"OBJECTIVE 1: Understanding and engineering the mechanism of reductive carboxylation.
We established methods to synthesize a diverse library of potential substrates to screen and engineer novel CO2-fixation reactions in the family of enoyl-CoA reductases/carboxylases (Peter et al. Molecules 2016). We used this library of substrates to assess the substrate specificity in enoyl-CoA carboxylases/reductases with a newly developed mass spectrometry based screen. Bioinformatics and site directed mutagenesis allowed us to identify three amino acid residues that determine substrate specificity in this class of CO2-fixing enzymes. This allowed us to engineer and extend the substrate spectrum of reductive carboxylases to create new CO2-fixation reactions that can provide new building blocks in polyketide biosynthesis (Vögeli, Cell Chem Biol 2018) as well as for the use in synthetic CO2-fixation pathways (Schwander et al. Science 2016). We further identified the molecular basis of CO2 binding in enoyl-CoA carboxylases/reductases (Stoffel et al. PNAS 2019) and succeeded to engineer the CO2-fixation capability into the scaffold of simple reductases (Bernhardgrütter et al. JACS 2019).
OBJECTIVE 2: Designing and realizing synthetic pathways for CO2-fixation based on reductive carboxylation.
We realized an artificial pathway for CO2-fixation, the CETCH cycle. The CETCH cycle was drafted by ‘metabolic retrosynthesis’, established with seventeen enzymes originating from nine different organisms of all three domains of life, and optimized in several rounds by enzyme engineering and metabolic proofreading. The CETCH cycle fixes CO2 in vitro at a rate of 5 nmol min-1 mg-1 protein (Schwander et al. Science 2016). We demon-strated that complex metabolic networks can be designed and realized by the principle of ‘metabolic retrosynthesis’ following basic (bio)chemical considerations, but that the operation of these synthetic networks needs further optimization by including biological design principles. Following these rules, we were able to realize a completely synthetic pathway for CO2-fixation that shows favorable thermodynamics compared to naturally evolved CO2-fixation pathways (requiring less ATP per CO2 fixed than photosynthetic CO2-fixation of plants). We also coupled the CETCH cycle to thylakoid membranes using microfluidics to generate an ""artificial chloroplast"" (Miller et al. Science 2020). The CETCH cycle was further optimized and developed to convert CO2 in to value added products, such as terpenes and polyketides (Sundaram in revision, Angewandte Chemie)
OBJECTIVE 3: Implementing synthetic CO2-fixation pathways based on reductive carboxylation into seleceted model organisms.
We started to establish microbial platforms to establish the CETCH cycle and other synthetic CO2-fixation pathways into Methylobacterium extorquens and other model bacteria. To that end, we developed novel tools for the genetic modification of Alphaproteobacteria (Carrillo et al. ACS Synthetic Biology 2018) and succesfully implemented the key sequence of the CETCH cycle in Methylobacterium extorquens (Carrillo, in preparation), as well as Escherichia coli (Sanchez-Pasquale, in preparation).
The resaerch has been disseminated in different formats and ways: for the scientific community through participation and presentation at multiple conferences; for the interested public through the press (TV and radio interviews), as well as through explanatory videos (youtube), amongst others."
