Complex Regulatory Networks Governing Production of the Glycopeptide A40926

Alduina, Rosa; Sosio, Margherita; Donadio, Stefano

doi:10.3390/antibiotics7020030

Cited by 15 publications

(13 citation statements)

References 45 publications

(71 reference statements)

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“…The data presented here and in previous work 2,5,6 allow us to refine a possible model for regulation of glycopeptide production in N. gerenzanensis 1 . When cells are actively growing, transcription of dbv3 and dbv4 is repressed by the direct action of Dbv6, as well as by other factors related to growth (nutritional factors, or quorum sensing systems could control A40926 biosynthesis) 5 .…”

Section: Strain or Plasmid Descriptionsupporting

confidence: 56%

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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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Section: Strain or Plasmid Descriptionsupporting

confidence: 56%

“…The dbv gene cluster for acA40926 biosynthesis contains 37 protein-coding sequences (Fig. 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 .…”

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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

Self Cite

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“…Although the global regulation of GPA biosynthesis is still largely unexplored, the pathway-specific regulation controlling the expression of BGCs is being elucidated in model systems (Bibb, 2013). The roles of cluster-situated regulatory genes have been investigated in Amycolatopsis balhimycina (Shawky et al, 2007), Nonomuraea gerenzanensis (Lo Grasso et al, 2015;Alduina et al, 2018), and Actinoplanes teichomyceticus (Horbal et al, 2014b;Yushchuk et al, 2019), producing balhimycin, A40926 (the natural precursor of dalbavancin), and teicoplanin, respectively. Overexpression of the teicoplanin cluster-situated regulatory genes (tei15*, coding for a StrR-like transcriptional regulator, and tei16*, coding for a LuxR-type regulator) in A. teichomyceticus markedly increased teicoplanin production in the wild type strain, representing one of the most successful examples of using molecular tools for improving antibiotic production (Horbal et al, 2012(Horbal et al, , 2014b.…”

Section: Introductionmentioning

confidence: 99%

New Molecular Tools for Regulation and Improvement of A40926 Glycopeptide Antibiotic Production in Nonomuraea gerenzanensis ATCC 39727

et al. 2020

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Genome sequencing has revealed that Nonomuraea spp. represent a still largely unexplored source of specialized metabolites. Nonomuraea gerenzanensis ATCC 39727 is the most studied representative species since it produces the glycopeptide antibiotic (GPA) A40926the precursor of the clinically relevant antibiotic dalbavancin, approved by the FDA in 2014 for the treatment of acute skin infections caused by multi-drug resistant Gram-positive pathogens. The clinical relevance of dalbavancin has prompted increased attention on A40926 biosynthesis and its regulation. In this paper, we investigated how to enhance the genetic toolkit for members of the Nonomuraea genus, which have proved quite recalcitrant to genetic manipulation. By constructing promoter-probe vectors, we tested the activity of 11 promoters (heterologous and native) using the GusA reporter system in N. gerenzanensis and in Nonomuraea coxensis; this latter species is phylogenetically distant from N. gerenzanesis and also possesses the genetic potential to produce A40926 or a very similar GPA. Finally, the strongest constitutive promoter analyzed in this study, aac(3) IVp, was used to overexpress the cluster-situated regulatory genes controlling A40926 biosynthesis (dbv3 and dbv4 from N. gerenzanensis and nocRI from N. coxensis) in N. gerenzanensis, and the growth and productivity of the best performing strains were assessed at bioreactor scale using an industrial production medium. Overexpression of positive pathway-specific regulatory genes resulted in a significant increase in the level of A40926 production in N. gerenzanensis, providing a new knowledge-based approach to strain improvement for this valuable glycopeptide antibiotic.

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“…Other important symbiotic relationship takes place between ants and Streptomyces. In recent years, new strains and compounds have been isolated from ant head, legs and nests [73][74][75][76].…”

Section: Symbiosis Relationshipsmentioning

confidence: 99%

“…Sometimes defined medium can be better for determined compounds. Optimization of nutritional requirements has been one of the most useful changes for secondary metabolite activities [73][74][75][76].…”

Section: Changing Media Componentsmentioning

confidence: 99%

Streptomyces as a Source of Antimicrobials: Novel Approaches to Activate Cryptic Secondary Metabolite Pathways

Manteca¹,

Yagüe²

2019

Antimicrobials, Antibiotic Resistance, Antibiofilm Strategies and Activity Methods

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Streptomyces is the most important bacterial genus for bioactive compound production. These soil bacteria are characterized by a complex differentiation cycle. Streptomyces is extremely important in biotechnology, producing approximately two thirds of all antibiotics, as well as many compounds of medical and agricultural interest. Drug discovery from streptomycetes became challenging once the most common compounds were discovered, and the system was basically abandoned by industry. Simultaneously, antibiotic resistance is increasing dramatically, and new antibiotics are required. Screening from nature is being resumed (exploring new environments, looking for elicitors, metagenome, etc.). Secondary metabolism is conditioned by differentiation; although the relationship between both has long remained elusive, differentiation as a trigger for antibiotic production remains basically unexplored. Most cultures used in screening campaigns for new bioactive molecules have been performed empirically, and workflow was extremely productive during the so-called golden age of antibiotics; however, currently there is a bottleneck. Streptomyces is still the most important natural source of antibiotics, and it also harbors many cryptic secondary metabolite pathways not expressed under laboratory conditions. In this chapter, we review strategies based on differentiation, one of the keys improving secondary metabolite production and activating cryptic pathways to face the challenges of drug discovery.

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Complex Regulatory Networks Governing Production of the Glycopeptide A40926

Cited by 15 publications

References 45 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

New Molecular Tools for Regulation and Improvement of A40926 Glycopeptide Antibiotic Production in Nonomuraea gerenzanensis ATCC 39727

Streptomyces as a Source of Antimicrobials: Novel Approaches to Activate Cryptic Secondary Metabolite Pathways

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