De novo biosynthesis of daptomycin through module construction and assembly in Escherichia coli

Abstract

Daptomycin (DAP) is a non-ribosomal peptide (NRP) produced by Streptomyces roseosporus and the fermentation process is time-consuming and labor-intensive. Since non-ribosomal peptide synthetase assembles DAP from simple amino acid building blocks, the heterologous expression of DAP in a robust and easily manipulated host is an attractive strategy to enhance its accessibility. In this study, we rationally designed and refactored the 73.8-kb DAP biosynthesis gene cluster (dpt cluster). Subsequently, the optimized 55.4-kb dpt cluster was divided into three rationally designed modules to respectively clone into pET28a. The three modules were then co-transformed into E . coli BL21 (DE3) to assemble and achieve heterologous expression of DAP. We further optimized the highly repetitive codons of the dptBC gene and reduced the copy number of dptBC gene, thereby stabilizing the gene to ensure consistent production of DAP in E. coli. We also refactored the dpt cluster to improve the translation efficiency of the DAP synthetic protein. Finally, we optimized and determined the most suitable fermentation conditions for DAP production in E. coli, which resulted in DAP yield up to 307.60 μg/L in a 5 L bioreactor. Our study demonstrates the inaugural successful peptide module construction and assembly to achieve de novo heterologous biosynthesis of DAP in E. coli, which establishes a proof-of-concept technology system for the engineered production of structurally diverse NRPs in prokaryotic hosts, advancing synthetic biology frameworks for complex natural product assembly.