| Erythromycins are a group of macrolide antibiotics produced by Saccharopolyspora erythraea with high economic value.The capacity of wild-type Saccharopolyspora erythraea to produce erythromycin is low,and erythromycin biosynthesis is limited by many factors.However,previous rational strain engineering mainly focused on primary metabolic genes and transcriptional regulation genes outside the gene cluster,and there was a lack of studies on erythromycin biosynthetic genes.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 ery cluster.In the previous work,we have characterized that six genes distributed in four transcription units of ery gene cluster,SACE0716-SACE0720 and SACE0731,played important roles in limiting erythromycin biosynthesis in the wild-type strain S.erythraea NRRL 23338.In this study,to relieve the potential bottlenecks of erythromycin biosynthesis caused by the low expression of these rate-limiting enzymes,we fine-tuned the expression of each key limiting ery gene by CRISPR/Cas9-mediated multi-locus promoter engineering.Firstly,the activities of four native promoters controlling rate-limiting gene transcription and three heterologous ones for promoter replacement were characterized by the GFP fluorescence reporting system.After that,the native promoters were replaced with different heterologous ones of various strengths using CRISPR/Cas9 genome editing method,generating ten engineered strains and erythromycin production of these strains was measured.The results showed that the erythromycin productions were 2.8-to 6.0-fold improved compared to that of the wild-type strain,and the accumulation of intermediate erythromycin D in the engineered strain was also changed.The results of real-time quantitative PCR experiments showed that the promoter with different strengths significantly affected the expression levels of the target genes in the early fermentation stage（day 3）,which consequently influenced the efficiency of erythromycin biosynthesis.Additionally,in this study,erythromycin production was also successfully increased by gene overexpression via φ31-mediated integration.The optimal expression pattern of multiple rate-limiting genes and preferred engineering strategies of each locus for maximizing erythromycin yield were also summarized by comparing the fermentation levels of engineered strains with different sites and promoter combinations,which lays a foundation for the multi-locus promoter engineering of ery cluster to further improve erythromycin production.Collectively,the experience of balancing multiple biosynthesis rate-limiting factors within a cluster is also promising to be applied in other actinomycetes to efficiently produce value-added natural products. |
