Thomas Paululat

Diffusion‐ordered NMR spectroscopy was used to monitor the diffusion of guest molecules in DNA hydrogels and related DNA matrices. As guest molecule the highly symmetric hollow‐spherical flavoprotein dodecin was studied. Thermoresponsive hydrogels were formed by self‐assembly via hybridization of linear double‐stranded DNA building blocks of 30 base pairs equipped with sticky ends, i. e. additional overhangs of 15 bases on both ends, which were complementary to each other. This resulted in hydrogels, in which dodecin was freely diffusing. When in contrast self‐assembly was performed with rather short building blocks (9 base pairs + sticky ends of 6 bases), the diffusion of the guest molecule was hampered, but as hybridization was reversible within the timescale of the experiment, the resulting DNA matrix did not behave as a true gel. Apparently true DNA hydrogels with small mesh size can be obtained only, when self‐assembly of short DNA building blocks via hybridization is combined with enzymatic ligation leading to a covalently linked network. In that case, the minimum achievable mesh size should be limited by the diameter of the ligase. While DNA hydrogels are an ideal matrix to host rather large molecules or even living cells, it is a challenge to design pristine DNA hydrogels with mesh sizes sufficiently small to capture guest molecules such as drugs or enzymes in the size of only a few nm.

Rishirilide B was isolated from Streptomyces rishiriensis and Streptomyces bottropensis on the basis of its inhibitory activity towards alpha-2-macroglobulin. The biosynthesis of rishirilide B was investigated by feeding experiments with different ¹³C labelled precursors using the heterologous host Streptomyces albus J1074::cos4 containing a cosmid encoding of the gene cluster responsible for rishirilide B production. NMR spectroscopic analysis of labelled compounds demonstrate that the tricyclic backbone of rishirilide B is a polyketide synthesized from nine acetate units. One of the acetate units is decarboxylated to give a methyl group. The origin of the starter unit was determined to be isobutyrate.

Simocyclinone D8 (1, SD8) has attracted attention due to its highly complex hybrid structure and the unusual way it inhibits bacterial DNA gyrase by preventing DNA binding to the enzyme. Although a hypothesis explaining simocyclinone biosynthesis has been previously proposed, little was proven in vivo due to the genetic inaccessibility of the producer strain. Herein, we report discovery of three new D-type simocyclinones (D9, D10, and D11) produced by Kitasatospora sp. and Streptomyces sp. NRRL B-24484, as well as the identification and annotation of their biosynthetic gene clusters. Unexpectedly, the arrangement of the newly discovered biosynthetic gene clusters is starkly different from the previously published one, despite the nearly identical structures of D8 and D9 simocyclinones. The gene inactivation and expression studies have disproven the role of a modular polyketide synthase (PKS) system in the assembly of the linear dicarboxylic acid. Instead, the new stand-alone ketosynthase genes were shown to be involved in the biosynthesis of the tetraene chain. Additionally, we identified the gene responsible for the conversion of simocyclinone D9 (2, SD9) into D8.