Methyltransferase Contingencies in the Pathway of Everninomicin D Antibiotics and Analogues

Everninomicins are orthoester oligosaccharide antibiotics with potent activity against multidrug-resistant bacterial pathogens. Everninomicins act by disrupting ribosomal assembly in a distinct region in comparison to clinically prescribed drugs. We employed microporous intergeneric conjugation with Escherichia coli to manipulate Micromonospora for targeted gene-replacement studies of multiple putative methyltransferases across the octasaccharide scaffold of everninomicin effecting the A₁, C, F, and H rings. Analyses of gene-replacement and genetic complementation mutants established the mutability of the everninomicin scaffold through the generation of 12 previously unreported analogues and, together with previous results, permitted assignment of the ten methyltransferases required for everninomicin biosynthesis. The in vitro activity of A₁- and H-ring-modifying methyltransferases demonstrated the ability to catalyze late-stage modification of the scaffold on an A₁-ring phenol and H-ring C-4’ hydroxy moiety. Together these results establish the potential of the everninomicin scaffold for modification through mutagenesis and in vitro modification of advanced biosynthetic intermediates.     

Effect of the cultivation mode on red pigments production from Monascus ruber

In submerged cultures performed in chemically defined fermentation medium containing glucose and glutamate, the growth and production of water‐soluble red pigments and citrinin by the filamentous fungus Monascus ruber were studied under various carbon/nitrogen (C/N) ratios. The specific production of the red pigments was optimal at a glucose/glutamate ratio of about 10 and then steadily decreased at higher C/N ratio. In contrast, the production of the mycotoxin increased with increased C/N with an optimum in the range of 30–45. In a fed‐batch mode, it was also found that the production of pigments was not favoured in fed‐batch mode by feeding the medium with glucose while keeping the C/N ratio lower than 10. This low production likely resulted from concurrent high accumulation of L‐malic acid that was reported to inhibit this production. In contrast, this mode of cultivation was rather favourable for the production of the mycotoxin.     

Crystallization of progesterone polymorphs using polymer-induced heteronucleation (PIHn) method

Progesterone is a natural hormone steroid used in humans for several treatments and in livestock for artificial insemination, which exhibits two polymorphic forms at ambient conditions: form 1 and form 2. Form 2 is metastable and more soluble than form 1; however, it is not suitable to use as powder raw material because it transforms into form 1 by the effects of grinding. A polymorphic screening of progesterone based on polymer-induced heteronucleation method was performed as an alternative to prepare the metastable form. Polyvinyl alcohol, hydroxypropyl methylcellulose (HPMC), dextran, gelatin, polyisoprene (PI) and acrylonitrile-butadiene (NBR) copolymer were used. Crystals were prepared from 0.5, 10 and 40?mg/mL solutions in acetone at room temperature by solvent evaporation. The samples were characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), scanning electron microcopy and attenuated total reflectance infrared Fourier transform spectroscopy. Form 1 was nucleated from 40?mg/mL solutions on the six polymers and from 10?mg/mL solutions on PI and NBR. The mixture of form 1 and form 2 was obtained from 10?mg/mL solution on HPMC, dextran and gelatin and from 0.5?mg/mL solution crystallizations. Therefore, the polymeric devices, which crystallized the metastable and more soluble polymorph (2) of progesterone, would be a promissory alternative for the pharmaceutical applications.     

Towards optimization of the osmotic drying process of alumina-gelatin objects: Regression analysis and verification

In this study, further analysis of the osmotic drying process was conducted to identify the optimum combination of parameters for drying rectangular alumina-gelatin beams. This study was designed to determine the effect of three variables related to the osmotic drying process (osmotic pressure, molecular weight, and immersion time) on the interaction between the liquid desiccant and the submerged alumina-gelatin samples. The water loss from the alumina-gelatin samples was positively correlated with the molecular weight, osmotic pressure, and immersion time. Up to 40% by weight of the initial water content was removed during the osmotic drying process. The samples also experienced solids gain due to the counterflow of solute from the liquid desiccant. The least amount of solids gain resulted from drying for the shortest immersion time at low osmotic pressure and high molecular weight. Evidence of possible interactions between variables was noted for the sintered density metric. Statistical methods were used to form regression equations for the measured responses (water loss, solids gain, bulk density). A verification experiment was conducted to compare the experimental outcomes to the predicted outcomes. The responses were simultaneously optimized to identify the combination of variable settings required to meet specified goals. In order to maximize water loss, minimize solids gain, and maximize bulk density, the ceramic-gelatin object should be immersed for approximately 60?min in an aqueous solution of 100,000?g/mol poly(ethylene oxide) at an osmotic pressure of 2.50?MPa. These values are valid for the range of parameter settings tested and the sample fabrication and drying methods used.