Graduate Students Mini Symposium XI - 2024

13:15 h Tristan Reif-Trauttmansdorff - AG Schuller
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13:45 h Ekaterina Jalomo Khayrova - AG Bange
tba

14:15 h Alina Schrodt - AG Erb
"Engineering Methylorubrum extorquens PA1 for the production of intermediates from Ethyl-Malonyl-CoA Pathway from methanol"

The development of methylotrophic microbial platforms for the production of value-added compounds is a crucial step towards a methanol-based bioindustry and a carbon-neutral bioeconomy. Methanol is a promising substrate for the synthesis of such compounds, as it is a cheap carbon source, which can be generated from syngas and CO₂, does not compete with the food industry and reduces the risk for contamination during fermentation. In this project, we aimed to engineer Methylorubrum extorquens PA1 for the production of crotonic acid during growth on methanol as a sole source of carbon and energy. For this, we selected a strain deleted in ccr (crotonyl-CoA reductase/carboxylase) to accumulate the precursor crotonyl-CoA, which is then converted to crotonic acid via the heterologously expressed thioesterase yciA from Escherichia coli. As the deletion of ccr would seize the ability of the strain to grow on C1 and C2 compounds due to a lack of glyoxylate, a glyoxylate-regeneration module was introduced on a plasmid. This strain was then evolved for improved growth on methanol through adaptive laboratory evolution, which yielded shorter doubling times and an increase of the final OD₆₀₀. Additionally, a CRISPR interference (CRISPRi) system was tested in the evolved strain to target polyydroxybutyrate (PHB) synthesis, a storage compound in many Alphaproteobacteria and a potential bottleneck in crotonic acid production. The CRISPRi system targeted phaC (PHB synthase) to reduce the accumulation of PHB to redirect the metabolic flux towards crotonyl-CoA.