Two Master Switch Regulators Trigger A40926 Biosynthesis in Nonomuraea sp. Strain ATCC 39727

Grasso, Letizia Lo; Maffioli, Sonia; Sosio, Margherita; Bibb, Mervyn J.; Puglia, Anna Maria; Alduina, Rosa

doi:10.1128/jb.00262-15

Cited by 38 publications

(64 citation statements)

References 36 publications

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“…1C) that participate in antibiotic biosynthesis, regulation, immunity, and export 1,4 . Specifically, the cluster encodes the positive regulators Dbv3 and Dbv4 that indirectly or directly activate different operons leading to acA40926 biosynthesis 5,6 . Dbv3 and Dbv4 belong to the LAL (large ATP binding regulators of LuxR) and the StrR family of regulators, respectively.…”

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confidence: 99%

A Two-Component regulatory system with opposite effects on glycopeptide antibiotic biosynthesis and resistance

Alduina

Tocchetti²,

Costa

et al. 2020

Sci Rep

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The glycopeptide A40926, produced by the actinomycete Nonomuraea gerenzanensis, is the precursor of dalbavancin, a second-generation glycopeptide antibiotic approved for clinical use in the USA and Europe in 2014 and 2015, respectively. The final product of the biosynthetic pathway is an O-acetylated form of A40926 (acA40926). Glycopeptide biosynthesis in N. gerenzanensis is dependent upon the dbv gene cluster that encodes, in addition to the two essential positive regulators Dbv3 and Dbv4, the putative members of a two-component signal transduction system, specifically the response regulator Dbv6 and the sensor kinase Dbv22. The aim of this work was to assign a role to these two genes. Our results demonstrate that deletion of dbv22 leads to an increased antibiotic production with a concomitant reduction in glycopeptide resistance. Deletion of dbv6 results in a similar phenotype, although the effects are not as strong as in the Δdbv22 mutant. Consistently, quantitative RT-PCR analysis showed that Dbv6 and Dbv22 negatively regulate the regulatory genes (dbv3 and dbv4), as well as some dbv biosynthetic genes (dbv23 and dbv24), whereas Dbv6 and Dbv22 positively regulate transcription of the single, cluster-associated resistance gene. Finally, we demonstrate that exogenously added acA40926 and its precursor A40926 can modulate transcription of dbv genes but with an opposite extent: A40926 strongly stimulates transcription of the Dbv6/Dbv22 target genes while acA40926 has a neutral or negative effect on transcription of those genes. We propose a model in which glycopeptide biosynthesis in N. gerenzanensis is modulated through a positive feedback by the biosynthetic precursor A40926 and a negative feedback by the final product acA40926. In addition to previously reported control systems, this sophisticated control loop might help the producing strain cope with the toxicity of its own product. This work, besides leading to improved glycopeptide producing strains, enlarges our knowledge on the regulation of glycopeptide biosynthesis in actinomycetes, setting N. gerenzanensis and its two-component system Dbv6-Dbv22 apart from other glycopeptide producers. The glycopeptide antibiotic A40926 (Fig. 1A) is the precursor for dalbavancin (Fig. 1B), a semisynthetic lipoglycopeptide clinically used for treatment of acute skin infections caused by methicillin-susceptible and methicillin-resistant Staphylococcus aureus and Streptococcus pyogenes. A40926 is produced by the actinomycete N. gerenzanensis as a complex of related compounds, differing mostly by the type of N-acyl chain attached to the glucuronic acid moiety. The core structure of A40926 consists of a heptapeptide containing the proteinogenic amino acid tyrosine and the non-proteinogenic amino acids 3,5-dihydroxyphenylglycine (DPG) and 4-hydroxyphenylglycine (HPG). The heptapeptide is assembled by a nonribosomal peptide synthetase (NRPS) and modified by the action of various enzymes: four oxygenases, one halogenase, one hydroxylase, two glycosyltransferases, one hexose ox...

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confidence: 99%

A Two-Component regulatory system with opposite effects on glycopeptide antibiotic biosynthesis and resistance

Alduina

Tocchetti²,

Costa

et al. 2020

Sci Rep

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“…In addition, the pathway-specific regulators are also important for the regulation of the biosynthesis of secondary metabolites. The large ATP-binding regulators of the LuxR family (LAL regulators) constitute a large family of pathway-specific regulators, and many regulators in this family have been identified in actinobacteria, such as AveR (20), PikD (21), RapH (22), GdmRI and GdmRII (23,24), SlnR (25), Dbv3 (26), and Tei16* (27). However, as no LuxI proteins have been found in actinobacteria and no N-acyl-L-homoserine lactone (AHL)-sensing system has been reported for Gram-positive bacteria, LuxR family regulators in actinobacteria are unlikely to be controlled by quorum-sensing signals similar to those in Vibrio fischeri (28).…”

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RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

Liu

Shao

et al. 2017

Appl Environ Microbiol

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Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium leprae. Although the biosynthetic pathway of rifamycin has been extensively studied in Amycolatopsis mediterranei, little is known about the regulation in rifamycin biosynthesis. Here, an in vivo transposon system was employed to identify genes involved in the regulation of rifamycin production in A. mediterranei U32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted in AMED_0655 (rifZ, encoding a LuxR family regulator). The rifZ gene was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster (rif cluster) was remarkably reduced in the rifZ null mutant. Based on the cotranscription assay results, genes within the rif cluster were grouped into 10 operons, sharing six promoter regions. By use of electrophoretic mobility shift assay and DNase I footprinting assay, RifZ was proved to specially bind to all six promoter regions, which was consistent with the fact that RifZ regulated the transcription of the whole rif cluster. The binding consensus sequence was further characterized through alignment using the RifZ-protected DNA sequences. By use of bionformatic analysis, another five promoters containing the RifZ box (CTACC-N8-GGATG) were identified, among which the binding of RifZ to the promoter regions of both rifK and orf18 (AMED_0645) was further verified. As RifZ directly regulates the transcription of all operons within the rif cluster, we propose that RifZ is a pathwayspecific regulator for the rif cluster.IMPORTANCE To this day, rifamycin and its derivatives are still the first-line antituberculosis drugs. The biosynthesis of rifamycin has been extensively studied, and most biosynthetic processes have been characterized. However, little is known about the regulation of the transcription of the rifamycin biosynthetic gene cluster (rif cluster), and no regulator has been characterized. Through the employment of transposon screening, we here characterized a LuxR family regulator, RifZ, as a direct transcriptional activator for the rif cluster. As RifZ directly regulates the transcription of the entire rif cluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation of rif cluster transcription, RifZ may provide a new clue for further engineering of highyield industrial strains.

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“…Heterologous expression provides a useful, but sometimes challenging approach to enable the production of a single series of compounds from a cryptic cluster of interest thereby enabling the compound's identification; such an approach represents the only way for accessing the chemical pallet of unculturable microbes. Metabolic switching in actinomycetes and the regulation of secondary metabolite production is highly complex and varied [120][121][122] and such expression systems often require careful tailoring in order to enable production of a specific compound of interest; this can often be a bottle-neck to the discovery process.…”

Section: Discussionmentioning

confidence: 99%

Prospecting for new bacterial metabolites: a glossary of approaches for inducing, activating and upregulating the biosynthesis of bacterial cryptic or silent natural products

et al. 2016

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Over the centuries, microbial secondary metabolites have played a central role in the treatment of human diseases and have revolutionised the pharmaceutical industry. With the increasing number of sequenced microbial genomes revealing a plethora of novel biosynthetic genes, natural product drug discovery is entering an exciting second golden age. Here, we provide a concise overview as an introductory guide to the main methods employed to unlock or up-regulate these so called 'cryptic', 'silent' and 'orphan' gene clusters, and increase the production of the encoded natural product. With a predominant focus on bacterial natural products we will discuss the importance of the bioinformatics approach for genome mining, the use of first different and simple culturing techniques and then the application of genetic engineering to unlock the microbial treasure trove.

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Two Master Switch Regulators Trigger A40926 Biosynthesis in Nonomuraea sp. Strain ATCC 39727

Cited by 38 publications

References 36 publications

A Two-Component regulatory system with opposite effects on glycopeptide antibiotic biosynthesis and resistance

A Two-Component regulatory system with opposite effects on glycopeptide antibiotic biosynthesis and resistance

RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

Prospecting for new bacterial metabolites: a glossary of approaches for inducing, activating and upregulating the biosynthesis of bacterial cryptic or silent natural products

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