SEQUENCING AND ANALYSIS OF PUTATIVE 3D-4H RING CYCLASE GENE lndF OF STREPTOMYCES GLOBISPORUS 1912 LANDOMYCIN E BIOSYNTHETIC GENE CLUSTER

DNA fragment of landomycin E biosynthesis gene cluster 700 bp in size has been completely sequenced. 3?-end of lndE (oxygenase) was identified, 5?-end of lndA (ketosynthase) and entire ORF for previously not sequenced lanF homologue, lndF. Analysis of lndF putative translation product revealed that it is highly conservative in comparison with other known cyclases from antibiotic biosynthesis gene clusters. Unlike urdamycin, jadomycin biosynthetic clusters, lndF and lndA are uncoupled, as well as genes lanF and lanA. Genes lndF and lndA are not preceded by direct or inverted repeats, putative sites for binding of transcriptional activator LndI. In contrast, lanF is flanked at it's 5?-end by three direct repeats, possible target for regulatory protein LanI.L. Castro is acknowledged for help in sequencing. This work was supported by INTAS grant YSF 00-208 to B.O

Design, development and application of whole cell based antibiotic specific biosensor

Synthetic biology techniques hold great promise for optimising the production of natural products by microorganisms. However, evaluating the phenotype of a modified bacterium represents a major bottleneck to the engineering cycle particularly for antibiotic producing actinobacteria strains, which grow slowly and are challenging to genetically manipulate. Here, we report the generation and application of antibiotic specific whole cell biosensor derived from TetR transcriptional repressor for use in identifying and optimising antibiotic producers. The constructed biosensor was successfully used to improve production of polyketide antibiotic pamamycin. However, an initial biosensor based on native genetic elements had inadequate dynamic and operating ranges. To overcome these limitations, we fine tuned biosensor performance through alterations of the promoter and operator of output module and the ligand affinity of transcription factor module, which enabled us to deduce recommendations for building and application of actinobacterial biosensor

Microparticles globally reprogram Streptomyces albus

Kuhl M, Gläser L, Rebets Y, et al. Microparticles globally reprogram Streptomyces albus toward accelerated morphogenesis, streamlined carbon core metabolism and enhanced production of the antituberculosis polyketide pamamycin. Biotechnology and Bioengineering. 2020.Streptomyces spp. are a rich source for natural products with recognized industrial value, explaining the high interest to improve and streamline production in these microbes. Here, we studied the production of pamamycins, macrodiolide homologues with a high activity against multi-resistant pathogenic microbes, using recombinant S. albus J1074/R2. Talc particles (hydrous magnesium silicate, 3MgO·4SiO2 ·H2 O) of micrometer size added to submerged cultures of the recombinant strain tripled pamamycin production up to 50 mg L-1 . Furthermore, they strongly affected morphology, reduced the size of cell pellets, formed by the filamentous microbe during the process, up to six-fold, and shifted the pamamycin spectrum to larger derivatives. Integrated analysis of transcriptome and precursor (CoA thioester) supply of particle-enhanced and control cultures provided detailed insights into the underlying molecular changes. The microparticles affected the expression of 3341 genes (56%), revealing a global and fundamental impact on metabolism. Morphology-associated genes, encoding major regulators such as SsgA, RelA, EshA, Factor C, as well as chaplins and rodlins, were found massively upregulated, indicating that the particles caused a substantially accelerated morphogenesis. In line, the pamamycin cluster was strongly upregulated (up to 1024-fold). Furthermore, the microparticles perturbed genes encoding for CoA-ester metabolism, which were mainly activated. The altered expression resulted in changes in the availability of intracellular CoA-esters, the building blocks of pamamycin. Notably, the ratio between methylmalonyl CoA and malonyl-CoA was increased four-fold. Both metabolites compete for incorporation into pamamycin so that the altered availability explained the pronounced preference for larger derivatives in the microparticle-enhanced process. The novel insights into the behavior of S. albus in response to talc appears of general relevance to further explore and upgrade the concept of microparticle enhanced cultivation, widely used for filamentous microbes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved