Transcriptional Regulation of the Daptomycin Gene Cluster in Streptomyces roseosporus by an Autoregulator, AtrA

Mao, Xu‐Ming; Luo, Shuai; Zhou, Ri-Cheng; Wang, Feng; Yu, Ping; Sun, Ning; Chen, Xiaoxia; Tang, Yi; Li, Yongquan

doi:10.1074/jbc.m114.608273

Cited by 67 publications

(85 citation statements)

References 27 publications

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“…Electrophoretic mobility shift assays (EMSAs) showed that DepR1 could specifically bind to dptEp and that this shift became even more significant as the amount of DepR1 was increased (Fig. 1C), which also was seen in other studies of, e.g., AtrA-dptEp interaction (21). Moreover, one binding site of DepR1 on dptEp was identified by DNase I footprinting assays ( Fig.…”

Section: Resultssupporting

confidence: 80%

“…However, the binding sequence had no consensus with other TetR family regulators or apparent palindrome sequence, which often are recognized by TetR family regulators (35). However, this observation is not unique, since we had identified another TetR family regulator, AtrA, which binds to nonpalindrome sequence either on dptEp or its own promoter (21). These results confirmed DepR1 was a transcriptional regulator interacting directly with the promoter of the daptomycin gene cluster.…”

Section: Resultsmentioning

confidence: 70%

“…This suggests that the daptomycin gene cluster does not contain the typical pathway-specific regulators within most other such clusters in secondary metabolites. To further understand the transcriptional regulatory mechanisms of daptomycin production, we used streptavidin-biotin interaction-based affinity purification to isolate the regulators binding to the promoter of the daptomycin gene cluster, dptEp (21). Using this approach, several DNA-binding proteins, including two TetR family transcriptional regulators, AtrA (21) and DepR1 (this study), were identified.…”

Section: Resultsmentioning

confidence: 99%

“…To further understand the transcriptional regulatory mechanisms of daptomycin production, we used streptavidin-biotin interaction-based affinity purification to isolate the regulators binding to the promoter of the daptomycin gene cluster, dptEp (21). Using this approach, several DNA-binding proteins, including two TetR family transcriptional regulators, AtrA (21) and DepR1 (this study), were identified. Here, we have demonstrated that DepR1 was bound to dptEp at a different site from that of AtrA (21) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Using this strategy, we identified AtrA, a TetR family regulator, which acts as one of the regulators of a conserved A-factor-mediated transcriptional cascade. This pathway positively regulates daptomycin gene cluster expression, and a more than 2-fold increase of daptomycin production was achieved by targeted genetic engineering to delete arpA, encoding the conserved A-factor receptor (21).…”

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confidence: 99%

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DepR1, a TetR Family Transcriptional Regulator, Positively Regulates Daptomycin Production in an Industrial Producer, Streptomyces roseosporus SW0702

Yuan

Zhou

Chen

et al. 2016

Appl Environ Microbiol

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Daptomycin is a potent cyclic lipopeptide antibiotic. It is widely used against various Gram-positive bacterial pathogens. Historically, a poor understanding of the transcriptional regulation of daptomycin biosynthesis has limited the options for targeted genetic engineering toward titer improvement. Here, we isolated a TetR family transcriptional regulator, DepR1, from the industrial producer Streptomyces roseosporus SW0702 using a biotinylated dptE promoter (dptEp) as a probe. The direct interaction between DepR1 and dptEp then was confirmed by electrophoretic mobility shift assays and DNase I footprinting assays. The deletion of depR1 led to a reduction in dptEp activity and the cessation of daptomycin production. The ⌬depR1 mutant produced less red pigment and failed to sporulate on R5 medium. This suggests that DepR1 plays a positive role in the control of morphological differentiation. Moreover, DepR1 was positively autoregulated by directly binding to its own promoter. This might account for the positive feedback regulation of daptomycin production. Based on these positive effects, genetic engineering by overexpression of depR1 raised daptomycin production and shortened the fermentation period both in flask and in fermentor. Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus via nonribosomal peptide synthases (1). It contains 13 amino acids and a straight-chain decanoic acid attached to the terminal amino group of Trp (2). In the presence of Ca 2ϩ , the lipid tail can insert into the bilayer membrane of pathogens. This results in potassium efflux, membrane depolarization, and eventual cell death (3). Daptomycin has been approved for the clinical treatment of diseases caused by drug-resistant Gram-positive bacterial pathogens. Such pathogens include vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, and others (4-7).Due to its importance in clinical medicine, several approaches have been employed for the production of daptomycin derivatives. These include semisynthetic modification (8, 9), chemoenzymatic synthesis (10, 11), and combinatorial biosynthesis (12)(13)(14), with the anticipation of altered pharmaceutical spectra and/or increased antimicrobial activities. In addition, many efforts have been made to increase daptomycin production. These have included random mutagenesis (15), in silico-based metabolic engineering (16,17), and attempts to increase precursor supply (18). However, up to the present, the poor understanding of the transcriptional regulation of daptomycin biosynthesis has limited options for targeted genetic engineering toward titer improvement.The gene cluster for daptomycin biosynthesis originally was identified in Streptomyces roseosporus NRRL 11379. Three putative cluster-situated transcriptional regulators, DptR1, DptR2, and DptR3 (1), also have been identified. We previously reported that the industrial strain S. roseosporus SW0702 also contained a similar daptomycin gene clus...

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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DepR1, a TetR Family Transcriptional Regulator, Positively Regulates Daptomycin Production in an Industrial Producer, Streptomyces roseosporus SW0702

Yuan

Zhou

Chen

et al. 2016

Appl Environ Microbiol

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Degradation mechanism of AtrA mediated by ClpXP and its application in daptomycin production in Streptomyces roseosporus

Sun

Gao

et al. 2023

Protein Science

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The efficiency of drug biosynthesis depends on different transcriptional regulatory pathways in Streptomyces, and the protein degradation system adds another layer of complexity to the regulatory processes. AtrA, a transcriptional regulator in the A‐factor regulatory cascade, stimulates the production of daptomycin by binding to the dptE promoter in Streptomyces roseosporus. Using pull‐down assays, bacterial two‐hybrid system and knockout verification, we demonstrated that AtrA is a substrate for ClpP protease. Furthermore, we showed that ClpX is necessary for AtrA recognition and subsequent degradation. Bioinformatics analysis, truncating mutation, and overexpression proved that the AAA motifs of AtrA were essential for initial recognition in the degradation process. Finally, overexpression of mutated atrA (AAA‐QQQ) in S. roseosporus increased the yield of daptomycin by 225% in shake flask and by 164% in the 15 L bioreactor. Thus, improving the stability of key regulators is an effective method to promote the ability of antibiotic synthesis.

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TetR and OmpR family regulators in natural product biosynthesis and resistance

Patil,

Sharma,

Bhaskarwar

et al. 2023

Proteins

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This article provides a comprehensive review and sequence‐structure analysis of transcription regulator (TR) families, TetR and OmpR/PhoB, involved in specialized secondary metabolite (SSM) biosynthesis and resistance. Transcription regulation is a fundamental process, playing a crucial role in orchestrating gene expression to confer a survival advantage in response to frequent environmental stress conditions. This process, coupled with signal sensing, enables bacteria to respond to a diverse range of intra and extracellular signals. Thus, major bacterial signaling systems use a receptor domain to sense chemical stimuli along with an output domain responsible for transcription regulation through DNA‐binding. Sensory and output domains on a single polypeptide chain (one component system, OCS) allow response to stimuli by allostery, that is, DNA‐binding affinity modulation upon signal presence/absence. On the other hand, two component systems (TCSs) allow cross‐talk between the sensory and output domains as they are disjoint and transmit information by phosphorelay to mount a response. In both cases, however, TRs play a central role. Biosynthesis of SSMs, which includes antibiotics, is heavily regulated by TRs as it diverts the cell's resources towards the production of these expendable compounds, which also have clinical applications. These TRs have evolved to relay information across specific signals and target genes, thus providing a rich source of unique mechanisms to explore towards addressing the rapid escalation in antimicrobial resistance (AMR). Here we focus on the TetR and OmpR family TRs, which belong to OCS and TCS, respectively. These TR families are well‐known examples of regulators in secondary metabolism and are ubiquitous across different bacteria, as they also participate in a myriad of cellular processes apart from SSM biosynthesis and resistance. As a result, these families exhibit higher sequence divergence, which is also evident from our bioinformatic analysis of 158 389 and 77 437 sequences from TetR and OmpR family TRs, respectively. The analysis of both sequence and structure allowed us to identify novel motifs in addition to the known motifs responsible for TR function and its structural integrity. Understanding the diverse mechanisms employed by these TRs is essential for unraveling the biosynthesis of SSMs. This can also help exploit their regulatory role in biosynthesis for significant pharmaceutical, agricultural, and industrial applications.

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Transcriptional Regulation of the Daptomycin Gene Cluster in Streptomyces roseosporus by an Autoregulator, AtrA

Cited by 67 publications

References 27 publications

DepR1, a TetR Family Transcriptional Regulator, Positively Regulates Daptomycin Production in an Industrial Producer, Streptomyces roseosporus SW0702

DepR1, a TetR Family Transcriptional Regulator, Positively Regulates Daptomycin Production in an Industrial Producer, Streptomyces roseosporus SW0702

Degradation mechanism of AtrA mediated by ClpXP and its application in daptomycin production in Streptomyces roseosporus

TetR and OmpR family regulators in natural product biosynthesis and resistance

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