In recent years, antimicrobial resistance has been on the rise. Glycopeptide antibiotics (GPAs), a distinct class of antibiotics, are essential for the treatment of infections caused by Gram-positive bacteria. GPAs and other antibiotics today are natural products derived from soil microbes. Unfortunately, with the emergence of glycopeptide-resistant enterococci and other resistant bacteria, and the decline in the discovery of new antibiotics, effective antibiotic treatment has become ever more difficult. Therefore, it is of utmost importance to create new or modify old GPAs in an effort to provide new treatments against infections caused by resistant bacteria. Many antibiotics are natural or semisynthetic compounds with structures too complex for chemical synthesis, meaning we are often reliant on bacterial fermentation for their production. Consequently, a long-standing goal for researchers has been to redesign the biosynthetic machinery responsible for the antibiotics production to enable the production of novel products. Nevertheless, recombinant biosynthetic machineries have almost consistently had disappointingly low yields.
Key to the peptide synthesis process is the actions of adenylation (A)-domains. These are responsible for the selection of amino acid substrates and their activation prior to incorporation of these residues into the growing peptide chain and are thus the main site for substrate selectivity. Using ancestral sequence reconstruction (ASR), we gain insight on the evolutionary process that allowed key adenylating domains to change substrate specificity. The ASR data in conjunction with obtained crystal structure provide invaluable insight on how to efficiently redesign the biosynthetic machinery responsible for the production of GPAs.