CRISPR/Cas9-Mediated Multi-Locus Promoter Engineering in Cluster to Improve Erythromycin Production in .

Erythromycins are a group of macrolide antibiotics produced by . Erythromycin biosynthesis, which is a long pathway composed of a series of biochemical reactions, is precisely controlled by the type I polyketide synthases and accessary tailoring enzymes encoded by cluster. In the previous work, we have characterized that six genes representing extremely low transcription levels, - and , played important roles in limiting erythromycin biosynthesis in the wild-type strain NRRL 23338. In this study, to relieve the potential bottlenecks of erythromycin biosynthesis, we fine-tuned the expression of each key limiting gene by CRISPR/Cas9-mediated multi-locus promoter engineering. The native promoters were replaced with different heterologous ones of various strengths, generating ten engineered strains, whose erythromycin productions were 2.8- to 6.0-fold improved compared with that of the wild-type strain. Additionally, the optimal expression pattern of multiple rate-limiting genes and preferred engineering strategies of each locus for maximizing erythromycin yield were also summarized. Collectively, our work lays a foundation for the overall engineering of cluster to further improve erythromycin production. The experience of balancing multiple rate-limiting factors within a cluster is also promising to be applied in other actinomycetes to efficiently produce value-added natural products.