Advanced Mutasynthesis Studies on the Natural α‐Pyrone Antibiotic Myxopyronin from Myxococcus fulvus

Myxopyronin is a natural α‐pyrone antibiotic from the soil bacterium Myxococcus fulvus Mx f50. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by binding to a part of the enzyme not targeted by the clinically used rifamycins. This mode of action makes myxopyronins promising molecules for the development of novel broad‐spectrum antibacterials. We describe the derivatization of myxopyronins by an advanced mutasynthesis approach as a first step towards this goal. Site‐directed mutagenesis of the biosynthetic machinery was used to block myxopyronin biosynthesis at different stages. The resulting mutants were fed with diverse precursors that mimic the biosynthetic intermediates to restore production. Mutasynthon incorporation and production of novel myxopyronin derivatives were analyzed by HPLC‐MS/MS. This work sets the stage for accessing numerous myxopyronin derivatives, thus significantly expanding the chemical space of f α‐pyrone antibiotics.     

Structure–Activity Relationships of 2‐Sufonylpyrimidines as Quorum‐Sensing Inhibitors to Tackle Biofilm Formation and eDNA Release of Pseudomonas aeruginosa

Drug‐resistant Pseudomonas aeruginosa (PA) strains are on the rise, making treatment with current antibiotics ineffective. Hence, circumventing resistance or restoring the activity of antibiotics by novel approaches is of high demand. Targeting the Pseudomonas quinolone signal quorum sensing (PQS‐QS) system is an intriguing strategy to abolish PA pathogenicity without affecting the viability of the pathogen. Herein we report the structure–activity relationships of 2‐sulfonylpyrimidines, which were previously identified as dual‐target inhibitors of the PQS receptor PqsR and the PQS synthase PqsD. The SAR elucidation was guided by a combined approach using ligand efficiency and ligand lipophilicity efficiency to select the most promising compounds. In addition, the most effective inhibitors were rationally modified by the guidance of QSAR using Hansch analyses. Finally, these inhibitors showed the capacity to decrease biofilm mass and extracellular DNA, which are important determinants for antibiotic resistance.     

Renewable linear alpha olefins by selective ethenolysis of decarboxylated unsaturated fatty acids

A two‐step concept for the production of linear alpha olefins from biomass is reported. As a starting material an internally unsaturated C17 alkene was used, which was obtained by the decarboxylation of oleic acid. Here, we report on the ethenolysis of this bio‐based product, using commercially available metathesis catalysts. The desired alpha olefin products, 1‐nonene and 1‐decene, were obtained in excellent yield (96%) and selectivity (96%). Practical applications: The two‐step conversion described in this contribution, starting from unsaturated fatty acids, provides a method for the production of industrially important linear alpha olefins. These valuable products are widely used as starting materials for the production of surfactants and polymers such as linear low density polyethylene (LLDPE).     

Direct Cloning, Genetic Engineering, and Heterologous Expression of the Syringolin Biosynthetic Gene Cluster in E. coli through Red/ET Recombineering

The reconstruction of a natural product biosynthetic pathway from bacteria in a vector and subsequent heterologous expression in a technically amenable microbial system represents an efficient alternative to empirical traditional methods for functional discovery, yield improvement, and genetic engineering to produce "unnatural" derivatives. However, the traditional cloning procedure based on genomic library construction and screening are complicated due to the large size (>10 kb) of most biosynthetic pathways. Here, we describe the direct cloning of a partial syringolin biosynthetic gene cluster (sylCDE, 19 kb) from a digested genomic DNA mixture of Pseudomonas syringae into a plasmid in which sylCDE is under the control of an inducible promoter by one step linear-plus-linear homologous recombination (LLHR) in Escherichia coli. After expression in E. coli GB05-MtaA, two new syringolin derivatives were discovered. The complete syringolin gene cluster was assembled by addition of sylAB and exchange of a synthetic bidirectional promoter against the native promoter to drive sylB and sylC expression by using Red/ET recombineering. The varying production distribution of syringolin derivatives showed the different efficiencies of native and synthetic promoters in E. coli. The successful reconstitution and expression of the syringolin biosynthetic pathway shows that Red/ET recombineering is an efficient tool to clone and engineer secondary metabolite biosynthetic pathways.     

Erythropoietin Modulates Autophagy Signaling in the Developing Rat Brain in an In Vivo Model of Oxygen-Toxicity

Autophagy is a self-degradative process that involves turnover and recycling of cytoplasmic components in healthy and diseased tissue. Autophagy has been shown to be protective at the early stages of programmed cell death but it can also promote apoptosis under certain conditions. Earlier we demonstrated that oxygen contributes to the pathogenesis of neonatal brain damage, which can be ameliorated by intervention with recombinant human erythropoietin (rhEpo). Extrinsic- and intrinsic apoptotic pathways are involved in oxygen induced neurotoxicity but the role of autophagy in this model is unclear. We analyzed the expression of autophagy activity markers in the immature rodent brain after exposure to elevated oxygen concentrations. We observed a hyperoxia-exposure dependent regulation of autophagy-related gene (Atg) proteins Atg3, 5, 12, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), LC3A-II, and LC3B-II which are all key autophagy activity proteins. Interestingly, a single injection with rhEpo at the onset of hyperoxia counteracted these oxygen-mediated effects. Our results indicate that rhEpo generates its protective effect by modifying the key autophagy activity proteins.     

Desktop manufacturing of complex objects,prototypes and biomedical scaffolds by means of computer‐assisted design combined with computer‐guided 3D plotting of polymers and reactive oligomers

Computer‐assisted design and image processing were combined with computer‐guided one‐ and two‐component air‐driven 3D dispensing of hotmelts, solutions, pastes, dispersions of polymers as well as monomers and reactive oligomers to produce solid objects with complex shapes and tailor‐made internal structures. During the 3D plotting process either individual microdots or microstrands were positioned in order to construct complex objects, fibers, tubes and scaffolds similar to non‐wovens. The resolution was in the range of 200 μm and depended upon inner nozzle diameter, air pressure, plotting speed, rheology, and plotting medium. Plotting in liquid media with densities similar to that of the dispensing liquid eliminated the need for construction of temporary support structures. The design capabilities of this computer‐guided 3D plotting process was demonstrated using conventional moisture‐curable silicone resin.     

Beneficial combination of wet oxidation,membrane separation and biodegradation processes for treatment of polymer processing wastewaters

The treatment of a model wastewater containing polyethylene glycol of molecular weight (MW) 10 000 by means of combined chemical oxidative pretreatment, membrane separation and biological post‐treatment was investigated. Wet oxidation was employed as a chemical pretreatment process to convert the original, high MW polymer to lower MW compounds in an attempt to improve the biotreatability of the waste‐water. The partially oxidized effluents formed during wet oxidation at temperatures up to 403 K were separated by nanofiltration where larger molecules were recycled into the wet oxidation reactor, while the permeate leaving the filtration step was treated biologically. At a biological residence time (τ_B) of 12 h and 3 h, the resulting total organic carbon (TOC) removal in the biological step was as high as 94% and 87%, respectively. Conversely, a continuous aerobic biological process was found inadequate to completely mineralize the original wastewater, since at τ_b of 96 h only about 60% to 70% TOC removal was achieved, while at τ_b of 12 h the original wastewater was practically non‐biockgradable.