Top-down synthetic biology approach for titer improvement of clinically important antibiotic daptomycin in Streptomyces roseosporus

Secondary metabolites are produced at low titers by native producers due to tight regulations of their productions in response to environmental conditions. Synthetic biology provides a rational engineering principle for transcriptional optimization of secondary metabolite BGCs (biosynthetic gene clusters). Here, we demonstrate the use of synthetic biology principles for the development of a high-titer strain of the clinically important antibiotic daptomycin. Due to the presence of large NRPS (non-ribosomal peptide synthetase) genes with multiple direct repeats, we employed a top-down approach that allows transcriptional optimization of genes in daptomycin BGC with the minimum inputs of synthetic DNAs. The repeat-free daptomycin BGC was created through partial codon-reprogramming of a NRPS gene and cloned into a shuttle BAC vector, allowing BGC refactoring in a host with a powerful recombination system. Then, transcriptions of functionally divided operons were sequentially optimized through three rounds of DBTL (design-build-test-learn) cycles that resulted in up to ~2300% improvement in total lipopeptide titers compared to the wild-type strain. Upon decanoic acid feeding, daptomycin accounted for ∼ 40% of total lipopeptide production. To the best of our knowledge, this is the highest improvement of daptomycin titer ever achieved through genetic engineering of S. roseosporus. The top-down engineering approach we describe here could be used as a general strategy for the development of high-titer industrial strains of secondary metabolites produced by BGCs containing genes of large multi-modular NRPS and PKS enzymes.