Genome Engineering and Editing
Genome Engineering using CRISPR activation
The development of the CRISPR-Cas system as modern gene scissors has revolutionized the field of gene technology and opened up novel strategies for genome engineering. In addition to its application as genome editing tool, silencing of the nuclease activity and fusion of the cas9 protein with transactivation domains resulted in a novel gene activation tool, known as CRISPR activation (CRISPRa). The functional separation of transcriptional activation on the protein level and genome targeting on the RNA level allows the simultaneous activation of multiple genes by the same transcriptional activator. Hence cellular expression profiles can be easily altered by expression of various target guide RNAs. Our research currently focusses on the development of CRISPR activation tools for the amoeba D. discoideum in order to activate cryptic biosynthetic pathways for the discovery of novel natural products with pharmaceutical application. Furthermore, we will employ CRISPRa to overcome bottlenecks in the heterologous production of plant-derived active molecules.
Discovery and characterisation of new secondary metabolites
The emergence of multi-resistant pathogens represents a serious threat to global health. Since conventional antibiotics are increasingly losing their effectiveness against these pathogens, there is great societal interest in the discovery of new antimicrobial agents. For the development of most antibiotics used in the medical field, microbial natural substances served as the starting point. Through the use of modern and efficient sequencing technologies, genomes of microorganisms are continuously decoded. Many of these genomes contain numerous genes for the synthesis of complex organic compounds with most of them exhibiting biological activity, the so-called secondary metabolites. However, there is often a large discrepancy between the number of annotated biosynthetic genes and the deciphered natural products of the respective microorganism, since the formation of these substances often does not occur under laboratory conditions. In particular, social amoebae such as D. discoideum have great potential for the discovery of new active ingredients due to their genetic repertoire. Using means of synthetic microbiology, we aim to disclose cryptic biosynthetic pathways in order to discover new natural products for drug discovery. This research includes the targeted activation or extrachromosomal expression of secondary metabolite routes followed by subsequent process optimization in bioreactors to generate sufficient quantities of target substances for structural and functional elucidation.
Metabolic engineering of social amoebae for microbial drug production
Plant secondary metabolites possess a plethora of biological activities whereby many are already used as conventional medicine. Phytocannabinoids from the plant Cannabis sativa, for instance, show strong therapeutic potential against many neuropathic disorders and pain. However, extraction and purification of such molecules from natural plant sources is cost intensive and elaborate as the respective plants often produce a broad spectrum of different compounds. Especially for the use as active pharmaceutical ingredient (API), compounds of interest have to fulfil highest quality standards. Hence, novel approaches aim to use microbial chassis organisms to produce desired products in the controlled environment of a bioreactor.
Here, we assess the general suitability of the amoeba D. discoideum to serve as a chassis for the synthesis of plant-derived active substances. Inspired by the fact that the amoeba possesses several enzymatic functions that are usually found in plants, we utilize multi-gene expression systems for the construction of heterologous biosynthetic pathways and microbial production of APIs in amoeba. We already demonstrated that amoeba-based processes can be scaled to industrial scales and will further exploit that knowledge to develop novel bioprocesses.
This research is funded by the BMBF GO-Bio project “ProDICAN”, FKZ 16LW0129.