Degradation mechanism of <scp>AtrA</scp> mediated by <scp>ClpXP</scp> and its application in daptomycin production in <i>Streptomyces roseosporus</i>

Xu, Wei-Feng; Sun, Cuirong; Gao, Wen-Li; Scharf, Daniel H.; Zhu, Chenyang; Bu, Qing-Ting; Zhao, Qinyu; Li, Yongquan

doi:10.1002/pro.4617

Cited by 3 publications

(2 citation statements)

References 31 publications

(41 reference statements)

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“…Recently, S. roseosporus AtrA was shown to be recognized by ClpX and directed to the ClpP protease for degradation. The authors also mutated the recognized AAA motifs of AtrA to increase the yield of daptomycin in the strain (35).…”

Section: Introductionmentioning

confidence: 99%

The gamma-butyrolactone receptors ScbR and AtrA form a quorum sensing switch between coelimycin and actinorhodin synthesis inStreptomyces coelicolorA3(2)

Bednarz,

Kotowska,

Wenecki

et al. 2024

Preprint

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BackgroundQuorum sensing enables gene expression regulation in response to changes in cell population density and controls diverse processes, such as biofilm formation, virulence and antibiotic production, in bacteria. In one of the largest, soil-dominant phylumActinobacteria, cell-to-cell communication occurs through the small, membrane-diffusible signalling molecules gamma-butyrolactones (GBLs). Their actions are exerted through receptor proteins that also act as response regulators in a one-component system manner. With only a few GBL systems characterized, most of them come from the large, antibiotic-producer genusStreptomyces. In the model organismStreptomyces coelicolorA3(2), two GBL receptors, ScbR and SlbR, which are both antibiotic production repressors, have been reported so far.ResultsIn this work, we identified a new GBL receptor protein, the conserved and pleiotropic regulator AtrA, which has an activating mode of action. Moreover, we elucidated the precise mechanism by which it controls the production of the antibiotic actinorhodin through the actinorhodin biosynthetic gene cluster activator ActII-orf4. GBL binding to AtrA prevents its binding to the promoter of theactII-orf4gene, thereby disabling its transcription, while at the same time, GBL binding to ScbR causes coelimycin antibiotic synthesis derepression.ConclusionsThe opposite modes of action of ScbR (repressor) and AtrA (activator) have opposite effects upon GBL binding, activating coelimycin and blocking actinorhodin production at the same time. This phenomenon constitutes an elegant regulatory mechanism that ensures that coelimycin and actinorhodin production are mutually exclusive. These findings also suggest that quorum sensing must be taken into account when designing efficient antibiotic production processes and can be manipulated to ensure both better yield and specificity.

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Section: Introductionmentioning

confidence: 99%

The gamma-butyrolactone receptors ScbR and AtrA form a quorum sensing switch between coelimycin and actinorhodin synthesis inStreptomyces coelicolorA3(2)

Bednarz,

Kotowska,

Wenecki

et al. 2024

Preprint

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“…Genes dptEFABCD comprise a large operon to produce a giant polycistronic transcript for daptomycin biosynthesis ( Miao et al, 2005 ; Baltz, 2009 ). Based on the genome mining of transcription factors and deciphering of regulatory mechanisms, the removal of the negative regulators ( Mao et al, 2015 ; Mao et al, 2017 ; Luo et al, 2018 ) and stabilization of the positive pleiotropic regulator AtrA ( Xu et al, 2023 ) have significantly increased daptomycin production. Meanwhile, targeting multi-level rational metabolic engineering by precursor supply, regulatory pathway reconstruction, byproduct removal, and gene cluster multiplicity have improved the yield by about 6.6 folds ( Lyu et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

Development of a native-locus dual reporter system for the efficient screening of the hyper-production of natural products in Streptomyces

Zhou

Zhao

et al. 2023

Front. Bioeng. Biotechnol.

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Streptomyces is renowned for its abundant production of bioactive secondary metabolites, but most of these natural products are produced in low yields. Traditional rational network refactoring is highly dependent on the comprehensive understanding of regulatory mechanisms and multiple manipulations of genome editing. Though random mutagenesis is fairly straightforward, it lacks a general and effective strategy for high throughput screening of the desired strains. Here in an antibiotic daptomycin producer S. roseosporus, we developed a dual-reporter system at the native locus of the daptomycin gene cluster. After elimination of three enzymes that potentially produce pigments by genome editing, a gene idgS encoding the indigoidine synthetase and a kanamycin resistant gene neo were integrated before and after the non-ribosomal peptidyl synthetase genes for daptomycin biosynthesis, respectively. After condition optimization of UV-induced mutagenesis, strains with hyper-resistance to kanamycin along with over-production of indigoidine were efficiently obtained after one round of mutagenesis and target screening based on the dual selection of the reporter system. Four mutant strains showed increased production of daptomycin from 1.4 to 6.4 folds, and significantly improved expression of the gene cluster. Our native-locus dual reporter system is efficient for targeting screening after random mutagenesis and would be widely applicable for the effective engineering of Streptomyces species and hyper-production of these invaluable natural products for pharmaceutical development.

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

Cited by 3 publications

References 31 publications

The gamma-butyrolactone receptors ScbR and AtrA form a quorum sensing switch between coelimycin and actinorhodin synthesis inStreptomyces coelicolorA3(2)

The gamma-butyrolactone receptors ScbR and AtrA form a quorum sensing switch between coelimycin and actinorhodin synthesis inStreptomyces coelicolorA3(2)

Development of a native-locus dual reporter system for the efficient screening of the hyper-production of natural products in Streptomyces

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

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