The Novel Transcriptional Regulator LmbU Promotes Lincomycin Biosynthesis through Regulating Expression of Its Target Genes in Streptomyces lincolnensis

Abstract

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 S. lincolnensis wild-type strain NRRL 2936. Both inactivation and overexpression of lmbU resulted 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=-CGCCG GCG-3=. However, LmbU cannot 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 to other known cluster-situated regulators (CSRs), which indicates that they belong to a new family of regulatory proteins. In conclusion, the present report identifies LmbU as a novel transcriptional regulator and provides new insights into regulation of lincomycin biosynthesis in S. lincolnensis. IMPORTANCE Although lincomycin biosynthesis has been extensively studied, its regulatory mechanism remains elusive. Here, a novel regulator, LmbU, which regulates transcription of its target genes in the lincomycin biosynthetic gene cluster (lmb gene cluster) and therefore promotes lincomycin biosynthesis, was identified in S. lincolnensis strain NRRL 2936. Importantly, we show that this new regulatory element is relatively widespread across diverse actinomycetes species. In addition, our findings provide a new strategy for improvement of yield of lincomycin through manipulation of LmbU, and this approach could also be evaluated in other secondary metabolite gene clusters containing this regulatory protein.

TetR-Type Regulator SLCG_2919 Is a Negative Regulator of Lincomycin Biosynthesis in Streptomyces lincolnensis

“…We suppose that an unknown regulator and SLCG_2919 might collaboratively or competitively modulate the transcription of lmbD. Recently, the CSR LmbU was shown to directly activate expression of the lmbA and lmbW genes and indirectly stimulate the expression of lmbC and lmbJ, while indirectly repressing the expression of lmbK and lmbU (10). These findings implied a more complex transcriptional regulatory mechanism of lincomycin biosynthesis than previously expected.…”

“…SLCG_2919 directly represses transcription of the CSR gene lmbU. The function of a positive cluster-situated regulator (CSR), LmbU, from the lincomycin biosynthetic pathway was recently described (10). To find a relationship between SLCG_2919 and LmbU, we compared transcriptional levels of lmbU between LCGL and the ΔSLCGL_2919 mutant.…”

“…Recently, Meng et al confirmed that the ABC1 transporter gene and the lincomycin export gene, lmrA, were transcriptionally activated by the global regulator GlnR with nitrate supplementation (9). Subsequently, Hou et al showed that LmbU from the lin cluster served as a cryptic cluster-situated regulator (CSR), promoting lincomycin production (10). Yet, the transcriptional regulatory mechanisms underlying lincomycin biosynthesis remain obscure, limiting production improvements for Lin-A.…”

Regulation of specialised metabolites in Actinobacteria – expanding the paradigms

“…Moreover, the incorporation of systems for the synchronisation of immunity mechanisms to ensure protection of the whole multicellular population is now being identified with increasing frequency. The discovery of the enormous array of signalling molecules, regulatory proteins, regulatory networks and pathways in Actinobacteria has shown that evolution has solved the problem in many different ways, and undoubtedly, there are novel mechanisms still to be elucidated, such as the recently discovered novel LmbU family of CSRs, which appears to be widespread in BGCs of Actinobacteria (Hou et al, 2018).…”

New Kid on the Block: LmbU Expands the Repertoire of Specialized Metabolic Regulators in Streptomyces

“…To date, genetic and biochemical studies have focused on elucidating the genes and enzymes responsible for synthesizing the precursors propylhygric acid (a nonproteogenic amino acid) and the sugar methylthiolincosamide and their subsequent conjugation to form the mature antibiotic (32). In this issue, Hou et al revisit the lincomycin biosynthetic pathway and shed light on its regulation (23). Bioinformatic analysis of the lincomycin gene cluster in S. lincolnensis suggested that gene lmbU encodes a novel cluster-situated regulator.…”