Evolutionary insights and guidelines to achieve effective and high-yield non-ribosomal peptide and polyketide engineering

Engineering of non-ribosomal peptide synthetase (NRPS) and/or polyketide synthase (PKS) assembly lines to generate modified products has long offered promise to produce novel antibiotics and other bioactive molecules. However, it is only in recent years that this promise has been realised with any consistency. Key to this has been a shift away from engineering approaches informed solely by structural data, and towards strategies that incorporate insights from evolutionary principles and datasets. Such analyses have not only guided the selection of optimal recombination boundaries for substitution of key subdomains, domains or modules, but also methods for increasing engineering throughput, often trading accuracy for volume. Diverse approaches have proven successful in NRPS systems, but a consistent theme has been that recombinant assembly lines are generally impaired in terms of product yield, and a meta-analysis of published results to date indicates that no one engineering strategy is significantly best for minimising yield losses. Evolution-inspired strategies have advanced the engineering of, and product yields for, PKS systems, and further breakthroughs appear imminent. Although no 'one size fits all' solution is apparent for either NRPS or PKS engineering, this review highlights important advances in synthetic biology that will support both discovery and production of next-generation antibiotics.