TY - JOUR A1 - Dehm, Daniel A1 - Krumbholz, Julia A1 - Baunach, Martin A1 - Wiebach, Vincent A1 - Hinrichs, Katrin A1 - Guljamow, Arthur A1 - Tabuchi, Takeshi A1 - Jenke-Kodama, Holger A1 - Süssmuth, Roderich D. A1 - Dittmann-Thünemann, Elke T1 - Unlocking the spatial control of secondary metabolism uncovers hidden natural product diversity in nostoc punctiforme JF - ACS chemical biology N2 - Filamentous cyanobacteria belong to the most prolific producers of structurally unique and biologically active natural products, yet the majority of biosynthetic gene clusters predicted for these multicellular collectives are currently orphan. Here, we present a systems analysis of secondary metabolite gene expression in the model strain Nostoc punctiforme PCC73102 using RNA-seq and fluorescence reporter analysis. Our data demonstrate that the majority of the cryptic gene clusters are not silent but are expressed with regular or sporadic pattern. Cultivation of N. punctiforme using high-density fermentation overrules the spatial control and leads to a pronounced upregulation of more than 50% of biosynthetic gene clusters. Our data suggest that a combination of autocrine factors, a high CO2 level, and high light account for the upregulation of individual pathways. Our overarching study not only sheds light on the strategies of filamentous cyanobacteria to share the enormous metabolic burden connected with the production of specialized molecules but provides an avenue for the genome-based discovery of natural products in multicellular cyanobacteria as exemplified by the discovery of highly unusual variants of the tricyclic peptide microviridin. Y1 - 2019 U6 - https://doi.org/10.1021/acschembio.9b00240 SN - 1554-8929 SN - 1554-8937 VL - 14 IS - 6 SP - 1271 EP - 1279 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Messerschmidt, Katrin A1 - Hochrein, Lena A1 - Dehm, Daniel A1 - Schulz, Karina A1 - Mueller-Roeber, Bernd T1 - Characterizing seamless ligation cloning extract for synthetic biological applications JF - Analytical biochemistry : methods in the biological sciences N2 - Synthetic biology aims at designing and engineering organisms. The engineering process typically requires the establishment of suitable DNA constructs generated through fusion of multiple protein coding and regulatory sequences. Conventional cloning techniques, including those involving restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments must be joined or scar-free fusions are mandatory. Overlap-based-cloning methods have the potential to overcome such limitations. One such method uses seamless ligation cloning extract (SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone (up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology regions are easily tolerated and the deletion of unwanted vector sequences concomitant with targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion method suitable for cloning projects in virtually all molecular. and synthetic biology projects. (C) 2016 Elsevier Inc. All rights reserved. KW - SLiCE KW - Seamless ligation cloning KW - Homologous recombination KW - Synthetic biology Y1 - 2016 U6 - https://doi.org/10.1016/j.ab.2016.05.029 SN - 0003-2697 SN - 1096-0309 VL - 509 SP - 24 EP - 32 PB - Elsevier CY - San Diego ER -