Helmholtz Institute for Pharmaceutical Research

Saarbrücken, Germany

Helmholtz Institute for Pharmaceutical Research

Saarbrücken, Germany
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Schaefer J.,University of Marburg | Schulze C.,Saarland University | Marxer E.E.J.,University of Marburg | Schaefer U.F.,Saarland University | And 4 more authors.
ACS Nano | Year: 2012

Upon contact with the human body, nanomaterials are known to interact with the physiological surroundings, especially with proteins. In this context, we explored analytical methods to provide biologically relevant information, in particular for manufactured nanomaterials as produced by the chemical industry. For this purpose, we selected two batches of SiO 2 nanoparticles as well as four batches of CeO 2 nanoparticles, each of comparably high chemical purity and similar physicochemical properties. Adsorption of serum proteins and bovine serum albumin (BSA) was quantified by SDS-PAGE in combination with densitometry and further investigated by atomic force microscopy (AFM) and analytical ultracentrifugation (AUC). The protein adsorption to SiO 2 nanoparticles was below the limit of detection, regardless of adjusting pH or osmolality to physiological conditions. In contrast, the four CeO 2 nanomaterials could be classified in two groups according to half-maximal protein adsorption. Measuring the work of adhesion and indention by AFM for the BSA-binding CeO 2 nanomaterials revealed the same classification, pointing to alterations in shape of the adsorbed protein. The same trend was also reflected in the agglomeration behavior/dispersibility of the four CeO 2 nanomaterials as revealed by AUC. We conclude that even small differences in physicochemical particle properties may nevertheless lead to differences in protein adsorption, possibly implicating a different disposition and other biological responses in the human body. Advanced analytical methods such as AFM and AUC may provide valuable additional information in this context. © 2012 American Chemical Society.


Myronovskyi M.,Albert Ludwigs University of Freiburg | Myronovskyi M.,Ivan Franko National University of Lviv | Welle E.,Albert Ludwigs University of Freiburg | Fedorenko V.,Ivan Franko National University of Lviv | Luzhetskyy A.,Helmholtz Institute for Pharmaceutical Research
Applied and Environmental Microbiology | Year: 2011

Here we describe a versatile and sensitive reporter system for actinomycetes that is based on gusA, which encodes the β-Glucuronidase enzyme. A series of gusA-containing transcriptional and translational fusion vectors were constructed and utilized to study the regulatory cascade of the phenalinolactone biosynthetic gene cluster. Furthermore, these vectors were used to study the efficiency of translation initiation at the ATG, GTG, TTG, and CTG start codons. Surprisingly, constructs using a TTG start codon showed the best activity, whereas those using ATG or GTG were approximately one-half or one-third as active, respectively. The CTG fusion showed only 5% of the activity of the TTG fusion. A suicide vector, pKGLP2, carrying gusA in its backbone was used to visually detect merodiploid formation and resolution, making gene targeting in actinomycetes much faster and easier. Three regulatory genes, plaR1, plaR2, and plaR3, involved in phenalinolactone biosynthesis were efficiently replaced with an apramycin resistance marker using this system. Finally, we expanded the genetic code of actinomycetes by introducing the nonproteinogenic amino acid N-epsiloncyclopentyloxycarbonyl-L-lysine with the GusA protein as a reporter. © 2011, American Society for Microbiology.


Horbal L.,Saarland University | Horbal L.,Ivan Franko National University of Lviv | Ostash B.,Ivan Franko National University of Lviv | Luzhetskyy A.,Saarland University | And 4 more authors.
Applied Microbiology and Biotechnology | Year: 2016

Moenomycins are phosphoglycolipid antibiotics notable for their extreme potency, unique mode of action, and proven record of use in animal nutrition without selection for resistant microflora. There is a keen interest in manipulation of structures of moenomycins in order to better understand their structure-activity relationships and to generate improved analogs. Only two almost identical moenomycin biosynthetic gene clusters are known, limiting our knowledge of the evolution of moenomycin pathways and our ability to genetically diversify them. Here, we report a novel gene cluster (tchm) that directs production of the phosphoglycolipid teichomycin in Actinoplanes teichomyceticus. Its overall genetic architecture is significantly different from that of the moenomycin biosynthesis (moe) gene clusters of Streptomyces ghanaensis and Streptomyces clavuligerus, featuring multiple gene rearrangements and two novel structural genes. Involvement of the tchm cluster in teichomycin biosynthesis was confirmed via heterologous co-expression of amidotransferase tchmH5 and moe genes. Our work sets the background for further engineering of moenomycins and for deeper inquiries into the evolution of this fascinating biosynthetic pathway. © 2016 Springer-Verlag Berlin Heidelberg


Ostash B.,Ivan Franko National University of Lviv | Ostash B.,Harvard University | Campbell J.,Harvard University | Campbell J.,Fraunhofer Institute for Manufacturing Innovation | And 2 more authors.
Molecular Microbiology | Year: 2013

Summary: The biosynthesis of the phosphoglycolipid antibiotic moenomycin A attracts the attention of researchers hoping to develop new moenomycin-based antibiotics against multidrug resistant Gram-positive infections. There is detailed understanding of most steps of this biosynthetic pathway in Streptomyces ghanaensis (ATCC14672), except for the ultimate stage, where a single pentasaccharide intermediate is converted into a set of unusually modified final products. Here we report that only one gene, moeH5, encoding a homologue of the glutamine amidotransferase (GAT) enzyme superfamily, is responsible for the observed diversity of terminally decorated moenomycins. Genetic and biochemical evidence support the idea that MoeH5 is a novel member of the GAT superfamily, whose homologues are involved in the synthesis of various secondary metabolites as well as K and O antigens of bacterial lipopolysaccharide. Our results provide insights into the mechanism of MoeH5 and its counterparts, and give us a new tool for the diversification of phosphoglycolipid antibiotics. © 2013 John Wiley & Sons Ltd.


Bilyk O.,Helmholtz Institute for Pharmaceutical Research | Luzhetskyy A.,Helmholtz Institute for Pharmaceutical Research | Luzhetskyy A.,Saarland University
Current Opinion in Biotechnology | Year: 2016

Actinomycetes are known to produce over two-thirds of all known secondary metabolites. We review here recent progress in the metabolic engineering of streptomycetes for natural product biosynthesis. Several examples of the yield improvement of polyketides (mithramycin and tylactone) and non-ribosomal peptides (balhimycin and daptomycin) demonstrate the power of precursor supply engineering. Another example is the manipulation of a regulatory network for increased production of nystatin and teicoplanin. The second part highlights new approaches in the derivatization of natural products via combination of mutasynthesis and genomic engineering. © 2016 Elsevier Ltd.


Horbal L.,Helmholtz Institute for Pharmaceutical Research | Horbal L.,Saarland University | Luzhetskyy A.,Helmholtz Institute for Pharmaceutical Research | Luzhetskyy A.,Saarland University
Metabolic Engineering | Year: 2016

Here, we present a novel scaffolding architecture of an inducible regulatory device. This dual control system is completely silent in the off stage and is coupled to the regulation of gene expression at both the transcriptional and translational levels. This system also functions as an AND gate. We demonstrated the effectiveness of the cumate-riboswitch dual control system for the control of pamamycin production in Streptomyces albus. Placing the cre recombinase gene under the control of this system permitted the construction of synthetic devices with non-volatile memory that sense the signal and respond by altering DNA at the chromosomal level, thereby producing changes that are heritable. In addition, we present a library of synthetic inducible promoters based on the previously described cumate switch. With only one inducer and different promoters, we demonstrate that simultaneous modulation of the expression of several genes to different levels in various operons is possible. Because all modules of the AND gates are functional in bacteria other than Streptomyces, we anticipate that these regulatory devices can be used to control gene expression in other Actinobacteria. The features described in this study make these systems promising tools for metabolic engineering and biotechnology in Actinobacteria. © 2016 International Metabolic Engineering Society.


Myronovskyi M.,Helmholtz Institute for Pharmaceutical Research | Rosenkranzer B.,Helmholtz Institute for Pharmaceutical Research | Luzhetskyy A.,Helmholtz Institute for Pharmaceutical Research
Applied Microbiology and Biotechnology | Year: 2014

The deletions of large genomic DNA fragments and consecutive gene knockouts are prerequisites for the generation of organisms with improved properties. One of the key issues in this context is the removal of antibiotic resistance markers from engineered organisms without leaving an active recombinase recognition site. Here, we report the establishment of an iterative marker excision system (IMES) that solves this problem. Based on the phiC31 integrase and its mutant att sites, IMES can be used for highly effective deletion of DNA fragments between inversely oriented B-CC and P-GG sites. The B-CC and P-GG sites are derived from attB and attP by substitution of the central core TT dinucleotide with CC and GG, respectively. An unnatural RR site that resides in the chromosome following deletion is the joining product of the right shoulders of B-CC and P-GG. We show that the RR sites do not recombine with each other as well as the RR site recombines with B-CC. The recombination efficiencies between RR and P-GG or RR and LL are only 0.1 % and 1 %, respectively. Thus, IMES can be used for multistep genomic engineering without risking unwanted DNA recombination. The fabrication of multi-purpose antibiotic cassettes and examples of the utilisation of IMES are described. © 2014 Springer-Verlag.


PubMed | Helmholtz Institute for Pharmaceutical Research
Type: Journal Article | Journal: Applied and environmental microbiology | Year: 2011

Here we describe a versatile and sensitive reporter system for actinomycetes that is based on gusA, which encodes the -glucuronidase enzyme. A series of gusA-containing transcriptional and translational fusion vectors were constructed and utilized to study the regulatory cascade of the phenalinolactone biosynthetic gene cluster. Furthermore, these vectors were used to study the efficiency of translation initiation at the ATG, GTG, TTG, and CTG start codons. Surprisingly, constructs using a TTG start codon showed the best activity, whereas those using ATG or GTG were approximately one-half or one-third as active, respectively. The CTG fusion showed only 5% of the activity of the TTG fusion. A suicide vector, pKGLP2, carrying gusA in its backbone was used to visually detect merodiploid formation and resolution, making gene targeting in actinomycetes much faster and easier. Three regulatory genes, plaR1, plaR2, and plaR3, involved in phenalinolactone biosynthesis were efficiently replaced with an apramycin resistance marker using this system. Finally, we expanded the genetic code of actinomycetes by introducing the nonproteinogenic amino acid N-epsilon-cyclopentyloxycarbonyl-l-lysine with the GusA protein as a reporter.

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