Merzhausen, Germany
Merzhausen, Germany

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Burgard C.,Helmholtz Center for Infection Research | Zaburannyi N.,Helmholtz Center for Infection Research | Nadmid S.,Helmholtz Center for Infection Research | Maier J.,IStLS Information Services to Life science | And 4 more authors.
ACS Chemical Biology | Year: 2017

Analysis of 122 myxobacterial genome sequences suggested 16 strains as producers of the myxochromide lipopeptide family. Detailed sequence comparison of the respective mch biosynthetic gene clusters informed a genome-mining approach, ultimately leading to the discovery and chemical characterization of four novel myxochromide core types. The myxochromide megasynthetase is subject to evolutionary diversification, resulting in considerable structural diversity of biosynthesis products. The observed differences are due to the number, type, sequence, and configuration of the incorporated amino acids. The analysis revealed molecular details on how point mutations and recombination events led to structural diversity. It also gave insights into the evolutionary scenarios that have led to the emergence of mch clusters in different strains and genera of myxobacteria. © 2017 American Chemical Society.


Grant
Agency: European Commission | Branch: H2020 | Program: RIA | Phase: BIOTEC-1-2014 | Award Amount: 8.06M | Year: 2015

Mycoplasmas are the smallest cell wall less, free-living microorganisms. The lack of a cell wall makes them resistant to many of the common antibiotics. Every year, infections caused by Mycoplasmas in poultry, cows, and pigs, result in multimillion euros losses in USA and Europe. Currently, there are vaccines against M hyopneumoniae in pigs and M gallisepticum and M synoviae in poultry. However, there is no vaccination against many Mycoplasma species infecting pets, humans and farm animals (ie M bovis cow infection). Mycoplasma species in many cases are difficult to grown in axenic culture and those that grow need a complex media with animal serum. In large scale production of Mycoplasma species for vaccination aside from the high cost of animal serum, more important is the high irreproducibility in the production process and the possible contamination with animal viruses. All this together highlights what European industry needs:i) a defined cheap reproducible medium that is animal serum free and ii) an universal Mycoplasma chassis that could be used in a pipeline to vaccinate against Mycoplasma species, as well as any pathogen. M pneumoniae is an ideal starting point for designing such a vaccine chassis. It has a small genome (860 kb) and it is probably the organism with the most comprehensive systems biology data acquired so far. By genome comparison, metabolic modeling and rationally engineering its genome, we will create a vaccine chassis that will be introduced into an industrial pipeline. The process will be guided by the second world largest industry on animal vaccination (MSD), as well as a SME specialized on peptide display and screening. This will ensure the exploitation and commercialization of our work contributing to maintain Europe privileged position in this field. Our ultimate goal is to meet the needs of the livestock industry,taking care of ethical issues, foreseeable risks, and prepare effective dissemination and training material for the public.


Osswald C.,Saarland University | Zipf G.,ATG-biosynthetics | Schmidt G.,ATG-biosynthetics | Maier J.,IStLS | And 3 more authors.
ACS Synthetic Biology | Year: 2014

Natural products of microbial origin continue to be an important source of pharmaceuticals and agrochemicals exhibiting potent activities and often novel modes of action. Due to their inherent structural complexity chemical synthesis is often hardly possible, leaving fermentation as the only viable production route. In addition, the pharmaceutical properties of natural products often need to be optimized for application by sophisticated medicinal chemistry and/or biosynthetic engineering. The latter requires a detailed understanding of the biosynthetic process and genetic tools to modify the producing organism that are often unavailable. Consequently, heterologous expression of complex natural product pathways has been in the focus of development over recent years. However, piecing together existing DNA cloned from natural sources and achieving efficient expression in heterologous circuits represent several limitations that can be addressed by synthetic biology. In this work we have redesigned and reassembled the 56 kb epothilone biosynthetic gene cluster from Sorangium cellulosum for expression in the high GC host Myxococcus xanthus. The codon composition was adapted to a modified codon table for M. xanthus, and unique restriction sites were simultaneously introduced and others eliminated from the sequence in order to permit pathway assembly and future interchangeability of modular building blocks from the epothilone megasynthetase. The functionality of the artificial pathway was demonstrated by successful heterologous epothilone production in M. xanthus at significant yields that have to be improved in upcoming work. Our study sets the stage for future engineering of epothilone biosynthesis and production optimization using a highly flexible assembly strategy. (Chemical Equation Presented). © 2012 American Chemical Society.


Gemperlein K.,Helmholtz Center for Infection Research | Zipf G.,ATG-biosynthetics | Bernauer H.S.,ATG-biosynthetics | Muller R.,Helmholtz Center for Infection Research | Wenzel S.C.,Helmholtz Center for Infection Research
Metabolic Engineering | Year: 2016

Long-chain polyunsaturated fatty acids (LC-PUFAs) can be produced de novo via polyketide synthase-like enzymes known as PUFA synthases, which are encoded by pfa biosynthetic gene clusters originally discovered from marine microorganisms. Recently similar gene clusters were detected and characterized in terrestrial myxobacteria revealing several striking differences. As the identified myxobacterial producers are difficult to handle genetically and grow very slowly we aimed to establish heterologous expression platforms for myxobacterial PUFA synthases. Here we report the heterologous expression of the pfa gene cluster from Aetherobacter fasciculatus (SBSr002) in the phylogenetically distant model host bacteria Escherichia coli and Pseudomonas putida. The latter host turned out to be the more promising PUFA producer revealing higher production rates of n-6 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). After several rounds of genetic engineering of expression plasmids combined with metabolic engineering of P. putida, DHA production yields were eventually increased more than threefold. Additionally, we applied synthetic biology approaches to redesign and construct artificial versions of the A. fasciculatus pfa gene cluster, which to the best of our knowledge represents the first example of a polyketide-like biosynthetic gene cluster modulated and synthesized for P. putida. Combination with the engineering efforts described above led to a further increase in LC-PUFA production yields. The established production platform based on synthetic DNA now sets the stage for flexible engineering of the complex PUFA synthase. © 2015 International Metabolic Engineering Society.


The paper reports on a study of carbon-based fullerenes C n (n = 60, 70 and 80) with the encapsulated trigonal bipyramid Co 5 cluster on the basis of the Perdew-Burke-Ernzerhof generalized gradient approximation functional (GGA-PBE) within the Density Functional Theory (DFT). It is revealed that magnetic moments of the Co 5@C n complexes depend on the fullerene size and shape, as well as orientation of Co 5 cluster in a carbon cage in the case of fullerene with D 5d point group symmetry. © 2011 Elsevier B.V. All rights reserved.


The present invention relates to a method for generating an RNA library or a (poly)peptide library comprising the steps of: (a) providing one or more nucleic acid molecules each comprising i) two or more coding elements (A) each giving rise to an RNA molecule upon transcription and/or a (poly)peptide upon transcription and translation; and ii) linking elements (B) arranged according to the general formula of B(AB)_(2+n), wherein said linking elements comprise one or more sequence motifs not found in said two or more coding elements allowing specific disruption of the linking elements (B); (b) cloning the nucleic acid molecule of step (a) into a vector; (c) transforming a host cell with the vector obtained in step (b) and propagating said transformed cell; (d) preparing vector DNA from the transformed and propagated cells of step (c); (e) (i) disrupting the vector DNA obtained in step (d) with one or more agents recognizing said one or more sequence motifs of the linking elements or (ii) performing an amplification step with the vector DNA obtained in step (d) and primers hybridizing to the sequence of said linking elements so that the sequences comprising the coding elements (A) are specifically amplified; (f) cloning the resulting coding elements (A) of step (e) into vectors; (g) transforming the vectors obtained in step (f) into host cells and establishing clonal colonies; and (h) culturing said clonal colonies under conditions suitable to express the coding elements. Also, the method relates to an RNA library or a (poly)peptide library obtainable or obtained according to the method of the invention. Moreover, the invention relates to a method for identifying a (poly)peptide epitope recognized by an antibody or a (poly)peptide-binding compound and a method for identifying a (poly)peptide epitope recognized by antibodies in serum. Further, the invention relates to a method for generating protein variants. Finally, the invention relates to a nucleic acid molecule a used in the method of the invention, a vector comprising the same, a cell comprising said nucleic acid molecule or said vector and a kit comprising one or more items selected from the group of said nucleic acid molecule, said vector, said cell, said RNA or a (poly)peptide library of the invention and, optionally, instructions for use.


Trademark
ATG-biosynthetics | Date: 2012-01-24

Chemicals used in industry, science and photography, as well as in agriculture, horticulture, and forestry, except fungicides, herbicides, insecticides and parasiticides. Scientific and technological services, namely, scientific research, analysis, testing and technical consulting in the field of molecular biology and product research relating thereto; industrial analysis and research services in the field of molecular biology.


PubMed | Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures, Helmholtz Center for Infection Research and ATG-biosynthetics
Type: Journal Article | Journal: Applied and environmental microbiology | Year: 2016

Here, we report the complete genome sequence of the type strain of the myxobacterial genus Chondromyces, Chondromyces crocatus Cm c5. It presents one of the largest prokaryotic genomes featuring a single circular chromosome and no plasmids. Analysis revealed an enlarged set of tRNA genes, along with reduced pressure on preferred codon usage compared to that of other bacterial genomes. The large coding capacity and the plethora of encoded secondary metabolite biosynthetic gene clusters are in line with the capability of Cm c5 to produce an arsenal of antibacterial, antifungal, and cytotoxic compounds. Known pathways of the ajudazol, chondramide, chondrochloren, crocacin, crocapeptin, and thuggacin compound families are complemented by many more natural compound biosynthetic gene clusters in the chromosome. Whole-genome comparison of the fruiting-body-forming type strain (Cm c5, DSM 14714) to an accustomed laboratory strain which has lost this ability (nonfruiting phenotype, Cm c5 fr-) revealed genetic changes in three loci. In addition to the low synteny found with the closest sequenced representative of the same family, Sorangium cellulosum, extensive genetic information duplication and broad application of eukaryotic-type signal transduction systems are hallmarks of this 11.3-Mbp prokaryotic genome.


PubMed | ATG-biosynthetics and Helmholtz Center for Infection Research
Type: | Journal: Metabolic engineering | Year: 2016

Long-chain polyunsaturated fatty acids (LC-PUFAs) can be produced de novo via polyketide synthase-like enzymes known as PUFA synthases, which are encoded by pfa biosynthetic gene clusters originally discovered from marine microorganisms. Recently similar gene clusters were detected and characterized in terrestrial myxobacteria revealing several striking differences. As the identified myxobacterial producers are difficult to handle genetically and grow very slowly we aimed to establish heterologous expression platforms for myxobacterial PUFA synthases. Here we report the heterologous expression of the pfa gene cluster from Aetherobacter fasciculatus (SBSr002) in the phylogenetically distant model host bacteria Escherichia coli and Pseudomonas putida. The latter host turned out to be the more promising PUFA producer revealing higher production rates of n-6 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). After several rounds of genetic engineering of expression plasmids combined with metabolic engineering of P. putida, DHA production yields were eventually increased more than threefold. Additionally, we applied synthetic biology approaches to redesign and construct artificial versions of the A. fasciculatus pfa gene cluster, which to the best of our knowledge represents the first example of a polyketide-like biosynthetic gene cluster modulated and synthesized for P. putida. Combination with the engineering efforts described above led to a further increase in LC-PUFA production yields. The established production platform based on synthetic DNA now sets the stage for flexible engineering of the complex PUFA synthase.

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