The β-lactam antibiotics remain the most commonly used to treat severe infections. Because of structural similarity between the β-lactam ring and the d-alanyl⁴-d-alanine⁵ extremity of bacterial cell wall precursors, the drugs act as suicide substrates of the dd-transpeptidases that catalyze the last cross-linking step of cell wall assembly. Here, we show that this mechanism of action can be defeated by a novel type of transpeptidase identified for the first time by reverse genetics in aβ-lactam-resistant mutant of Enterococcus faecium. The enzyme, Ldt_fm, catalyzes in vitro the cross-linking of peptidoglycan subunits in a β-lactam-insensitive ld-transpeptidation reaction. The specificity of Ldt_fm for the l-lysyl³-d-alanine⁴ peptide bond of tetrapeptide donors accounts for resistance because the substrate does not mimic β-lactams in contrast to d-alanyl⁴-d-alanine⁵ in the pentapeptide donors required for dd-transpeptidation. Ldt_fm homologues are encountered sporadically among taxonomically distant bacteria, indicating that ld-transpeptidase-mediated resistance may emerge in various pathogens.

The biosynthetic gene cluster of the antifungal metabolite sporothriolide 1 was identified from three producing ascomycetes: Hypomontagnella monticulosa MUCL 54604, H. spongiphila CLL 205 and H. submonticulosa DAOMC 242471. A transformation protocol was established, and genes encoding a fatty acid synthase subunit and a citrate synthase were simultaneously knocked out which led to loss of sporothriolide and sporochartine production. In vitro reactions showed that the sporochartines are derived from non-enzymatic Diels–Alder cycloaddition of 1 and trienylfuranol A 7 during the fermentation and extraction process. Heterologous expression of the spo genes in Aspergillus oryzae then led to the production of intermediates and shunts and delineation of a new fungal biosynthetic pathway originating in fatty acid biosynthesis. Finally, a hydrolase was revealed by in vitro studies likely contributing towards self-resistance of the producer organism.