1st Gießen Symposium of Insect Biotechnology, October 9-10, 2017 (published online)

Abstract

The genome of entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus encodes for up to 25 biosynthesis gene clusters (BGCs), representing up to 7.5% of the total genome. While several natural products have been identified and isolated from these bacteria during the last ten years due to efforts from several labs (Bode 2009, Challinor and Bode 2016, Vizcaino 2013, the majority of these compounds are still unknown. Therefore, these natural products are not accessible for a detailed analysis of their function in the natural ecosystem or as drugs in a more applied way (antibiotics, anticancer compounds, biotechnology etc) and methods for faster access to these natural products is desirable.We have analysed the deletion of known global regulators from Gram-negative bacteria on the natural product biosynthesis in Xenorhabdus and Photorhabdus (Engel 2017) and did identify the RNA chaperon Hfq playing a major role in natural product biosynthesis. In hfq mutants of several strains analysed so far, all natural products were reduced 10 to 100-fold, most of them below the limit of detection (Tobias 2017). Hfq is known for binding small regulatory RNAs and deliver them to target mRNAs and thereby can activate or reduce protein levels. When we applied our well-established promoter exchange approach (Bode 2015) to these hfq mutants, we were able to produce only the expected compound class upon activation of the promoter. We could transform the wildtype strains (as producers of multiple compounds) via the hfq strains (as no-producers of any compound) into single producers of a desired natural product class. Since an extract generated from these strains contains just one compound class, it can be tested directly against a variety of different targets allowing a simplified functional assignment of its biological function and without actual isolation of the natural product.Moreover, since the natural product can be produced on-demand in its natural environment due to its inducible promoter, the determination of the individual compound's contribution and role in the overall ecosystem can be addressed easily.As an additional way to generate chemical diversity from microorganisms, we have developed the eXchange Unit (XU) technology that allows the simple assembly of building blocks derived from natural non-ribosomal peptide synthetases (NRPS) to generate cyclic, linear, acylated peptides and/or peptides containing D-amino acids. Due to the definition of the XU and their assembly at a specific sequence, the yields even for non-natural peptides are between 1-40 mg/L in E. coli enabling detailed bioactivity testing. The XU technology can also be applied to the original producers enabling the simple modification of NRPS encoding BGCs as well as the artificial fusion of adjacent BGCs.Both, the latest results about hfq mediated production of individual compound classes and the XU technology will be presented. Justus-Liebig-University Giessen, Bioinformatics and Systems Biology, 35392 Gießen, Germany Corresponding author...