Lincomycin Biosynthesis Research Articles

TetR family regulator AbrT controls lincomycin production and morphological development in Streptomyces lincolnensis

BackgroundThe TetR family of transcriptional regulators (TFRs), serving as crucial regulators of diverse cellular processes, undergo conformational changes induced by small-molecule ligands, which either inhibit or activate them to modulate target gene expression. Some ligands of TFRs in actinomycetes and their regulatory effects have been identified and studied; however, regulatory mechanisms of the TetR family in the lincomycin-producing Streptomyces lincolnensis remain poorly understood.ResultsIn this study, we found that AbrT (SLCG_1979), a TetR family regulator, plays a pivotal role in regulating lincomycin production and morphological development in S. lincolnensis. Deletion of abrT gene resulted in increased lincomycin A (Lin-A) production, but delayed mycelium formation and sporulation on solid media. AbrT directly or indirectly repressed the expression of lincomycin biosynthetic (lin) cluster genes and activated that of the morphological developmental genes amfC, whiB, and ftsZ. We demonstrated that AbrT bound to two motifs (5′-CGCGTACTCGTA-3′ and 5′-CGTACGATAGCT-3′) present in the bidirectional promoter between abrT and SLCG_1980 genes. This consequently repressed abrT itself and its adjacent gene SLCG_1980 that encodes an arabinose efflux permease. D-arabinose, not naturally occurring as L-arabinose, was identified as the effector molecule of AbrT, reducing its binding affinity to abrT-SLCG_1980 intergenic region. Furthermore, based on functional analysis of the AbrT homologue in Saccharopolyspora erythraea, we inferred that the TetR family regulator AbrT may play an important role in regulating secondary metabolism in actinomycetes.ConclusionsAbrT functions as a regulator for governing lincomycin production and morphological development of S. lincolnensis. Our findings demonstrated that D-arabinose acts as a ligand of AbrT to mediate the regulation of lincomycin biosynthesis in S. lincolnensis. Our findings provide novel insights into ligand-mediated regulation in antibiotic biosynthesis.

Effects of the pleiotropic regulator DasR on lincomycin production in Streptomyces lincolnensis

The lincoamide antibiotic lincomycin, derived from Streptomyces lincolnensis, is widely used for the treatment of infections caused by gram-positive bacteria. As a common global regulatory factor of GntR family, DasR usually exists as a regulatory factor that negatively regulates antibiotic synthesis in Streptomyces. However, the regulatory effect of DasR on lincomycin biosynthesis in S. lincolnensis has not been thoroughly investigated. The present study demonstrates that DasR functions as a positive regulator of lincomycin biosynthesis in S. lincolnensis, and its overexpression strain OdasR exhibits a remarkable 7.97-fold increase in lincomycin production compared to the wild-type strain. The effects of DasR overexpression could be attenuated by the addition of GlcNAc in the medium in S. lincolnensis. Combined with transcriptome sequencing and RT-qPCR results, it was found that most structural genes in GlcNAc metabolism and central carbon metabolism were up-regulated, but the lincomycin biosynthetic gene cluster (lmb) were down-regulated after dasR knock-out. However, DasR binding were detected with the DasR responsive elements (dre) of genes involved in GlcNAc metabolism pathway through electrophoretic mobility shift assay, while they were not observed in the lmb. These findings will provide novel insights for the genetic manipulation of S. lincolnensis to enhance lincomycin production.Key points• DasR is a positive regulator that promotes lincomycin synthesis and does not affect spore production• DasR promotes lincomycin production through indirect regulation• DasR correlates with nutrient perception in S. lincolnensis

Coupled strategy based on regulator manipulation and medium optimization empowers the biosynthetic overproduction of lincomycin

The biosynthesis of bioactive secondary metabolites, specifically antibiotics, is of great scientific and economic importance. The control of antibiotic production typically involves different processes and molecular mechanism. Despite numerous efforts to improve antibiotic yields, joint engineering strategies for combining genetic manipulation with fermentation optimization remain finite. Lincomycin A (Lin-A), a lincosamide antibiotic, is industrially fermented by Streptomyces lincolnensis. Herein, the leucine-responsive regulatory protein (Lrp)-type regulator SLCG_4846 was confirmed to directly inhibit the lincomycin biosynthesis, whereas indirectly controlled the transcription of SLCG_2919, the first reported repressor in S. lincolnensis. Inactivation of SLCG_4846 in the high-yield S. lincolnensis LA219X (LA219XΔ4846) increases the Lin-A production and deletion of SLCG_2919 in LA219XΔ4846 exhibits superimposed yield increment. Given the effect of the double deletion on cellular primary metabolism of S. lincolnensis, Plackett-Burman design, steepest ascent and response surface methodologies were utilized and employed to optimize the seed medium of this double mutant in shake flask, and Lin-A yield using optimal seed medium was significantly increased over the control. Above strategies were performed in a 15-L fermenter. The maximal yield of Lin-A in LA219XΔ4846-2919 reached 6.56 g/L at 216 h, 55.1 % higher than that in LA219X at the parental cultivation (4.23 g/L). This study not only showcases the potential of this strategy to boost lincomycin production, but also could empower the development of high-performance actinomycetes for other antibiotics.

Three new LmbU targets outside lmb cluster inhibit lincomycin biosynthesis in Streptomyces lincolnensis

BackgroundAntibiotics biosynthesis is usually regulated by the cluster-situated regulatory gene(s) (CSRG(s)), which directly regulate the genes within the corresponding biosynthetic gene cluster (BGC). Previously, we have demonstrated that LmbU functions as a cluster-situated regulator (CSR) of lincomycin. And it has been found that LmbU regulates twenty non-lmb genes through comparative transcriptomic analysis. However, the regulatory mode of CSRs’ targets outside the BGC remains unknown.ResultsWe screened the targets of LmbU in the whole genome of Streptomyces lincolnensis and found fourteen candidate targets, among which, eight targets can bind to LmbU by electrophoretic mobility shift assays (EMSA). Reporter assays in vivo revealed that LmbU repressed the transcription of SLINC_0469 and SLINC_1037 while activating the transcription of SLINC_8097. In addition, disruptions of SLINC_0469, SLINC_1037, and SLINC_8097 promoted the production of lincomycin, and qRT-PCR showed that SLINC_0469, SLINC_1037, and SLINC_8097 inhibited transcription of the lmb genes, indicating that all the three regulators can negatively regulate lincomycin biosynthesis.ConclusionsLmbU can directly regulate genes outside the lmb cluster, and these genes can affect both lincomycin biosynthesis and the transcription of lmb genes. Our results first erected the cascade regulatory circuit of LmbU and regulators outside lmb cluster, which provides the theoretical basis for the functional research of LmbU family proteins.

PAS domain containing regulator SLCG_7083 involved in morphological development and glucose utilization in Streptomyces lincolnensis

BackgroundStreptomyces lincolnensis is well known for producing the clinically important antimicrobial agent lincomycin. The synthetic and regulatory mechanisms on lincomycin biosynthesis have been deeply explored in recent years. However, the regulation involved in primary metabolism have not been fully addressed.ResultsSLCG_7083 protein contains a Per-Arnt-Sim (PAS) domain at the N-terminus, whose homologous proteins are highly distributed in Streptomyces. The inactivation of the SLCG_7083 gene indicated that SLCG_7083 promotes glucose utilization, slows mycelial growth and affects sporulation in S. lincolnensis. Comparative transcriptomic analysis further revealed that SLCG_7083 represses eight genes involved in sporulation, cell division and lipid metabolism, and activates two genes involved in carbon metabolism.ConclusionsSLCG_7083 is a PAS domain-containing regulator on morphological development and glucose utilization in S. lincolnensis. Our results first revealed the regulatory function of SLCG_7083, and shed new light on the transcriptional effects of SLCG_7083-like family proteins in Streptomyces.

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.

LcbR1, a newly identified GntR family regulator, represses lincomycin biosynthesis in Streptomyces lincolnensis.

The Actinomycetes Streptomyces lincolnensis is the producer of lincosamide-type antibiotic lincomycin, a widely utilized drug against Gram-positive bacteria and protozoans. In this work, through gene knockout, complementation, and overexpression experiments, we identified LcbR1 (SLINC_1595), a GntR family transcriptional regulator, as a repressor for lincomycin biosynthesis. Deletion of lcbR1 boosted lincomycin production by 3.8-fold, without obvious change in morphological development or cellular growth. The homologues of LcbR1 are widely distributed in Streptomyces. Heterologous expression of SCO1410 from Streptomyces coelicolor resulted in the reduction of lincomycin yield, implying that the function of LcbR1 is conserved across different species. Alignment among sequences upstream of lcbR1 and their homologues revealed a conserved 16-bp palindrome (-TTGAACGATCCTTCAA-), which was further proven to be the recognition motif of LcbR1 by electrophoretic mobility shift assays (EMSAs). Via this motif, LcbR1 suppressed the transcription of lcbR1 and SLINC_1596 sharing the same bi-directional promoter. SLINC_1596, one important target of LcbR1, exerted a positive effect on lincomycin production. As detected by quantitative real-time PCR (qRT-PCR) analyses, the expressions of all selected structural (lmbA, lmbC, lmbJ, lmbV, and lmbW), resistance (lmrA and lmrB) and regulatory genes (lmrC and lmbU) from lincomycin biosynthesis cluster were upregulated in deletion strain ΔlcbR1 at 48h of fermentation, while the mRNA amounts of bldD, glnR, ramR, SLCG_Lrp, and SLCG_2919, previously characterized as the regulators on lincomycin production, were decreased in strain ΔlcbR1, although the regulatory effects of LcbR1 on the above differential expression genes seemed to be indirect. Besides, indicated by EMSAs, the expression of lcbR1 might be regulated by GlnR, SLCG_Lrp, and SLCG_2919, which shows the complexity of the regulatory network on lincomycin biosynthesis. KEY POINTS: • LcbR1 is a novel and conservative GntR family regulator regulating lincomycin production. • LcbR1 modulates the expressions of lcbR1 and SLINC_1596 through a palindromic motif. • GlnR, SLCG_Lrp, and SLCG_2919 can control the expression of lcbR1.

New targets of TetR-type regulator SLCG_2919 for controlling lincomycin biosynthesis in Streptomyces lincolnensis.

The transcription factor (TF)-mediated regulatory network controlling lincomycin production in Streptomyces lincolnensis is yet to be fully elucidated despite several types of associated TFs having been reported. SLCG_2919, a tetracycline repressor (TetR)-type regulator, was the first TF to be characterized outside the lincomycin biosynthetic cluster to directly suppress the lincomycin biosynthesis in S. lincolnensis. In this study, improved genomic systematic evolution of ligands by exponential enrichment (gSELEX), an in vitro technique, was adopted to capture additional SLCG_2919-targeted sequences harboring the promoter regions of SLCG_6675, SLCG_4123-4124, SLCG_6579, and SLCG_0139-0140. The four DNA fragments were confirmed by electrophoretic mobility shift assays (EMSAs). Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) showed that the corresponding target genes SLCG_6675 (anthranilate synthase), SLCG_0139 (LysR family transcriptional regulator), SLCG_0140 (beta-lactamase), SLCG_6579 (cytochrome P450), SLCG_4123 (bifunctional DNA primase/polymerase), and SLCG_4124 (magnesium or magnesium-dependent protein phosphatase) in ΔSLCGL_2919 were differentially increased by 3.3-, 4.2-, 3.2-, 2.5-, 4.6-, and 2.2-fold relative to those in the parental strain S. lincolnensis LCGL. Furthermore, the individual inactivation of these target genes in LCGL reduced the lincomycin yield to varying degrees. This investigation expands on the known DNA targets of SLCG_2919 to control lincomycin production and lays the foundation for improving industrial lincomycin yields via genetic engineering of this regulatory network.

Structure-Function Analysis of the S-Glycosylation Reaction in the Biosynthesis of Lincosamide Antibiotics.

The S-glycosyltransferase LmbT, involved in the biosynthesis of lincomycin A, is the only known enzyme that catalyzes the enzymatic incorporation of rare amino acid L-ergothioneine (EGT) into secondary metabolites. Here, we show the structure and function analyses of LmbT. Our in vitro analysis of LmbT revealed that the enzyme shows promiscuous substrate specificity toward nitrogenous base moieties in the generation of unnatural nucleotide diphosphate (NDP)-D-α-D-lincosamides. Furthermore, the X-ray crystal structures of LmbT in its apo form and in complex with substrates indicated that the large conformational changes of the active site occur upon binding of the substrates, and that EGT is strictly recognized by salt-bridge and cation-π interactions with Arg260 and Trp101, respectively. The structure of LmbT in complex with its substrates, the docking model with the EGT-S-conjugated lincosamide, and the structure-based site-directed mutagenesis analysis revealed the structural details of the LmbT-catalyzed SN 2-like S-glycosylation reaction with EGT.

Structure‐Function Analysis of the S‐Glycosylation Reaction in the Biosynthesis of Lincosamide Antibiotics

AbstractThe S‐glycosyltransferase LmbT, involved in the biosynthesis of lincomycin A, is the only known enzyme that catalyzes the enzymatic incorporation of rare amino acid L‐ergothioneine (EGT) into secondary metabolites. Here, we show the structure and function analyses of LmbT. Our in vitro analysis of LmbT revealed that the enzyme shows promiscuous substrate specificity toward nitrogenous base moieties in the generation of unnatural nucleotide diphosphate (NDP)‐D‐α‐D‐lincosamides. Furthermore, the X‐ray crystal structures of LmbT in its apo form and in complex with substrates indicated that the large conformational changes of the active site occur upon binding of the substrates, and that EGT is strictly recognized by salt‐bridge and cation‐π interactions with Arg260 and Trp101, respectively. The structure of LmbT in complex with its substrates, the docking model with the EGT‐S‐conjugated lincosamide, and the structure‐based site‐directed mutagenesis analysis revealed the structural details of the LmbT‐catalyzed SN2‐like S‐glycosylation reaction with EGT.

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.

Transcriptomics-Guided Investigation of the SLCG_Lrp Regulon Provides New Insights into Its Role for Lincomycin Biosynthesis

Lincomycin industrially produced by Streptomyces lincolnensis can be adopted to treat infections caused by Gram-positive bacteria. SLCG_Lrp, a transcriptional regulator of the Lrp family, was first identified to positively regulate lincomycin biosynthesis. However, the regulatory role of SLCG_Lrp is yet to be elucidated. This study utilized RNA-seq for comparing the transcriptome profile of original-strain LCGL and the ΔSLCGL_Lrp mutant. A total of 244 genes comprising 116 downregulated and 128 upregulated genes were differentially expressed between LCGL and ΔSLCGL_Lrp. An in-depth analysis revealed that SLCG_Lrp promotes nitrate assimilation but inhibits fatty acid metabolism, as well as directly regulates five regulators participating in the modulation of multiple cellular processes. With individual inactivation of those regulatory genes in S. lincolnensis LCGL, we confirmed the FadR transcriptional regulator SLCG_2185 was obviously correlated with lincomycin production and found it to transcriptionally stimulate the lincomycin biosynthetic cluster. Furthermore, SLCG_2185 overexpression in the high-yield S. lincolnensis LA219X promoted lincomycin production by 17.8%, and SLCG_2185 being co-overexpressed with SLCG_Lrp in LA219X increased lincomycin production by 28.1% compared to LA219X. Therefore, this investigation not only provides a direction for further investigations regarding the regulation mechanism of SLCG_Lrp, but also provides a basis for guiding the further improvement of lincomycin levels.

AflQ1-Q2 represses lincomycin biosynthesis via multiple cascades in Streptomyces lincolnensis.

Lincomycin is a broad-spectrum antibiotic and particularly effective against Gram-positive pathogens. Albeit familiar with the biosynthetic mechanism of lincomycin, we know less about its regulation, limiting the rational design for strain improvement. We therefore analyzed two-component systems (TCSs) in Streptomyces lincolnensis, and selected eight TCS gene(s) to construct their deletion mutants utilizing CRISPR/Cas9 system. Among them, lincomycin yield increased in two strains (Δ3900-3901 and Δ5290-5291) while decreased in other four strains (Δ3415-3416, Δ4153-4154, Δ4985, and Δ7949). Considering the conspicuous effect, SLINC_5291-5290 (AflQ1-Q2) was subsequently studied in detail. Its repression on lincomycin biosynthesis was further proved by gene complementation and overexpression. By binding to a 16-bp palindromic motif, the response regulator AflQ1 inhibits the transcription of its encoding gene and the expression of eight operons inside the lincomycin synthetic cluster (headed by lmbA, lmbJ, lmbK, lmbV, lmbW, lmbU, lmrA, and lmrC), as demonstrated by quantitative RT-PCR and electrophoretic mobility shift assays. Besides, the regulatory genes including bldD, glnR, lcbR1, and ramR are also regulated by the TCS. According to the screening towards nitrogen sources, aspartate affects the regulatory behavior of histidine kinase AflQ2. And in return, AflQ1 accelerates aspartate metabolism via ask-asd, asd2, and thrA. In summary, we acquired six novel regulators related to lincomycin biosynthesis, and elucidated the regulatory mechanism of AflQ1-Q2. This highly conserved TCS is a promising target for the construction of antibiotic high-yield strains. KEY POINTS: • AflQ1-Q2 is a repressor for lincomycin production. • AflQ1 modulates the expression of lincomycin biosynthetic and regulatory genes. • Aspartate affects the behavior of AflQ2, and its metabolism is promoted by AflQ1.

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.

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. 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. Altogether, these results indicated that, beyond promoting the morphological development, RamR regulates negatively lincomycin biosynthesis and positively the expression of the nitrogen regulator GlnR. 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.

Insight into L‐DOPA dioxygenase mechanism with 6‐substituted L‐DOPA derivatives

Dioxygenase enzymes are essential protein catalysts for the breakdown of catecholic rings, structural components of plant woody tissue. This powerful chemistry is used in nature to make natural products as well as degrade plant material, but we have a limited understanding of substrate space and mechanism across the superfamily. To this end, we report the syntheses, redox potentials and pKas of L‐3,4‐dihydroxyphenylalanine (L‐DOPA) derivatives substituted at the 6‐position and their characterization as substrates of L‐DOPA dioxygenase from lincomycin biosynthesis in Streptomyces lincolnensis. In particular, the spectroscopic properties of 6‐nitroDOPA provide insight into the steps of the enzymatic mechanism. The applications for dioxygenase cleavage of 6‐substituted L‐DOPA derivatives to natural product biosynthesis will be discussed.

Beyond Self-Resistance: ABCF ATPase LmrC Is a Signal-Transducing Component of an Antibiotic-Driven Signaling Cascade Accelerating the Onset of Lincomycin Biosynthesis.

ABSTRACTIn natural environments, antibiotics are important means of interspecies competition. At subinhibitory concentrations, they act as cues or signals inducing antibiotic production; however, our knowledge of well-documented antibiotic-based sensing systems is limited. Here, for the soil actinobacterium Streptomyces lincolnensis, we describe a fundamentally new ribosome-mediated signaling cascade that accelerates the onset of lincomycin production in response to an external ribosome-targeting antibiotic to synchronize antibiotic production within the population. The entire cascade is encoded in the lincomycin biosynthetic gene cluster (BGC) and consists of three lincomycin resistance proteins in addition to the transcriptional regulator LmbU: a lincomycin transporter (LmrA), a 23S rRNA methyltransferase (LmrB), both of which confer high resistance, and an ATP-binding cassette family F (ABCF) ATPase, LmrC, which confers only moderate resistance but is essential for antibiotic-induced signal transduction. Specifically, antibiotic sensing occurs via ribosome-mediated attenuation, which activates LmrC production in response to lincosamide, streptogramin A, or pleuromutilin antibiotics. Then, ATPase activity of the ribosome-associated LmrC triggers the transcription of lmbU and consequently the expression of lincomycin BGC. Finally, the production of LmrC is downregulated by LmrA and LmrB, which reduces the amount of ribosome-bound antibiotic and thus fine-tunes the cascade. We propose that analogous ABCF-mediated signaling systems are relatively common because many ribosome-targeting antibiotic BGCs encode an ABCF protein accompanied by additional resistance protein(s) and transcriptional regulators. Moreover, we revealed that three of the eight coproduced ABCF proteins of S. lincolnensis are clindamycin responsive, suggesting that the ABCF-mediated antibiotic signaling may be a widely utilized tool for chemical communication.

A putative redox-sensing regulator Rex regulates lincomycin biosynthesis in Streptomyces lincolnensis.

Lincomycin is an important antimicrobial agent which is widely used in clinical and animal husbandry. The biosynthetic pathway of lincomycin comes to light in the past 10 years, however, the regulatory mechanism is still unclear. In this study, a redox-sensing regulator Rex from Streptomyces lincolnensis (Rexlin ) was identified and characterized to affect cell growth and lincomycin biosynthesis. Disruption of rex resulted in an increase in cell growth, but a decrease in lincomycin production. The results of quantitative real-time polymerase chain reaction showed that Rexlin can promote transcription of the regulatory gene lmbU and the structural genes lmbA, lmbC, lmbJ, lmbV, and lmbW. However, electrophoretic mobility shift assay analysis demonstrated that Rexlin can not bind to the promoter regions of these genes above. Findings in this study broadened our horizons in the regulatory mechanism of lincomycin production and laid a foundation for strain improvement of antibiotic producers.

Studies of lincosamide formation complete the biosynthetic pathway for lincomycin A

The structure of lincomycin A consists of the unusual eight-carbon thiosugar core methyllincosamide (MTL) decorated with a pendent N-methylprolinyl moiety. Previous studies on MTL biosynthesis have suggested GDP-ᴅ-erythro-α-ᴅ-gluco-octose and GDP-ᴅ-α-ᴅ-lincosamide as key intermediates in the pathway. However, the enzyme-catalyzed reactions resulting in the conversion of GDP-ᴅ-erythro-α-ᴅ-gluco-octose to GDP-ᴅ-α-ᴅ-lincosamide have not yet been elucidated. Herein, a biosynthetic subpathway involving the activities of four enzymes-LmbM, LmbL, CcbZ, and CcbS (the LmbZ and LmbS equivalents in the closely related celesticetin pathway)-is reported. These enzymes catalyze the previously unknown biosynthetic steps including 6-epimerization, 6,8-dehydration, 4-epimerization, and 6-transamination that convert GDP-ᴅ-erythro-α-ᴅ-gluco-octose to GDP-ᴅ-α-ᴅ-lincosamide. Identification of these reactions completes the description of the entire lincomycin biosynthetic pathway. This work is significant since it not only resolves the missing link in octose core assembly of a thiosugar-containing natural product but also showcases the sophistication in catalytic logic of enzymes involved in carbohydrate transformations.