Engineering a Highly Efficient Carboligase for Synthetic One-Carbon Metabolism

Area of Science:

• Biocatalysis
• Synthetic biology
• Metabolic engineering

Background:

• Circular carbon economy relies on synthesizing complex molecules from one-carbon (C1) building blocks.
• Natural C1-C1 condensation pathways are limited, necessitating the development of robust biocatalysts.
• Thiamine diphosphate-dependent enzymes show promise for C-C bond formation.

Purpose of the Study:

• To engineer a novel biocatalyst for direct C1-C1 condensation.
• To convert oxalyl-CoA decarboxylase (OXC) into a highly active glycolyl-CoA synthase (GCS).
• To facilitate the synthesis of complex carbon compounds from C1 feedstocks.

Main Methods:

• Structure-guided iterative saturation mutagenesis was used to modify OXC from *Methylobacterium extorquens*.
• A quadruple variant (MeOXC4) was generated and characterized for its enzymatic activities.
• In vitro and in vivo assays were performed to evaluate catalytic efficiency and application.

Main Results:

• MeOXC4 exhibited a 100,000-fold switch from OXC to GCS activity, with a 200-fold increase in GCS activity.
• The engineered enzyme demonstrated high formaldehyde affinity and catalytic efficiency, outcompeting natural and other engineered enzymes.
• MeOXC4 enabled in vivo production of glycolate from formaldehyde in *Escherichia coli*.

Conclusions:

• Engineered MeOXC4 significantly advances C1-C1 condensation for circular carbon economy applications.
• This biocatalyst overcomes bottlenecks in C1 assimilation and whole-cell bioconversions.
• The study paves the way for synthetic C1 assimilation routes and the production of valuable chemicals from C1 feedstocks.