysis) during the late stationary growth phase.

Endoglucanases from Paenibacillus spp. form a new clan in glycoside hydrolase family 5.

Endoglucanase (Egl)-producing bacteria from soil samples were screened using insoluble cellulosic substrates as sole carbon sources at alkaline pH (pH 9-10). Four Egls with Avicelase activity at alkaline pH were found in the culture broth of each isolate. The Egl genes of the isolates (all Paenibacillus spp.) were shotgun cloned and sequenced-all had a 1752bp open reading frame (584 amino acids) with a putative signal sequence (33 amino acids), and encoded mature enzymes of 551 amino acids (58,360-58,672Da). The mature enzymes showed a high degree of similarity to each other (>93% identity), with the next closest similarity to Egl3a of a patented strain of Paenibacillus lautus NCIMB 40250 (81.5-87.3% identity). These enzymes showed low similarity to other known Egls with less than 50% identity. A representative recombinant enzyme degraded lichenan, carboxymethylcellulose (CMC), glucomannan, acid or alkaline swollen celluloses, and microcrystalline cellulose (Avicel). The optimal pH and temperature of the recombinant enzyme for degrading CMC and Avicel were pH 6.0-8.5 and 45-55 degrees C, respectively. Egls belong to glycoside hydrolase family 5 and form a distinct clan based on the phylogenetic analysis of their amino acid sequences.