Enzyme engineering for the microbial production of enhanced pyoverdines for biomedical applications. – PYOCLICK

Combinatorial biosynthesis relies on enzyme and metabolic engineering approaches aimed at modifying the production of natural peptides, particularly by incorporating non-canonical amino acids (ncAAs) into their sequence. This strategy generates peptides carrying functional groups for bioconjugation, thereby diversifying their structures, properties, and applications. The PYOCLICK project applies this approach to the biosynthesis of pyoverdine (Pyo), the primary siderophore of Pseudomonas aeruginosa, whose metal-chelating and specific transport properties are particularly valuable for biomedical applications targeting this critical pathogen. PYOCLICK builds upon recent work published by three partners of the consortium, describing the engineering of one enzyme of the Pyo biosynthesis pathway, enabling the incorporation of an amino acid carrying an azide functional group into Pyo structure. This leads to the synthesis, by the mutant strain, of a functionalized Pyo (azido-Pyo), which can be efficiently conjugated via click chemistry to an antibiotic and transported into P. aeruginosa. However, the low production yield, due to the enzyme's low specificity for the functionalized amino acid, limits its broader application. The goal of the PYOCLICK project is to use cutting-edge innovative methodologies to optimize azido-Pyo production and enable the synthesis of new conjugates. To achieve this, we will use advanced molecular modeling tools to design an enzyme with improved specificity for the functionalized amino acid. We will develop a higher throughput and innovative screening method using a cell-free system to identify the most specific enzyme variants. Then, we will construct azido-Pyo-producing strains and optimize their production through bioreactor scale-up. Two types of conjugates will then be synthesized to target P. aeruginosa infections i) Pyo-antibiotic conjugates for targeted and specific antibiotic delivery to treat infections and ii) Pyo-octadentate siderophore conjugates for infection diagnosis via in vivo imaging. This project is based on an integrated approach combining synthetic biology, protein engineering, and chemobiology, opening broad application opportunities for Pyo and potentially other peptides. It harnesses the ability of cells to function as true biological factories, offering a sustainable solution for producing high-value molecules under environmentally friendly conditions.