| The macrolide antibiotic erythromycin has broad-spectrum antibacterial activity,and its biosynthesis is controlled by a cluster of genes on the chromosome of Saccharopolyspora erthraea,but the gene cluster does not contain any regulatory genes,suggesting that its transcriptional regulatory mechanism is unusual.Currently,several Tet R family transcriptional factors had been found to be able to control erythromycin biosynthesis through different regulation modes,but the regulatory network of erythromycin synthesis modulated by them was still poorly understood.Our laboratory had previously discovered a Tet R family transcriptional factor SACE＿0303 in Sac.erthraea,which could directly inhibit its own genes and activate the expression of its neighbor gene SACE＿0304,encoding for a Mar R family regulator,but indirectly activated the transcription of the structural gene ery AI and the resistance gene erm E in the erythromycin synthesis gene cluster,resulting in the biosynthesis of erythromycin;It was found that SACE＿0304 was negatively correlated with the yield of erythromycin biosynthesis.Based on the previous study,the regulatory mechanism of SACE＿0304 was explored,and the regulatory network mediated by SACE＿0303 was preliminarily constructed by identifying its other target genes,and I systematically modified these genes to enhance erythromycin yield.First,through the phenotypic analysis of the starting strain A226 andΔSACE＿0304,it was found that deletion of SACE＿0304 did not affect the growth and morphological differentiation of Sac.erthraea.In order to determine whether SACE＿0304 affects the transcription of erythromycin synthesis gene cluster,RT-q PCR was used to analyze the transcription levels of ery AI and erm E in A226 andΔSACE＿0304,and the results showed that SACE＿0304 could inhibit their transcriptional expression.In vivo analysis with the green fluorescence reporting system found that SACE＿0304 could directly act on the promoter region of ery AI to inhibit the expression of ery AI,thereby negatively regulating the biosynthesis of erythromycin.Further analysis by RT-q PCR and in vivo green fluorescence reporting system confirmed that SACE＿0304 directly activates its own genes but inhibits the transcriptional expression of SACE＿0303.This indicated that there was a mutual regulation between SACE＿0303 and SACE＿0304.Therefore,SACE＿0303 might positively regulate the biosynthesis of erythromycin by directly inhibiting the transcription of SACE＿0304.In order to identify other target genes regulated by SACE＿0303,based on the motif for SACE＿0303 to bind,a virtual footprint software was used to search the whole genome of A226,and the upstream regions of 83 genes including SACE＿0303 and SACE＿0304promoter regions(P0303-0304)were found.Among them,10 promoter regions with high predicted scores of genes having clear function were selected for subsequent EMSA analysis.The results showed that SACE＿0303 could specifically bind to the promoter regions of SACE＿2467（cation-transporting ATPase）,SACE＿3156（large transcriptional regulator）and SACE＿5222（alpha-ketoglutarate permease）,and through RT-q PCR analysis,it was confirmed that SACE＿0303 directly inhibited the transcriptional expression of those three genes.Furthermore,their overexpression in A226 strain was each constructed,and the fermentation results showed that the erythromycin A production of A226/p IB139-2467,A226/p IB139-3156or A226/p IB139-5222 was reduced to lower degrees compared with A226,indicating that those genes were negatively related to erythromycin A production.Based on the above-established mini-regulatory network of SACE＿0303,systematically modified strain derived from erythromycin high-producing strain WB were built.First,SACE＿0304 was deleted in WB,and the yield of erythromycin A in constructed mutant WBΔSACE＿0304 was 24%higher than that of WB.Then,the plasmid p SETd Cas9-0303 was introduced into WBΔSACE＿0304 to over-express SACE＿0303,and the yield of erythromycin A in WBΔSACE＿0304/p SETd Cas9-0303 was improved by 35%compared with that in WB.With the CRISPRi technology,WBΔ0304/p0303-sg2467-3156-5222 was constructed to co-inhibit SACE＿0303 target genes,SACE＿2467,SACE＿3156 and SACE＿5222,and the yield of erythromycin A in the constructed strain was 67%higher than that of WB.In summary,through a series of in vivo and in vitro experimental techniques,this study confirmed that SACE＿0304,the adjacent target gene of SACE＿0303 in Sac.erthraea,could directly inhibit the transcriptional expression of ery AI,and found that the two transcription factors of Tet R and Mar R families had interactive transcriptional regulation effects.Subsequently,three other target genes of SACE＿0303 were identified,and the mini-regulatory network around SACE＿0303 was combined to improve the yield of erythromycin.This study laid the foundation for revealing the regulation network of erythromycin biosynthesis and increasing its fermentation yield.. |
