Development of the computational antibiotic screening platform (CLASP) to aid in the discovery of new antibiotics

The CLASP is a freely-distributed script for screening potential drug molecules through bacterial outer membrane porins. The automated scripts provide comprehensive thermodynamic and kinetic output data within a few hours of wall-clock time.

Homologous Overexpression of Genes in Cordyceps militaris Improves the Production of Polysaccharide

Background Cordyceps polysaccharides have been used around the globe for its bioactivity for millennia. However, the study and medicinal of Cordyceps militaris polysaccharides has been hampered by the low in natural abundances. Recently, the genetic engineered C. militaris developed for production of exopolysaccharides (EPS) had received extensive attention.Results In this study, based on the biosynthetic pathway and metabolization mechanism of exopolysaccharides, the crucial biosynthetic genes of Cordyceps polysaccharides were introduced by Agrobacterium transformation to provide a high flux of EPS. 21 mutants of C. militaris were identified through antibiotic screening and DNA sequencing. The maximum yield of EPS produced by mutant CM-pgm-H was 4.63 ± 0.23 g/L, while the yield of wild-type strain was 3.43 ± 0.26 g/L. And the data obtained in the present study indicated that the yield of EPS produced by the engineered strain treated with co-overexpression of phosphoglucomutase and UDP-glucose 6-dehydrogenase genes achieved 6.11 ± 0.21 g/L, which was increased by 78.13% compared with the wild-type strain.Conclusions CM-pgm-H obtained the highest EPS content than that of mutants glucokinase, UDP-glucose pyrophosphorylase, UDP-glucose 6-dehydrogenase. It indicated that the content of protein phosphoglucomutase was the most critical influencing factor on the CP production in C.militaris. Furthermore, the EPS production of CM-ugdh-pgm-M was significantly improved 1.78-fold by co-overexpression. It anticipated that our engineering strategies will play an important role in the development of C. militaris for sustainable production of Cordyceps polysaccharides.

Activity-Related Conformational Changes in d,d-Carboxypeptidases Revealed by In Vivo Periplasmic Förster Resonance Energy Transfer Assay in Escherichia coli

ABSTRACT One of the mechanisms of β-lactam antibiotic resistance requires the activity of d,d-carboxypeptidases (d,d-CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development. The activity of PG-synthesizing enzymes is often correlated with their association with other proteins. The PG layer is maintained in the periplasm between the two membranes of the Gram-negative cell envelope. Because no methods existed to detect in vivo interactions in this compartment, we have developed and validated a Förster resonance energy transfer assay. Using the fluorescent-protein donor-acceptor pair mNeonGreen-mCherry, periplasmic protein interactions were detected in fixed and in living bacteria, in single samples or in plate reader 96-well format. We show that the d,d-CPases PBP5, PBP6a, and PBP6b of Escherichia coli change dimer conformation between resting and active states. Complementation studies and changes in localization suggest that these d,d-CPases are not redundant but that their balanced activity is required for robust PG synthesis. IMPORTANCE The periplasmic space between the outer and the inner membrane of Gram-negative bacteria contains many essential regulatory, transport, and cell wall-synthesizing and -hydrolyzing proteins. To date, no assay is available to determine protein interactions in this compartment. We have developed a periplasmic protein interaction assay for living and fixed bacteria in single samples or 96-well-plate format. Using this assay, we were able to demonstrate conformation changes related to the activity of proteins that could not have been detected by any other living-cell method available. The assay uniquely expands our toolbox for antibiotic screening and mode-of-action studies. IMPORTANCE The periplasmic space between the outer and the inner membrane of Gram-negative bacteria contains many essential regulatory, transport, and cell wall-synthesizing and -hydrolyzing proteins. To date, no assay is available to determine protein interactions in this compartment. We have developed a periplasmic protein interaction assay for living and fixed bacteria in single samples or 96-well-plate format. Using this assay, we were able to demonstrate conformation changes related to the activity of proteins that could not have been detected by any other living-cell method available. The assay uniquely expands our toolbox for antibiotic screening and mode-of-action studies.