A rational multi-target combination strategy for synergistic improvement of non-ribosomal peptide producing strains

Non-ribosomal peptides (NRPs) constitute a biomedically significant class of natural products, encompassing a plethora of antibiotics and other clinically utilized drugs. However, biosynthesis of these NRPs is governed by a sophisticated regulatory network, and to data, strain improvement through manipulation of multiple regulatory targets has remained an empirical process. We here developed a screening-based, multi-target rational combination strategy and demonstrated its effectiveness in enhancing the titers of two NRP drugs ˗ daptomycin and thaxtomin A ˗ in different strains. Initially, we devised a reliable colormetric analog co-expression and co-biosynthesis reporter system for screening high-yield phenotypes. Subsequently, through coupling CRISPR interference to induce genome-wide differential expression, we identified dozens of repressors that inhibit daptomycin or thaxtomin A biosynthesis, however, randomly combining these targets did not consistently increase titers further. We therefore developed a rational multi-target combination strategy by introducing an interplay map based on the synergy coefficient of each dual-target interaction. Employing this strategy, we engineered the final strains with multi-target synergistic combination that achieved the highest reported titers of daptomycin in native host and thaxtomin A in heterologous host, respectively. Our work represents the pioneering efforts in implementing a rational multi-target combination strategy for microbial strain improvement.