METABOTUNE · Developing CRISPR activation/CRISPR interference toolset for fine-tuning production of metabolites in plants

Plants produce a great variety of specialised metabolites. In plants they are often involved in interactions with their environment, but many of them have applications in pharmaceutical, cosmetic, and food industries, making them targets for metabolic engineering efforts aiming to increase their production. Plant metabolic engineering is usually based on metabolic pathway reconstitution in microbes, but recently direct activation of biosynthetic genes has been done in plants using CRISPR tools. METABOTUNE aims to develop a CRISPR toolset for simultaneous activation and inactivation of metabolic genes to fine-tune biosynthesis and maximise yields of specialised metabolites. First, I will develop a CRISPR interference (CRISPRi) tool for inactivating genes that channel metabolites into undesired metabolic pathways. Second, I will identify genes involved in the biosynthesis of a coumarin metabolite with medicinal properties, esculetin. Finally, CRISPRi will be applied together with the already available CRISPR activation (CRISPRa) tool in development of plant cell cultures specifically producing esculetin and the minimal amount of other coumarins. To achieve these objectives, I will combine my expertise in molecular biology, genetics, specialised metabolites, and biochemistry with the expertise in metabolic engineering and coumarins available at the host institute. Allowing precise channelling of metabolites into pathways of interest, as proposed here, will open new venues for highly specific plant metabolic engineering efforts and allow a shift from production in microbes to increasing production in plant species that already have genes required for biosynthesis of high-value specialised metabolites.