Metabolic engineering of Streptomyces roseosporus for increased production of clinically important antibiotic daptomycin

Abstract

Abstract Daptomycin (DAP), a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, is clinically important for treatment of infections caused by multidrug‐resistant Gram‐positive pathogens, but the low yield hampers its large‐scale industrial production. Here, we describe a combination metabolic engineering strategy for constructing a DAP high‐yielding strain. Initially, we enhanced aspartate (Asp) precursor supply in S. roseosporus wild‐type (WT) strain by separately inhibiting Asp degradation and competitive pathway genes using CRISPRi and overexpressing Asp synthetic pathway genes using strong promoter kasOp*. The resulting strains all showed increased DAP titre. Combined inhibition of acsA4, pta, pyrB, and pyrC increased DAP titre to 167.4 μg/mL (73.5% higher than WT value). Co‐overexpression of aspC, gdhA, ppc, and ecaA led to DAP titre 168 μg/mL (75.7% higher than WT value). Concurrently, we constructed a chassis strain favourable for DAP production by abolishing by‐product production (i.e., deleting a 21.1 kb region of the red pigment biosynthetic gene cluster (BGC)) and engineering the DAP BGC (i.e., replacing its native dptEp with kasOp*). Titre for the resulting chassis strain reached 185.8 μg/mL. Application of our Asp precursor supply strategies to the chassis strain further increased DAP titre to 302 μg/mL (2.1‐fold higher than WT value). Subsequently, we cloned the engineered DAP BGC and duplicated it in the chassis strain, leading to DAP titre 274.6 μg/mL. The above strategies, in combination, resulted in maximal DAP titre 350.7 μg/mL (2.6‐fold higher than WT value), representing the highest reported DAP titre in shake‐flask fermentation. These findings provide an efficient combination strategy for increasing DAP production and can also be readily applied in the overproduction of other Asp‐related antibiotics.