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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 (Neor) 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 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. lincolnensisIMPORTANCE 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.

SUBMITTER: Hou B

PROVIDER: S-EPMC5738732 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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

Hou Bingbing B Lin Yanwei Y Wu Haizhen H Guo Meijin M Petkovic Hrvoje H Tao Liyuan L Zhu Xiaoyu X Ye Jiang J Zhang Huizhan H

Journal of bacteriology 20171220 2

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 (Neor</su ...[more]

PMID: 29038257

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Project description:Lincomycin A (Lin-A) is a widely used antibacterial antibiotic fermented by Streptomyces lincolnensis However, the transcriptional regulatory mechanisms underlying lincomycin biosynthesis have seldom been investigated. Here, we first identified a TetR family transcriptional regulator (TFR), SLCG_2919, which negatively modulates lincomycin biosynthesis in S. lincolnensis LCGL. SLCG_2919 was found to specifically bind to promoter regions of the lincomycin biosynthetic gene cluster (lin cluster), including 25 structural genes, three resistance genes, and one regulatory gene, and to inhibit the transcription of these genes, demonstrating a directly regulatory role in lincomycin biosynthesis. Furthermore, we found that SLCG_2919 was not autoregulated, but directly repressed its adjacent gene, SLCG_2920, which encodes an ATP/GTP binding protein whose overexpression increased resistance against lincomycin and Lin-A yields in S. lincolnensis The precise SLCG_2919 binding site within the promoter region of SLCG_2920 was determined by a DNase I footprinting assay and by electrophoretic mobility shift assays (EMSAs) based on base substitution mutagenesis, with the internal 10-nucleotide (nt) AT-rich sequence (AAATTATTTA) shown to be essential for SLCG_2919 binding. Our findings indicate that SLCG_2919 is a negative regulator for controlling lincomycin biosynthesis in S. lincolnensis The present study improves our understanding of molecular regulation for lincomycin biosynthesis.IMPORTANCE TetR family transcriptional regulators (TFRs) are generally found to regulate diverse cellular processes in bacteria, especially antibiotic biosynthesis in Streptomyces species. However, knowledge of their function in lincomycin biosynthesis in S. lincolnensis remains unknown. The present study provides a new insight into the regulation of lincomycin biosynthesis through a TFR, SLCG_2919, that directly modulates lincomycin production and resistance. Intriguingly, SLCG_2919 and its adjoining gene, SLCG_2920, which encodes an ATP/GTP binding protein, were extensively distributed in diverse Streptomyces species. In addition, we revealed a new TFR binding motif, in which SLCG_2919 binds to the promoter region of SLCG_2920, dependent on the intervening AT-rich sequence rather than on the flanking inverted repeats found in the binding sites of other TFRs. These insights into transcriptional regulation of lincomycin biosynthesis by SLCG_2919 will be valuable in paving the way for genetic engineering of regulatory elements in Streptomyces species to improve antibiotic production.

Project description:BackgroundAdpA is a global regulator of morphological differentiation and secondary metabolism in Streptomyces, but the regulatory roles of the Streptomyces AdpA family on the biosynthesis of the natural product ε-poly-L-lysine (ε-PL) remain unidentified, and few studies have focused on increasing the production of ε-PL by manipulating transcription factors in Streptomyces.ResultsIn this study, we revealed the regulatory roles of different AdpA homologs in ε-PL biosynthesis and morphological differentiation and effectively promoted ε-PL production and sporulation in Streptomyces albulus NK660 by heterologously expressing adpA from S. neyagawaensis NRRLB-3092 (adpASn). First, we identified a novel AdpA homolog named AdpASa in S. albulus NK660 and characterized its function as an activator of ε-PL biosynthesis and morphological differentiation. Subsequently, four heterologous AdpA homologs were selected to investigate their phylogenetic relationships and regulatory roles in S. albulus, and AdpASn was demonstrated to have the strongest ability to promote both ε-PL production and sporulation among these five AdpA proteins. The ε-PL yield of S. albulus heterologously expressing adpASn was approximately 3.6-fold higher than that of the control strain. Finally, we clarified the mechanism of AdpASn in enhancing ε-PL biosynthesis and its effect on ε-PL polymerization degree using real-time quantitative PCR, microscale thermophoresis and MALDI-TOF-MS. AdpASn was purified, and its seven direct targets, zwf, tal, pyk2, pta, ack, pepc and a transketolase gene (DC74_2409), were identified, suggesting that AdpASn may cause the redistribution of metabolic flux in central metabolism pathways, which subsequently provides more carbon skeletons and ATP for ε-PL biosynthesis in S. albulus.ConclusionsHere, we characterized the positive regulatory roles of Streptomyces AdpA homologs in ε-PL biosynthesis and their effects on morphological differentiation and reported for the first time that AdpASn promotes ε-PL biosynthesis by affecting the transcription of its target genes in central metabolism pathways. These findings supply valuable insights into the regulatory roles of the Streptomyces AdpA family on ε-PL biosynthesis and morphological differentiation and suggest that AdpASn may be an effective global regulator for enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces.

Dataset Information

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

Publications

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

Similar Datasets

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