hhu.de Cyanobacteria as platform organisms for biotechnology Cyanobacteria are photoautotrophic bacteria capable of photosynthesis and high rates of CO2 fixation. Their metabolic potential makes them promising candidates for biotechnological production of industrially relevant compounds such as plant secondary metabolites. Valencene - an important aroma and more Valencene is a sesquiterpene that can be found in many citrus fruits. While it can be used in various applications in industry, including cosmetics and flavor, it also serves as a precursor for nootkatone, which has been shown to act as a powerful insect repellant, but can also inhibit growth of cancer cells. ValenCell: Large-scale production of sesquiterpenes from microalgae Anna Behle1, Ilka Axmann1, Lars Bähr2, Arne Wüstenberg2 CO2BioTech Kick-Off Meeting Lars Bähr (CellDEG) Anna Behle (HHU) Heinrich-Heine-Universität Düsseldorf Institute for Synthetic Microbiology 1 Institute for Synthetic Microbiology, HHU Düsseldorf; 2 CellDEG GmbH Berlin synmikrobiologie.hhu.de CO2 CO2 CO2 NADPH Valencene A WP1: Engineering the bacterial metabolism We previously showed that increasing metabolic flux through the terpenoid biosynthesis pathway had positive effects on strain productivity. Since sesquiterpene production competes with pigment biosynthesis, it is important to supply the cell with sufficient precursor molecules, as well as cofactors. We were able to achieve, rerouting carbon supply from pigment biosynthesis towards product formation with inducible systems1. We aim to build a stable production strain without costly inducers using the following strategies. Titration of expression strength by testing multiple genetic cassettes Testing different sesquiterpene synthases Variation of cultivation conditions Reinforcement of carbon and cofactor supply by rerouting the metabolism (Fig. 1) WP3: Scaling up with CellDEG One main challenge when scaling up the cultivation volume of cyanobacterial strains is ensuring a sufficient availability of nutrients such as light and CO2. The CellDEG technology is specifically designed to enable fast growth of photoautotrophic organisms to a high cell density (Fig. 2B). To identify strains that perform best in such reactors, promising strains will be screened in HD10 reactors early on. Upscaling of the best production strains will take place in the HD1500 production platform (Fig. 2A). The final aim of this project is to achieve a strain with a growth rate of 2.5 g DCW/L/day while maintaining a production rate of 200 mg valencene / g DCW. WP2: Alternative extraction methods Valencene, along with many sesqui- and monoterpenes, is a volatile compound. This is especially advantageous for downstream processing, as it eliminates the need for expensive and time- consuming extraction from the biomass. The current state-of-the-art for quantification of valencene production makes use of a thin layer of dodecane on top of the culture, which captures the volatile product (Fig. 2C). This is favorable for analysis, as dodecane can be analyzed via GC/MS directly. In terms of cost, scalability and sustainability it is of high interest to find alternative extraction- or capturing methods for valencene. A B C Figure 1: Relevant metabolic pathways in the cyanobacterium Synechocystis sp. PCC 6803. Relevant genes for overexpression or deletion are boxed. Involvement of ATP and NADPH is shown in red and blue, respectively. Figure 2 A. From left to right: HD10, HD100, HD1500 reactor setup. B. Growth curves of Synechocystis sp. PCC 6803 WT in the three different reactors. C. Schematic setup for growth of production strains to high cell densities using the HD10 screening platform (image modified from2 Scientific reports, 10(1), 5932 https://doi.org/10.1038/s41598-020-62681-w) 1 OD₇₅₀ = 0,322 gDW L⁻¹     1Dietsch, Behle et al.: Metabolic engineering of Synechocystis sp. PCC 6803 for the photoproduction of the sesquiterpene valencene. Metab. Eng. Commun. 2021 Aug 13