ORCA: Redesigning Enzyme Biochemistry towards Orthogonal CoA Analogs

• Applicant: Dr. Maren Nattermann
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Biochemistry
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 581057063

Engineered microbes offer exciting new opportunities for the sustainable production of value-added chemicals from cheap feedstock without the need for high temperatures, pressure, or toxic solvents. However, the metabolic integration of synthetic pathways can be challenging. One mayor concern is the competition of natural and synthetic pathways for the same core metabolites. So far, these conflicts have been approached by spatial or temporal separation of synthetic metabolism. In the former, synthetic pathways are sequestered into distinct compartments to avoid interactions with core metabolism, while in the latter, synthetic pathways are only activated once cells have left exponential growth and have thus become less metabolically active. Orthogonal pathways offer a third possibility for metabolic separation – one on the biochemical level. Pathways proceeding via non-natural intermediates lower the cross-talk between synthetic and natural metabolism, and thus the overall burden on the cell. Such biochemical separation is abundant in nature, perhaps most prominently in the orthogonal use of the redox cofactors NADH and NADPH. While they catalyze the same chemical redox reactions, their use in metabolism is highly distinct. NADH is used primarily in catabolism, pushing oxidative cascades, while NADPH is allocated to anabolism, and reducing reactions. In this project, we aim to expand this concept of orthogonal cofactors by developing enzymatic cascades based around a non-natural cofactor allocated exclusively to synthetic metabolism, offering an opportunity for strict separation from native pathways. Many synthetic pathways rely on coenzyme A (CoA) as a carrier of acyl moieties, leading to frequent crosstalk with native CoA metabolism. We will therefore re-design a short, fully CoA-dependent pathway for the exclusive use of the synthetic analog N-acetyl cysteamine (SNAC), making it entirely bio-orthogonal. We plan to lay a solid foundation by investigating the CoA binding domains of the enzymes involved, before applying the knowledge gained to binding site re-design for selective use of SNAC. Finally, we will re-constitute the cascade in vitro and test its resilience to of CoA. This project will therefore advance our understanding of CoA-enzyme interaction, demonstrate for the first time that enzymes can be redesigned for selective use of SNAC, and open up future opportunities to implement this orthogonal cascade in vivo, expanding the use of SNAC in biotechnological applications.

• DFG Programme: Research Grants