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Project Topic
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Society urgently needs a sustainable production processes for key platform chemicals with various applications in several industries, in light of resource scarcity. Developing a bioeconomy is a cornerstone to meet this grand challenge. The bioeconomy can make major socioeconomic contributions, lead to health improvement, boost the productivity of agriculture and industrial processes, and enhance environmental sustainability. The C1Pro project aims to establish a sustainable platform for methanol-based production of value-added products (GABA, 5AVA, L-Pro and L-PA) with proven industrial applications. Methanol is an attractive and alternative raw material for biotechnological processes because of its chemical properties, relatively low price and availability from both fossil and renewable sources. The Gram-positive, methylotrophic and thermophilic bacterium Bacillus methanolicus was chosen as model organism in this project for several reasons: it utilizes methanol as raw material for growth and energy, it grows at elevated temperatures (50 – 55 °C), it naturally overproduces L-glutamate, and its classical mutants have demonstrated a high potential to overproduce L-lysine. We propose to apply innovative technologies in the fields of modelling, development of genetic tools, strain engineering, fermentation technology and downstream processing to establish and develop methanol-based production. Four different products, GABA, 5AVA, L-Pro and L-PA, were chosen deliberately as they share a) biosynthesis pathways, b) functional characteristics and c) industrial applications. The omega-amino acids GABA and 5AVA can be cyclized by lactamization, while L-Pro and L-PA are cyclic amino acids. Biosynthesis of L-Pro and GABA starts from L-glutamate, whereas L-PA and 5AVA derive from L-lysine. The targeted products serve as building blocks of polymers or precursors of pharmaceuticals and other biologically active substances. The commonalities of the chosen product pairs GABA/5AVA and L-Pro/L-PA enable transferability: process intensification and DSP development for one example (GABA) can be carried out in parallel with initial strain and fermentation development for the other products. Later in the project, the most promising processes will be scaled up. Systems and synthetic biology approaches are key to the proposed strain and process development, which is facilitated by common biosynthesis pathways. Novel genetic tools will simplify regulated gene expression on different levels via CRISPR/Cas9 for genome modifications and riboswitches for regulatory circuits. Pathway design will be guided by the genome-scale metabolic model which will be iteratively fine-tuned based on experimental test results. Strain performance in methanol-based fermentations will be characterized in-depth by RNAseq, metabolome and/or flux analyses. This will guide iterative optimization of strains and fermentation conditions in subsequent rounds. All data will be collected in an LCA-compliant way and used to select the most promising strain(s) for up-scaling where methanol-based production at 20-150 L scale will be performed in a clean bioprocess. Finally, GABA isolation to at least 80% purity will be demonstrated.
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