Author: Philipp StephanPublisher:
ISBN:
Category :
Languages : de
Pages : 0
Book Description
Over the last decades, discovery rates of new antibiotics have declined while the occurrence of multi-resistant pathogens has increased, leading towards a post-antibiotic era. Many current antibiotics are natural products or derivatives, known for their excellent bioactivities but complex structures that complicate synthetic production. Many antibiotics, like penicillin and vancomycin, are peptides produced by nonribosomal peptide synthetases (NRPS). These multi-modular enzyme complexes function like assembly lines, with each module adding a specific amino acid to the peptide through the biocatalytic activities of distinct domains. Targeted NRPS engineering to alter structures of the produced peptides has faced challenges, but directed evolution of NRPS domains seems to offer a promising solution. This thesis presents a straightforward method for the directed evolution of adenylation (A) domains using LC-MS/MS detection of dipeptides in cell lysates, demonstrating its power by engineering the GrsB1 A domain from gramicidin S biosynthesis to select for L-piperazic acid (Piz) instead of its native substrate L-Pro without reducing enzyme activity. The modified antibiotic, Piz-gramicidin S, showed improved bioactivity. Despite compatibility issues between NRPS domains, kinetic analyses revealed that A domain substrate preferences ultimately determine final product ratios. Additionally, the thesis proposes a novel DNA-based approach for NRPS engineering, using zinc finger domains that bind specific DNA sequences to simplify module rearrangement by making NRPSs DNA-templated. This method offers a more efficient alternative to traditional cloning techniques by replacing the handling of large NRPS genes with much shorter sequences of the zinc finger domains. Overall, this research advances the engineering of NRPSs for developing new antibiotics with better efficacy against resistant pathogens and highlights the usefulness of directed evolution in this context.
