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Example of the graphaligner FSM and scoring matrix. a. A theoretical next-generation sequencing read is shown, where the numbers represent sequential positional reads and the letters represent the nucleotides read at those positions. b. The state model is a representation of the FSM for a reference where the numbers represent the state of the model, the arrows are the transitions between the states, and the letters represent possible states, with ε representing an empty state indicating a deletion or an insertion of various length. c. Scoring matrices for the state
Source publication
The fields of antibody engineering, enzyme optimization and pathway construction rely increasingly on screening complex variant DNA libraries. These highly diverse libraries allow researchers to sample a maximized sequence space; and therefore, more rapidly identify proteins with significantly improved activity. The current state of the art in synt...
Citations
... The biosynthetic machinery seemed intolerant to the macrocycle variations, which is in agreement with recent findings using an in vitro system. (20) Additionally, biosynthesis is also intolerant to N-or C-terminal extension, i.e. shorter peptides were not detected while longer peptides were trimmed down to heptamers. Furthermore, a comprehensive one-by-one aminoacid (AA) exchange of the DAR core peptide was performed to test if (i) the macrocycles were formed and (ii) resulting compounds exhibit antibacterial activity (Table S5). ...
Increasing numbers of multi-drug resistant pathogens call for new chemical scaffolds, addressing novel targets, that can serve as lead structures for the development of life-saving drugs. For antibiotics, natural product-inspired molecules represent a most promising resource. Natural products evolved to high chemical complexity and occupy a chemical space different than synthetic libraries. However, clinical translation of promising natural products is often impeded by their relative inaccessibility to medicinal chemistry optimization, e.g. iterative synthesis of large series of derivatives. Here, this limitation is addressed with a randomized library of bicyclic heptapeptides based on the natural product darobactin that hits the clinically not addressed target BamA. Variants of the ribosomally synthesized and post-translationally modified peptides were generated using heterologous mutasynthesis. A parallelized screening assay is adapted in nanoliter-scale beads to test the darobactin derivatives against our sensor strain. Loss of fluorescence sorting prioritized 563 events out of the analyzed ~500k beads. Re-testing confirmed 48 hit events, of which 40 proved to produce distinct darobactin-type molecules. Most promising structures were isolated and the growth inhibitory effects against Gram-negative pathogens validated. One of our current frontrunner compounds (i.e., darobactin B) was reinforced by the randomized screen. While microbiological investigations of the new derivatives is ongoing, darobactin B was profiled in later tier assays and compared to another promising, rationally-designed analog (i.e., darobactin B9, (also named D22)). Early ADMET profiling and efficacy tests in a mouse pneumonia model were performed. Darobactin B reduced bacterial load of Pseudomonas aeruginosa and Klebsiella pneumoniae by intraperitoneal, as well as intratracheal administration. Our study showcases the potential of mutasynthetic libraries for high-throughput screening and identification of functional peptides for drug lead discovery.
