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.
Bibliographical noteFunding Information:
This work was supported by the Grants from the National Research Foundation of Korea (NRF-2018R1C1B3001028, and NRF-2019R1A4A1020626). C.-H.J. received the financial support from the NRF-Korea through Global Ph.D. Fellowship program (NRF-2019H1A2A1073592).
This work was supported by the Grants from the National Research Foundation of Korea ( NRF-2018R1C1B3001028 , and NRF-2019R1A4A1020626 ). C.-H.J. received the financial support from the NRF-Korea through Global Ph.D. Fellowship program ( NRF-2019H1A2A1073592 ).
© 2021 International Metabolic Engineering Society
- Biosynthetic gene clusters
- Production titer
- Secondary metabolites
- Synthetic biology
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology