A synthetic cell-free pathway for biocatalytic upgrading of formate from electrochemically reduced CO2

ABSTRACT

Electrochemical reduction of carbon dioxide (CO2) can produce important one-carbon (C1) feedstocks for sustainable biomanufacturing, such as formate. Unfortunately, natural formate assimilation pathways are inefficient and constrained to organisms that are difficult to engineer. Here we establish a synthetic reductive formate pathway (ReForm) in vitro. ReForm is a six-step pathway consisting of five engineered enzymes catalyzing nonnatural reactions to convert formate into the universal biological building block acetyl-CoA. We establish ReForm by selecting enzymes among 66 candidates from prokaryotic and eukaryotic origins. Through iterative cycles of engineering, we create and evaluate 3,173 sequence-defined enzyme mutants, tune cofactor concentrations and adjust enzyme loadings to increase pathway activity toward the model end product malate. We demonstrate that ReForm can accept diverse C1 substrates, including formaldehyde, methanol and formate produced from the electrochemical reduction of CO2. Our work expands the repertoire of synthetic C1 utilization pathways, with implications for synthetic biology and the development of a formate-based bioeconomy.