Haizhen Wu, Hrvoje Petkovic, Liyuan Tao, Huizhan Zhang, Xiaoyu Zhu, Yanwei Lin, Jiang Ye, Meijin Guo, Bingbing Hou
Lincomycin A is a clinically important antimicrobial agent produced by Streptomyces lincolnensis In this study, a new regulator designated LmbU (Genbank accession no. ABX00623.1) was identified and characterized to regulate lincomycin biosynthesis in a S. lincolnensis wild-type strain NRRL 2936. Both inactivation and overexpression of lmbU result in significant influences on lincomycin production. Transcriptional analysis and in vivo neomycin resistance (neo(r) ) reporter assays demonstrated that LmbU activates expression of the lmbA, lmbC, lmbJ and lmbW genes, and represses expression of the lmbK and lmbU genes. Electrophoretic mobility shift assays (EMSAs) demonstrated that LmbU can bind to the regions upstream of the lmbA and lmbW genes through the consensus and palindromic sequence 5' -CGCCGGCG-3' . However, LmbU can not bind to the regions upstream of the lmbC, lmbJ, lmbK and lmbU genes as they lack this motif. These data indicate a complex transcriptional regulatory mechanism of LmbU. LmbU homologues are present in the biosynthetic gene clusters of secondary metabolites of many other actinomycetes. Furthermore, the LmbU homologue from Saccharopolyspora erythraea (Genbank accession no. WP_009944629.1) also binds to the regions upstream of lmbA and lmbW, which suggests widespread activity for this regulator. LmbU homologues have no significant structural similarities with other known cluster-situated regulator (CSRs), which indicates that they belong to a new family of regulatory proteins. In conclusion, the present study identifies LmbU as a novel transcriptional regulator, and provides new insights into regulation of lincomycin biosynthesis in S. lincolnensisImportance Although lincomycin biosynthesis has been extensively studied, its regulatory mechanism remains elusive. Here, a novel regulator LmbU, was identified in S. lincolnensis strain NRRL 2936, which regulates transcription of its target genes in the lincomycin biosynthetic gene cluster (lmb gene cluster), and therefore promotes lincomycin biosynthesis. Importantly, we show that this new regulatory element is relatively widespread across diverse actinomycetes species. In addition, our findings provide a new strategy for yield improvement of lincomycin through manipulation of LmbU, and this approach could also be evaluated in other secondary metabolite gene clusters containing this regulatory protein.
