DeoR regulates lincomycin production in Streptomyces lincolnensis.

Regulators belonging to the DeoR family are widely distributed among the bacteria. Few studies have reported that DeoR family proteins regulate secondary metabolism of Streptomyces. This study explored the function of DeoR (SLINC_8027) in Streptomyces lincolnensis. Deletion of deoR in NRRL 2936 led to an increase in cell growth. The lincomycin production of the deoR deleted strain ΔdeoR was 3.4-fold higher than that of the wild strain. This trait can be recovered to a certain extent in the deoR complemented strain ΔdeoR::pdeoR. According to qRT-PCR analysis, DeoR inhibited the transcription of all detectable genes in the lincomycin biosynthesis cluster and repressed the expression of glnR, bldD, and SLCG_Lrp, which encode regulators outside the cluster. DeoR also inhibited the transcription of itself, as revealed by the XylE reporter. Furthermore, we demonstrated that DeoR bound directly to the promoter region of deoR, lmbA, lmbC-D, lmbJ-K, lmrA, lmrC, glnR, and SLCG_Lrp, by recognizing the 5'-CGATCR-3' motif. This study found that versatile regulatory factor DeoR negatively regulates lincomycin biosynthesis and cellular growth in S. lincolnensis, which expanded the regulatory network of lincomycin biosynthesis.

Characterization of a TetR-type positive regulator AtrA for lincomycin production in Streptomyces lincolnensis.

AtrA belongs to the TetR family and has been well characterized for its roles in antibiotic biosynthesis regulation. Here, we identified an AtrA homolog (AtrA-lin) in Streptomyces lincolnensis. Disruption of atrA-lin resulted in reduced lincomycin production, whereas the complement restored the lincomycin production level to that of the wild-type. In addition, atrA-lin disruption did not affect cell growth and morphological differentiation. Furthermore, atrA-lin disruption hindered the transcription of regulatory gene lmbU, structural genes lmbA and lmbW inside the lincomycin biosynthesis gene cluster, and 2 other regulatory genes, adpA and bldA. Completement of atrA-lin restored the transcription of these genes to varying degrees. Notably, we found that AtrA-lin directly binds to the promoter region of lmbU. Collectively, AtrA-lin positively modulated lincomycin production via both pathway-specific and global regulators. This study offers further insights into the functional diversity of AtrA homologs and the mechanism of lincomycin biosynthesis regulation.

AdpAlin regulates lincomycin and melanin biosynthesis by modulating precursors flux in Streptomyces lincolnensis.

Lincomycin is one of the most important antibiotics. However, transcriptional regulation network of secondary metabolism in Streptomyces lincolnensis, the lincomycin producer, remained obscure. AdpA from S. lincolnensis (namely AdpAlin ) has been proved to activate lincomycin biosynthesis. Here we found that both lincomycin and melanin took l-tyrosine as precursor, and AdpAlin activated melanin biosynthesis as well. Three tyrosinases, MelC2, MelD2, and MelE, and one tyrosine peroxygenase, LmbB2, participated in lincomycin and melanin biosynthesis in different ways. For melanin biosynthesis, MelC2 was the only key enzyme required. For lincomycin biosynthesis, MelD2 and LmbB2 were positive factors and were suggested to convert l-tyrosine to l-dihydroxyphenylalanine (l-DOPA). Otherwise, MelC2 and MelE were negative factors for lincomycin biosynthesis and they were supposed to oxidize l-DOPA to generate melanin and certain unknown metabolite, respectively. Based on in silico analysis combined with electrophoretic mobility shift assays (EMSAs), we proved that AdpAlin directly interacted with promoters of melC, melD, and melE by binding to putative AdpA-binding sites in vitro. Moreover, in vivo experiments revealed that AdpAlin positively regulated the transcription of melC and melE, but negatively regulated melD. In conclusion, AdpAlin was the switch of secondary metabolism in S. lincolnensis, and it modulated precursor flux of lincomycin and melanin biosynthesis by directly activating melC, melE, and lmbB1/lmbB2 or repressing melD.

An alternative σ factor σL sl regulates lincomycin production in Streptomyces lincolnensis.

Lincomycin produced by Streptomyces lincolnensis is a critical antibacterial antibiotic in the clinical. To further understand the regulatory mechanism of lincomycin biosynthesis, we identified an alternative σ factor, σL sl , in Streptomyces lincolnensis NRRL 2936. Deletion of sigLsl resulted in an increase in cell growth but a decrease in lincomycin production. σL sl boosted lincomycin biosynthesis by directly stimulating the transcription of four genes (lmbD, lmbV, lmrC, and lmbU) within the lincomycin biosynthetic lmb gene cluster. Besides, σL sl participated in lincomycin biosynthesis by directly stimulating the transcription of mshC, a gene responsible for MSH synthesis. In conclusion, our findings demonstrated that σL sl plays a direct regulatory role in lincomycin biosynthesis. This study extends the understanding of molecular mechanisms of lincomycin biosynthetic regulation.

Developmental regulator RamRsl controls both morphological development and lincomycin biosynthesis in Streptomyces lincolnensis.

AIMS: Assessing the role of ramRsl , a gene absent in a lincomycin over-producing strain, in the regulation of morphological development and lincomycin biosynthesis in Streptomyces lincolnensis. METHODS AND RESULTS: The gene ramRsl was deleted from the wild-type strain NRRL 2936 and the ΔramR mutant strain was characterized by a slower growth rate and a delayed morphological differentiation compared to the original strain NRRL 2936. Furthermore, the ΔramR produced 2.6-fold more lincomycin than the original strain, and consistently the level of expression of all lincomycin cluster located genes was enhanced at 48 and 96 h in the ΔramR. Complementation of ΔramR with an intact copy of ramRsl restored all wild-type features, whereas the over-expression of ramRsl led to a reduction of 33% of the lincomycin yield. Furthermore, the level of expression of glnR, bldA and SLCG_2919, three of known lincomycin biosynthesis regulators, was lower in the ΔramR than in the original strain at the early stage of fermentation and we demonstrated, using electrophoretic mobility shift assay and XylE reporter assay, that glnR is a novel direct target of RamR. CONCLUSIONS: Altogether, these results indicated that, beyond promoting the morphological development, RamR regulates negatively lincomycin biosynthesis and positively the expression of the nitrogen regulator GlnR. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrated that RamR plays a negative role in the regulation of lincomycin biosynthesis in S. lincolnensis. Interestingly, the deletion of this gene in other antibiotic-producing Streptomyces strains might also increase their antibiotic-producing abilities.

Complete genome sequence of high-yield strain S. lincolnensis B48 and identification of crucial mutations contributing to lincomycin overproduction.

The lincosamide family antibiotic lincomycin is a widely used antibacterial pharmaceutical generated by Streptomyces lincolnensis, and the high-yield strain B48 produces 2.5 g/L lincomycin, approximately 30-fold as the wild-type strain NRRL 2936. Here, the genome of S. lincolnensis B48 was completely sequenced, revealing a ~10.0 Mb single chromosome with 71.03% G + C content. Based on the genomic information, lincomycin-related primary metabolism network was constructed and the secondary metabolic potential was analyzed. In order to dissect the overproduction mechanism, a comparative genomic analysis with NRRL 2936 was performed. Three large deletions (LDI-III), one large inverted duplication (LID), one long inversion and 80 small variations (including 50 single nucleotide variations, 13 insertions and 17 deletions) were found in B48 genome. Then several crucial mutants contributing to higher production phenotype were validated. Deleting of a MarR-type regulator-encoding gene slinc377 from LDI, and the whole 24.7 kb LDII in NRRL 2936 enhanced lincomycin titer by 244% and 284%, respectively. Besides, lincomycin production of NRRL 2936 was increased to 7.7-fold when a 71 kb supercluster BGC33 from LDIII was eliminated. As for the duplication region, overexpression of the cluster situated genes lmbB2 and lmbU, as well as two novel transcriptional regulator-encoding genes (slinc191 and slinc348) elevated lincomycin titer by 77%, 75%, 114% and 702%, respectively. Furthermore, three negative correlation genes (slinc6156, slinc4481 and slinc6011) on lincomycin biosynthesis, participating in regulation were found out. And surprisingly, inactivation of RNase J-encoding gene slinc6156 and TPR (tetratricopeptide repeat) domain-containing protein-encoding gene slinc4481 achieved lincomycin titer equivalent to 83% and 68% of B48, respectively, to 22.4 and 18.4-fold compared to NRRL 2936. Therefore, the comparative genomics approach combined with confirmatory experiments identified that large fragment deletion, long sequence duplication, along with several mutations of genes, especially regulator genes, are crucial for lincomycin overproduction.

AdpAlin, a Pleiotropic Transcriptional Regulator, Is Involved in the Cascade Regulation of Lincomycin Biosynthesis in Streptomyces lincolnensis.

Lincomycin is one of the most important antibiotics in clinical practice. To further understand the regulatory mechanism on lincomycin biosynthesis, we investigated a pleiotropic transcriptional regulator AdpAlin in the lincomycin producer Streptomyces lincolnensis NRRL 2936. Deletion of adpA lin (which generated ΔadpA lin ) interrupted lincomycin biosynthesis and impaired the morphological differentiation. We also found that putative AdpA binding sites were unusually scattered in the promoters of all the 8 putative operons in the lincomycin biosynthetic gene cluster (BGC). In ΔadpA lin , transcript levels of structural genes in 8 putative operons were decreased with varying degrees, and electrophoretic mobility shift assays (EMSAs) confirmed that AdpAlin activated the overall putative operons via directly binding to their promoter regions. Thus, we speculated that the entire lincomycin biosynthesis is under the control of AdpAlin. Besides, AdpAlin participated in lincomycin biosynthesis by binding to the promoter of lmbU which encoded a cluster sited regulator (CSR) LmbU of lincomycin biosynthesis. Results of qRT-PCR and catechol dioxygenase activity assay showed that AdpAlin activated the transcription of lmbU. In addition, AdpAlin activated the transcription of the bldA by binding to its promoter, suggesting that AdpAlin indirectly participated in lincomycin biosynthesis and morphological differentiation. Uncommon but understandable, AdpAlin auto-activated its own transcription via binding to its own promoter region. In conclusion, we provided a molecular mechanism around the effect of AdpAlin on lincomycin biosynthesis in S. lincolnensis, and revealed a cascade regulation of lincomycin biosynthesis by AdpAlin, LmbU, and BldA.

LmbU, a Cluster-Situated Regulator for Lincomycin, Consists of a DNA-Binding Domain, an Auto-Inhibitory Domain, and Forms Homodimer.

Few studies were reported about the regulatory mechanism of lincomycin biosynthesis since it was found in 1962. Although we have proved that a cluster-situated regulator (CSR) LmbU (GenBank Accession No. ABX00623.1) positively modulates lincomycin biosynthesis in Streptomyces lincolnensis NRRL 2936, the molecular mechanism of LmbU regulation is still unclear. In this study, we demonstrated that LmbU binds to the target lmbAp by a central DNA-binding domain (DBD), which interacts with the binding sites through the helix-turn-helix (HTH) motif. N-terminal of LmbU includes an auto-inhibitory domain (AID), inhibiting the DNA-binding activity of LmbU. Without the AID, LmbU variant can bind to its own promoter. Interestingly, compared to other LmbU homologs, the homologs within the biosynthetic gene clusters (BGCs) of known antibiotics generally contain N-terminal AIDs, which offer them the abilities to play complex regulatory functions. In addition, cysteine 12 (C12) has been proved to be mainly responsible for LmbU homodimer formation in vitro. In conclusion, LmbU homologs naturally exist in hundreds of actinomycetes, and belong to a new regulatory family, LmbU family. The present study reveals the DBD, AID and dimerization of LmbU, and sheds new light on the regulatory mechanism of LmbU and its homologs.