Evaluation of heterologous promoters for genetic analysis of Actinoplanes teichomyceticus—Producer of teicoplanin, drug of last defense

Horbal, Liliya; Kobylyanskyy, Anton; Yushchuk, Oleksandr; Zaburannyi, Nestor; Luzhetskyy, Andriy; Ostash, Bohdan; Marinelli, Flavia; Fedorenko, Victor

doi:10.1016/j.jbiotec.2013.10.018

Cited by 25 publications

(32 citation statements)

References 26 publications

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“…To induce P21-cmt-driven gusA-expression, cumate was added at the final concentration of 50 μM to cultures carrying pGCymRP21 24 h after inoculation. After 120 h of cultivation, mycelial lysates were prepared as previously reported by Horbal et al, 2013. Glucuronidase activity was measured as previously described (Myronovskyi et al, 2011;Horbal et al, 2013) (Marcone et al, 2014).…”

Section: β-Glucuronidase Activity Assaymentioning

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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Section: β-Glucuronidase Activity Assaymentioning

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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“…As a result, plasmid pSETmoeEtchmH5 (Table 1) was obtained. A 2.4 kb XbaI/EcoRI fragment, containing the tchmH5 gene fused with the moeE5p promoter, was retrieved from pSETmoeE5Asp and cloned into the respective site of pOOB47a (Ostash et al 2013). This yielded pOOBPmoetchmH5 ( Table 1).…”

Section: Identification Of the Tchm Gene Clustermentioning

confidence: 99%

“…Flasks were incubated at 30°C and 220 rpm for 5 days. Moenomycin was extracted from biomass as previously described (Ostash et al 2013). Obtained samples were directly used for the LC-MS or MS 2 .…”

Section: Identification Of the Tchm Gene Clustermentioning

confidence: 99%

“…For the complementation experiments, we constructed plasmids pOOBPmoetchmH5 and pOOBPmoetchmH5-hyg (Table 1) where tchmH5 was placed under the control of the NDP-hexose epimerase gene moeE5 promoter (moeE5p) (Horbal et al 2013). The pOOBPmoetchmH5 and pOOBPmoetchmH5-hyg were transferred into S. coelicolor M1152 moeno 38-6 + and S. ghanaensis dH5, respectively.…”

Section: )mentioning

confidence: 99%

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A gene cluster for the biosynthesis of moenomycin family antibiotics in the genome of teicoplanin producer Actinoplanes teichomyceticus

Horbal

Ostash

Luzhetskyy

et al. 2016

Appl Microbiol Biotechnol

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“…24,[26][27][28][29][30] Recently, significant progress has been achieved in the development of a molecular toolkit for NRRL-B16726, which enables the deletion or overexpression of genes, and the analysis of promoter activity. [31][32][33][34] The biosynthesis of Tcp can be divided into a three-stage process: (a) production of dedicated non-proteinogenic amino acid precursors; 35,36 (b) peptide assembly, halogenation and oxidative crosslinking of the heptapeptide scaffold; 22,30,36-39 (c) modification of the scaffold via glycosylation and acylation. 24,26,40-42 These late-stage tailoring reactions are essential for the optimal biological activity of Tcp; 9,15,17,44,45 the acyl chain is even a major determinant of Tcp activity against certain vancomycin-resistant strains.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the Post-Assembly Line Tailoring Processes in Teicoplanin Biosynthesis

et al. 2016

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Actinoplanes teichomyceticus produces teicoplanin (Tcp), a "last resort" lipoglycopeptide antibiotic used to treat severe multidrug resistant infections such as methicillin-resistant Staphylococcus aureus (MRSA). A number of studies have addressed various steps of Tcp biosynthesis using in vitro assays, although the exact sequence of Tcp peptide core tailoring reactions remained speculative. Here, we describe the generation and analysis of a set of A. teichomyceticus mutant strains that have been used to elucidate the sequence of reactions from the Tcp aglycone to mature Tcp. By combining these results with previously published data, we propose an updated order of post-assembly line tailoring processes in Tcp biosynthesis. We also demonstrate that the acyl-CoA-synthetase Tei13* and the type II thioesterase Tei30* are dispensable for Tcp production. Five Tcp derivatives featuring hitherto undescribed combinations of glycosylation and acylation patterns are described. The generation of strains that produce novel Tcp analogues now provides a platform for the production of additional Tcp-like molecules via combinatorial biosynthesis or chemical derivatization.

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Evaluation of heterologous promoters for genetic analysis of Actinoplanes teichomyceticus—Producer of teicoplanin, drug of last defense

Cited by 25 publications

References 26 publications

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

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

A gene cluster for the biosynthesis of moenomycin family antibiotics in the genome of teicoplanin producer Actinoplanes teichomyceticus

Characterization of the Post-Assembly Line Tailoring Processes in Teicoplanin Biosynthesis

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