Engineering Streptomyces clavuligerus deacetoxycephalosporin C synthase for optimal ring expansion activity toward penicillin G

The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k(cat)/K(m) ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k(cat)/K(m) values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k(cat)/K(m) ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.     

Clinical and Prognostic Implications of Roundabout 4 (Robo4) in Adult Patients with Acute Myeloid Leukemia

Background

Robo4 is involved in hematopoietic stem/progenitor cell homeostasis and essential for tumor angiogenesis. Expression of Robo4 was recently found in solid tumors and leukemia stem cells. However, the clinical implications of Robo4 expression in patients with acute myeloid leukemia (AML) remain unclear.

Methods

We investigated the clinical and prognostic relevance of mRNA expression of Robo4 in bone marrow (BM) mononuclear cells from 218 adult patients with de novo AML. We also performed immunohistochemical staining to assess the Robo4 protein expression in the BM biopsy specimens from 30 selected AML patients in the cohort.

Results

Higher Robo4 expression was closely associated with lower white blood cell counts, expression of HLA-DR, CD13, CD34 and CD56 on leukemia cells, t(8;21) and ASXL1 mutation, but negatively correlated with t(15;17) and CEBPA mutation. Compared to patients with lower Robo4 expression, those with higher expression had significantly shorter disease-free survival (DFS) and overall survival (OS). This result was confirmed in an independent validation cohort. Furthermore, multivariate analyses showed that higher Robo4 expression was an independent poor prognostic factor for DFS and OS in total cohort and patients with intermediate-risk cytogenetics, irrespective of age, WBC count, karyotype, and mutation status of NPM1/FLT3-ITD, and CEBPA.

Conclusions

BM Robo4 expression can serve as a new biomarker to predict clinical outcomes in AML patients and Robo4 may serve as a potential therapeutic target in patients with higher Robo4 expression.     

Directed Evolution of Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Enhancement of Penicillin G Expansion

The deacetoxycephalosporin C synthase from Streptomyces clavuligerus was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the k_cat/K_m ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a k_cat/K_m ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering.     

Resveratrol Modulates Mitochondria Dynamics in Replicative Senescent Yeast Cells

Mitochondria form a reticulum network dynamically fuse and divide in the cell. The balance between mitochondria fusion and fission is correlated to the shape, activity and integrity of these pivotal organelles. Resveratrol is a polyphenol antioxidant that can extend life span in yeast and worm. This study examined mitochondria dynamics in replicative senescent yeast cells as well as the effects of resveratrol on mitochondria fusion and fission. Collecting cells by biotin-streptavidin sorting method revealed that majority of the replicative senescent cells bear fragmented mitochondrial network, indicating mitochondria dynamics favors fission. Resveratrol treatment resulted in a reduction in the ratio of senescent yeast cells with fragmented mitochondria. The readjustment of mitochondria dynamics induced by resveratrol likely derives from altered expression profiles of fusion and fission genes. Our results demonstrate that resveratrol serves not only as an antioxidant, but also a compound that can mitigate mitochondria fragmentation in replicative senescent yeast cells.     

Production and Further Characterization of an Alkaline Elastase Produced by Alkalophilic Bacillus Strain Ya-B

The characteristics of the obligate alkalophilic Bacillus sp. strain Ya-B, which produces alkaline elastase extracellularly, were examined. This strain grew at pH 7.0 only in the presence of 1% or more NaCl. Its fatty acid distribution pattern was similar to that of other Bacillus species in which iso-C₁₅ and anteiso-C₁₅ were the most abundant fatty acids. About 120 mg of enzyme was recovered from 1 liter of culture broth in a medium (pH 10.1) containing mainly glucose, soymeal, and glycerol. The antiserum against this enzyme did not recognize microbial proteinases, such as subtilisins, but reacted with proteinase C, which was purified from commercial pronase. Chemical modification studies revealed that certain histidine and tyrosine residues might be involved in the enzyme activity. This enzyme underwent a partial unfolding at pHs higher than 12.0, as indicated by the circular dichroism study.     

Effect of triton X-100 on alkaline lipase production by Pseudomonas pseudoalcaligenes F-111

Summary An extracellular alkaline lipase was evaluated from Pseudomonas pseudoalcaligenes F-111, which was isolated from soil using a rhodamine B agar plate with Na₂CO₃. For enzyme production, olive oil, soymeal, triton X-100 and sodium ion were found to be essential. The addition of triton X-100 increased the alkaline lipase by 50- fold. The effect of additives as well as the development of suitable culture conditions for lipase production is discussed.     

Prokaryotic expression and action mechanism of antimicrobial LsGRP1C recombinant protein containing a fusion partner of small ubiquitin-like modifier

Applied Microbiology and Biotechnology - Antimicrobial peptides (AMPs) are peptides exhibiting broad-spectrum antimicrobial activities and considered as potential therapeutic agents. LsGRP1C, a...     

Characterization of a Novel PepF-Like Oligopeptidase Secreted by Bacillus amyloliquefaciens 23-7A

An oligopeptidase from Bacillus amyloliquefaciens 23-7A was characterized along with its biochemical activities and structural gene. The protein's amino acid sequence and enzymatic activities were similar to those of other bacterial PepFs, which belong to metallopeptidase family M3. While most bacterial PepFs are cytoplasmic endopeptidases, the identified PepF_Ba oligopeptidase is a secreted protein and may facilitate the process of sporulation.     

Trypsin-induced proteome alteration during cell subculture in mammalian cells

Background

It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions.

Methods

In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting.

Results

36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization.

Conclusions

In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.