Biology subjects

Borrero del Pino, J.

Publications and source records attributed to Borrero del Pino, J..

3 recordsLinked to original sources

bioRxiv2026

Gfa, a new integron-borne fosfomycin resistance family

Fosfomycin is increasingly used to treat multidrug-resistant Gram-negative infections, yet resistance surveillance largely relies on homology-based detection of known fos genes. Here, we identify Gfa, a previously unrecognized integron-borne fosfomycin resistance determinant uncovered through functional screening of 129 integron gene cassettes. Gfa confers high-level fosfomycin resistance across Enterobacterales and Pseudomonas aeruginosa and is resistant to phosphonoformate inhibition. Structural modelling and mutagenesis reveal a conserved cysteine-rich, zinc-organized architecture essential for activity and distinct from canonical Fos enzymes. Crude-extract assays confirm glutathione-dependent fosfomycin inactivation under metal-chelating conditions. Distant Gfa homologs also conferred resistance, although activity was restricted to a subset of proteins tested. Together, these findings establish Gfa as a previously unrecognized fosfomycin resistance family and highlight the limitations of homology-based mining for discovering novel resistance determinants and predicting which family members are functional.

microbiology↗

bioRxiv2026

An Enhanced Split Intein-Mediated Ligation (SIML) Platform for Rapid Discovery and Functional Screening of Circular Bacteriocins

Bacteriocins are ribosomally synthesized antimicrobial peptides with promising applications in biotechnology, particularly in food preservation and animal and human health. Circular bacteriocins are especially attractive due to their head-to-tail cyclized structure, which confers enhanced stability and antimicrobial potency relative to linear peptides. Here, we report an in vitro cell-free protein synthesis system coupled with an enhanced Split Intein-Mediated Ligation platform (IV-CFPS/SIML) for the efficient synthesis of circular bacteriocins through systematic evaluation of cyclization sites and alternative split inteins. Using enterocin AS-48 as a model, we systematically evaluated multiple serine-based cyclization sites in combination with three split inteins, NpuDnaE, Gp41-1, and SspGyrB, to identify configurations supporting efficient splicing and high antimicrobial activity. Gp41-1 emerged as the most effective intein and was subsequently applied to the production of garvicin ML, amylocyclicin, and 27 naturally occurring sequence variants, demonstrating that cyclization site selection, intein identity, and minor sequence variations strongly influence antimicrobial potency and target range. Finally, SIML expression cassettes encoded in pUC-derived vectors enabled in vivo production and functional expression of selected circular bacteriocins in recombinant Escherichia coli. Collectively, these results establish SIML as a versatile platform for in vitro and in vivo production, screening, and functional characterization of known and putative circular bacteriocins.

synthetic biology↗

bioRxiv2024

Multiplexing bacteriocin synthesis to kill and prevent antimicrobial resistance

Antibiotic resistance represents an emergency for global public health. This calls for using alternative drugs and developing innovative therapies based on a clear understanding of their mechanisms of action and resistance in bacteria. Bacteriocins represent a unique class of natural molecules selectively eliminating bacteria. These secreted proteins exhibit a narrower spectrum of activity compared to conventional broad-spectrum antimicrobials by interacting with specific protein and lipid receptors on bacterial cell envelopes. Despite their diverse molecular structures, the commonality of being genetically encoded makes bacteriocins amenable to synthetic biology design. In using cell-free gene expression (CFE) and continuous-exchange CFE (CECFE), we produced controlled combinations (cocktails) of bacteriocins in single synthesis reactions for the first time. A first set of bacteriocin cocktails comprising both linear and circular proteins allowed the targeting of different bacterial species. Other cocktails were designed to target one bacterial species and considering bacteriocins pathways to cross the cell-envelope. Such combinations demonstrated efficient bacterial eradication and prevention of resistance. We illustrate the effectiveness of these bacteriocin mixtures in eradicating various human pathogenic-multiresistant--isolates. Finally, we highlight their potential as targeted and versatile tools in antimicrobial therapy by testing a combination of bacteriocins for treatment in vivo in the animal model Galleria mellonella.

synthetic biology↗