Genombasierte Modellbildung zur Biosynthese von Acarviostatin-Metaboliten in drei Actinoplanes sp. SE50/110-Stämmen

Ortseifen V. Genombasierte Modellbildung zur Biosynthese von Acarviostatin-Metaboliten in drei Actinoplanes sp. SE50/110-Stämmen. Bielefeld: Universität Bielefeld; 2016.Das Sekundärmetabolit Acarbose wird weltweit in der klinischen Behandlung von Diabetes mellitus Typ 2 Patienten eingesetzt. Acarbose ist ein α-Glucosidase Inhibitor und hilft Patienten, ihren Blutzucker besser zu kontrollieren. Das Medikament ist seit 1990 kommerziell erhältlich und zählt zu den meistverkauften Antidiabetika. Das Sekundärmetabolit wird von Stämmen der Klasse Actinobacteria produziert, insbesondere von Actinoplanes sp. SE50/110 sowie vom Wildtyp abgeleiteten Stämmen. In einem ersten Teil wurde die Biosynthese von Acarviostatin-Metaboliten im Acarboseproduzenten 1 (AP1) in Abhängigkeit zur C-Quelle analysiert. Dazu konnte eine Detektionsmethode entwickelt werden, um anschließend die Synthese von Acarviostatin- Metaboliten in AP1 bei Anzucht auf Minimalmedium mit unterschiedlichen Zuckern, respektive Maltose, Glucose, Galactose sowie Zuckermischungen, zu charakterisieren. Es wurde gezeigt, dass die Bildung von Acarbose und deren Homologen in Interdepen- denz zur Kohlenstoffquelle erfolgt. Außerdem werden immer mehrere Acarviostatin- Metabolite in unterschiedlichen Konzentrationen von der Zelle gebildet. Basierend auf den Messungen wurde ein Modell zur Bildung von Haupt- und Nebenkomponenten der Acarviostatin-Metabolite in AP1 entwickelt. Im zweiten Teil der Arbeit wurde die Acarviostatin-Metabolit-Biosynthese in drei Acarboseproduzenten bei Anzucht auf Maltose- (Mal-MM) - sowie Maltotriose-Minimalmedium (MT-MM) betrachtet, sprich in Interdependenz zum Genotyp. Durch Vergleich der Genomsequenzen von AP1, AP2 und AP3 wurden Punktmutationen im Acarbosebiosynthesecluster identifiziert. Das Gen der Acarviosyltransferase AcbD ist von zwei Basen- transitionen betroffen, wobei eine in der Kodierung eines Stopcodons resultiert. Die iden- tifizierten Mutationen in acbD konnten auf Proteom-Ebene bestätigt werden. Aufgrund der zuvor etablierten Detektionsmethode konnte das Acarviostatin-Metabolit-Spektrum in AP1, AP2 und AP3 bei Anzucht auf Maltose- sowie Maltotriose-Minimalmedium charakterisiert werden. Diskrepanzen zur vorher postulierten C-Quellen-assoziierten Produktion der Acarviostatin-Metabolite zeigten sich für den Stamm AP3. Einerseits wurden unter Wachstum der Kultur auf Mal-MM (B) sowohl Acarviosyl-Maltose (Acarbose) als auch Acarviosyl-Maltotriose als Hauptkomponenten gebildet und andererseits zeigte sich bei Wuchs auf MT-MM (B) Acarviosyl-Maltose als einzige Hauptkomponente. Die in Teil 1 entwickelte Modellvorstellung wurde anschließend für die in der Arbeit betrachteten Stämme AP2 und AP3 erweitert. Dem Modell liegen dabei drei Grundannahmen zu Grunde: I) die extrazelluläre Acarviosyltransferaseaktivität fehlt im Acarboseproduzenten 3, II) innerhalb der Zelle erfolgt der Aufbau von Maltotriose aus Maltose durch eine Maltodextrin-Glucosidase und III) Maltotriose kann im Acarboseproduzenten 2 und 3 nicht mehr aufgenommen werden. Für die im Modell beschriebenen Stoffwechselwege konnten in den Genomsequenzen der drei Acarboseproduzenten Gene identifiziert werden, die für die gesuchten Synthese- und Transportprozesse kodieren können. Die vorliegende Dissertation analysiert damit erstmalig die Biosynthese von Acarviostatin-Metaboliten in Abhängigkeit von der C- Quelle im Kulturmedium sowie vom Genotyp des produzierenden Actinoplanes sp. SE50/110-Stammes

On the AAVenue to success: Advances in technologies for AAV production

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Factores que influyen en la dispersión espacio-temporal de broca del café Hypothenemus hampei (Coleoptera: Curculionidae)

La broca del café, Hypothenemus hampei (Ferrari), es la plaga más importante del café, Coffea spp., en el mundo. Este insecto ha sido motivo de numerosos estudios, sin embargo, muchos aspectos de su dispersión se desconocen o requieren ser determinados. La presente investigación tuvo el objetivo de estudiar los factores que influyen en la dispersión espacio-temporal de la broca del café. Dado que la dispersión es un proceso complejo, para ser estudiada se desarrollaron técnicas para la obtención del material biológico con condiciones adecuadas para el estudio, para facilitar el estudio de la morfología del complejo espermático de H. hampei y de marcaje-liberación-recaptura para estudiar la capacidad de vuelo y los factores que la afectan. Los resultados más sobresalientes indican que la espermateca de H. hampei está ubicada en la parte final del abdomen entre la glándula accesoria y el oviducto común y está compuesta por un ducto espermático, músculos espermáticos y una glándula espermática; además, posee músculos al final de su curvatura extrema y su cutícula está finamente reticulada. Los espermatozoides se observaron como filamentos minúsculos, endebles y homogéneos, congregados longitudinalmente dentro de la espermateca. La emergencia masiva de las CH se relacionó con las lluvias y se presentó entre febrero y junio, que corresponde al periodo intercosecha. Todas las CH examinadas tenían espermatozoides en la espermateca, lo q ue sugiere que se habían apareado antes de abandonar el fruto natal. La sobrevivencia de las hembras colonizadoras fue mayor en ambientes humedos y en sustratos diferentes a su hospedero. Durante el periodo de fructificación del café, las hembras se dispersaron mediante vuelo a los glomérulos de frutos adyacentes. Después de colonizar un fruto, la CH perdió su capacidad de vuelo de forma gradual en el transcurso de 5 a 6 días; durante este tiempo, las hembras evaluadas realizar más de un vuelo. Algunas CH marcadas y liberadas se recapturaron a 75 m de distancia del sitio de liberación a las 24 h después de haber sido liberadas. Finalmente, se discute la importancia de los hallazgos de la dispersión espacio-temporal de la broca del café, como información clave para mejorar la comprensión del complejo café-broca, y el manejo de la broca. (Résumé d'auteur

The MalR type regulator AcrC is a transcriptional repressor of acarbose biosynthetic genes in Actinoplanes sp. SE50/110

Wolf T, Droste J, Gren T, et al. The MalR type regulator AcrC is a transcriptional repressor of acarbose biosynthetic genes in Actinoplanes sp. SE50/110. BMC Genomics. 2017;18(1): 562.Background Acarbose is used in the treatment of diabetes mellitus type II and is produced by Actinoplanes sp. SE50/110. Although the biosynthesis of acarbose has been intensively studied, profound knowledge about transcription factors involved in acarbose biosynthesis and their binding sites has been missing until now. In contrast to acarbose biosynthetic gene clusters in Streptomyces spp., the corresponding gene cluster of Actinoplanes sp. SE50/110 lacks genes for transcriptional regulators. Results The acarbose regulator C (AcrC) was identified through an in silico approach by aligning the LacI family regulators of acarbose biosynthetic gene clusters in Streptomyces spp. with the Actinoplanes sp. SE50/110 genome. The gene for acrC, located in a head-to-head arrangement with the maltose/maltodextrin ABC transporter malEFG operon, was deleted by introducing PCR targeting for Actinoplanes sp. SE50/110. Characterization was carried out through cultivation experiments, genome-wide microarray hybridizations, and RT-qPCR as well as electrophoretic mobility shift assays for the elucidation of binding motifs. The results show that AcrC binds to the intergenic region between acbE and acbD in Actinoplanes sp. SE50/110 and acts as a transcriptional repressor on these genes. The transcriptomic profile of the wild type was reconstituted through a complementation of the deleted acrC gene. Additionally, regulatory sequence motifs for the binding of AcrC were identified in the intergenic region of acbE and acbD. It was shown that AcrC expression influences acarbose formation in the early growth phase. Interestingly, AcrC does not regulate the malEFG operon. Conclusions This study characterizes the first known transcription factor of the acarbose biosynthetic gene cluster in Actinoplanes sp. SE50/110. It therefore represents an important step for understanding the regulatory network of this organism. Based on this work, rational strain design for improving the biotechnological production of acarbose can now be implemented

COVID19 Disease Map, a computational knowledge repository of virus-host interaction mechanisms.

Funder: Bundesministerium für Bildung und ForschungFunder: Bundesministerium für Bildung und Forschung (BMBF)We need to effectively combine the knowledge from surging literature with complex datasets to propose mechanistic models of SARS-CoV-2 infection, improving data interpretation and predicting key targets of intervention. Here, we describe a large-scale community effort to build an open access, interoperable and computable repository of COVID-19 molecular mechanisms. The COVID-19 Disease Map (C19DMap) is a graphical, interactive representation of disease-relevant molecular mechanisms linking many knowledge sources. Notably, it is a computational resource for graph-based analyses and disease modelling. To this end, we established a framework of tools, platforms and guidelines necessary for a multifaceted community of biocurators, domain experts, bioinformaticians and computational biologists. The diagrams of the C19DMap, curated from the literature, are integrated with relevant interaction and text mining databases. We demonstrate the application of network analysis and modelling approaches by concrete examples to highlight new testable hypotheses. This framework helps to find signatures of SARS-CoV-2 predisposition, treatment response or prioritisation of drug candidates. Such an approach may help deal with new waves of COVID-19 or similar pandemics in the long-term perspective

Analysis of Gum proteins involved in xanthan biosynthesis throughout multiple cell fractions in a "single-tube"

Hahn J, Koch D, Niehaus K, Ortseifen V. Analysis of Gum proteins involved in xanthan biosynthesis throughout multiple cell fractions in a "single-tube". Journal of Proteomics . 2022;257: 104513.Xanthomonas is a phytopathogenic bacterium and of industrial interest due to its capability to produce xanthan, used as a thickener and emulsifier in the food and non-food industry. Until now, proteome analyses of Xcc lacking a detailed view on the proteins involved in xanthan biosynthesis. The proteins involved in the biosynthesis of this polysaccharide are located near, in or at the cell membrane. This study aims to establish a robust and rapid protocol for a comprehensive proteome analysis of Xcc strains, without the need to isolate different cell fractions. Therefore, a method for the analysis of the whole cell proteome was compared to the isolation of specific fractions regarding the total number of identified proteins, the overlap, and the differences between the approaches. The whole cell proteome analysis with extended peptide separation methods resulted in more than 3254 identified proteins covering 73.1% of the whole proteome. The protocol was used to study xanthan production in a label-free quantification approach. Expression profiles of 8 Gum proteins were compared between the stationary and logarithmic growth phase. Differential expression levels within the operon structure indicate a complex regulatory mechanism for xanthan biosynthesis. Data are available via ProteomeXchange with identifier PXD027261. SIGNIFICANCE: Bacteria are metabolite factories with a wide variety of natural products. Thus, proteome analyses play a crucial role to understand the biological processes within a cell behind the biosynthesis of those metabolites. Proteins involved in the biosynthesis of secreted products are often organised on, in or around the membrane allowing metabolite channelling. Experiments targeting those biosynthesis pathways on protein level often require the analysis of multiple cell fractions like cytosolic, inner, and outer membrane. This is time consuming and demands different protocols. The protocol presented here is a rapid and robust solution to study biosynthetic pathways of biological or biotechnological interest in a single approach on protein level, where gene products are partitioned across multiple cell fractions. The use of a single method also simplifies the comparison of different experiments, for example, production vs. nonproduction conditions. Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved

Microfluidics for Biotechnology: Bridging Gaps to Foster Microfluidic Applications

Ortseifen V, Viefhues M, Wobbe L, Grünberger A. Microfluidics for Biotechnology: Bridging Gaps to Foster Microfluidic Applications. Frontiers in Bioengineering and Biotechnology. 2020;8: 589074.Microfluidics and novel lab-on-a-chip applications have the potential to boost biotechnological research in ways that are not possible using traditional methods. Although microfluidic tools were increasingly used for different applications within biotechnology in recent years, a systematic and routine use in academic and industrial labs is still not established. For many years, absent innovative, ground-breaking and “out-of-the-box” applications have been made responsible for the missing drive to integrate microfluidic technologies into fundamental and applied biotechnological research. In this review, we highlight microfluidics’ offers and compare them to the most important demands of the biotechnologists. Furthermore, a detailed analysis in the state-of-the-art use of microfluidics within biotechnology was conducted exemplarily for four emerging biotechnological fields that can substantially benefit from the application of microfluidic systems, namely the phenotypic screening of cells, the analysis of microbial population heterogeneity, organ-on-a-chip approaches and the characterisation of synthetic co-cultures. The analysis resulted in a discussion of potential “gaps” that can be responsible for the rare integration of microfluidics into biotechnological studies. Our analysis revealed six major gaps, concerning the lack of interdisciplinary communication, mutual knowledge and motivation, methodological compatibility, technological readiness and missing commercialisation, which need to be bridged in the future. We conclude that connecting microfluidics and biotechnology is not an impossible challenge and made seven suggestions to bridge the gaps between those disciplines. This lays the foundation for routine integration of microfluidic systems into biotechnology research procedures

The complete genome sequence of the actinobacterium Streptomyces glaucescens GLA.O (DSM 40922) carrying gene clusters for the biosynthesis of tetracenomycin C, 5`-hydroxy streptomycin, and acarbose.

Ortseifen V, Kalinowski J, Pühler A, Rückert C. The complete genome sequence of the actinobacterium Streptomyces glaucescens GLA.O (DSM 40922) carrying gene clusters for the biosynthesis of tetracenomycin C, 5`-hydroxy streptomycin, and acarbose. J Biotechnol. 2017;262:84-88.The secondary metabolite acarbose is used worldwide in the clinical treatment of diabetes mellitus type 2 patients. Acarbose is a - glucosidase inhibitor and supports patients to control their blood glucose as well as their serum insulin levels. The secondary metabolite is produced by strains of the class Actinobacteria, in particular from Actinoplanes sp. SE50/110, which is a progenitor of today`s production strains. Moreover, secondary metabolite clusters could also be identified in Streptomyces coelicoflavus ZG0656 as well as Streptomyces glaucescens GLA.O. In this study, the genome S. glaucescens GLA.O with focus on the acarbose biosynthesis cluster (gac-cluster) was analyzed. First, the tetracenomycin C and the 5`-hydroxy streptomycin gene clusters could be described completely. Then the gac gene region in S. glaucescens GLA.O is compared to the other known biosynthesis gene cluster. In comparison to Actinoplanes sp. SE50/110 the gac-cluster showed structural variances, like the missing homolog of the glycosyltransferase AcbD in the whole genome of S. glaucescens GLA.O. Due to the lack of the glycosyltransferase, it was of particular interest whether additional acarviose metabolites other than acarbose could be formed. For detection of acarviose metabolites biosynthesis the supernatant of S. glaucescens GLA.O grown in starch supplemented complex media was harvested at 72 and 96 hours. Although a homolog of the known glycosyltransferase is absent, the LC-MS-supported analysis revealed that a spectrum of acarviose metabolites was formed

Corynebacterium glutamicum ggtB encodes a functional gamma-glutamyl transpeptidase with gamma-glutamyl dipeptide synthetic and hydrolytic activity

Walter F, Grenz S, Ortseifen V, Persicke M, Kalinowski J. Corynebacterium glutamicum ggtB encodes a functional gamma-glutamyl transpeptidase with gamma-glutamyl dipeptide synthetic and hydrolytic activity. Journal of Biotechnology. 2016;232:99-109.In this work the role of gamma-glutamyl transpeptidase in the metabolism of gamma-glutamyl dipeptides produced by Corynebacterium glutamicum ATCC 13032 was studied. The enzyme is encoded by the gene ggtB (cg1090) and synthesized as a 657 amino acids long preprotein. Gamma-glutamyl transpeptidase activity was found to be associated with intact cells of C. glutamicum and was abolished upon deletion of ggtB. Bioinformatic analysis indicated that the enzyme is a lipoprotein and is attached to the outer side of the cytoplasmic membrane. Biochemical parameters of recombinant GgtB were determined using the chromogenic substrate gamma-glutamyl-p-nitroanilide. Highest activity of the enzyme was measured in sodium bicarbonate buffer at pH 9.6 and 45 degrees C. The K-M value was 123 mu M. GgtB catalyzed the concentration-dependent synthesis and hydrolysis of gamma-glutamyl dipeptides and showed strong glutaminase activity. The intracellular concentrations of five gamma-glutamyl dipeptides (gamma-Glu-Glu,gamma-Glu-Glu,gamma-Glu-Val, gamma-Glu-Met) were determined by HPLC-MS and ranged from 0.15 to 0.4 mg/g CDW after exponential growth in minimal media. Although deletion and overexpression of ggtB had significant effects on intracellular dipeptide concentrations, it was neither essential for biosynthesis nor catabolism of these dipeptides in vivo. (C) 2015 Elsevier B.V. All rights reserved

Flavin-Dependent Halogenases from Xanthomonas campestris pv. campestris B100 Prefer Bromination over Chlorination

Ismail M, Frese M, Patschkowski T, Ortseifen V, Niehaus K, Sewald N. Flavin-Dependent Halogenases from Xanthomonas campestris pv. campestris B100 Prefer Bromination over Chlorination. ADVANCED SYNTHESIS & CATALYSIS. 2019;361(11):2475-2486.Flavin-dependent halogenases selectively introduce halogen substituents into (hetero-)aromatic substrates and require only molecular oxygen and halide salts for this regioselective oxidative CH-functionalization. Genomic analysis of Xanthomonas campestris pv. campestris B100 identified three novel putative members of this enzyme class. They were shown to introduce halogen substituents into, e. g., substituted indoles, while preferring bromide over chloride