Assessing the engineering potential of polymyxin antibiotics via heterologous expression in Bacillus subtilis Hanne Put 1,2 , Maarten Fauvart 1,3 , Jan Michiels 1,2 1 Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium, 2 Center for Microbiology, VIB-KU Leuven, Leuven, Belgium, 3 imec, Leuven, Belgium Polymyxins are non-ribosomal peptides, originally produced by Paenibacillus polymyxa and currently considered as last-resort antibiotics against Gram-negative infections. Unfortunately, significant side effects, including nefro- and neurotoxicity, complicate treatments, and the recent identification of plasmid-borne mcr resistance genes raises many concerns about the future of these antibiotics. Although the modular structure of non-ribosomal peptide-synthetases (NRPS) has great potential for engineering new variants with improved characteristics, a deeper understanding of the biosynthetic pathway and the specificity-determining regions is necessary to achieve this goal. Therefore, our research aims to study the polymyxin biosynthetic gene cluster (BGC) using heterologous expression and site-directed mutagenesis. We have cloned the BGC on a new BAC/YAC vector with increased stability and flexibility, followed by heterologous expression in a Bacillus subtilis host strain. Production of polymyxin antibiotics was confirmed via high-sensitivity LC/MS analyses. Ongoing work assesses the effect of promoter exchange on polymyxin production in B. subtilis and further optimization of polymyxin production.
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© The Author(s), 2022
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