Engineering of two thiamine diphosphate-dependent enzymes for the regioselective condensation of C1-formaldehyde into C4-erythrulose

A number of carboligases, which catalyze condensation of C1- and/or C2-aldehydes into multi-carbon products, have been reported. However, their catalytic activities and/or regioselectivities remained rather low. Thereby, this study has focused on engineering of C1 and C2 carboligases for the regioselective condensation of C1-formaldehyde into C4-erythrulose via C2-glycolaldehyde. The crystal structure of the glyoxylate carboligase from Escherichia coli (EcGCL) was elucidated in complex with glycolaldehyde. A structure-guided rationale generated several mutants, one of whose catalytic activity reached 15.6 M-1s(-1), almost 10 times greater than the wild-type enzyme. Another variant (i.e., EcGCL(_R484M/N283Q/L478M/M488L/R284K)) has shown significantly increased stability to the glycolaldehyde toxicity, enabling production of glycolaldehyde to 31 mM from 75 mM formaldehyde (conversion: 83 %). Besides, the E1 subunit of alpha-ketoglutarate dehydrogenase complex from Vibrio vulnificus (VvSucA) was engineered as a regiospecific C2 carboligase for condensation of glycolaldehyde into erythrulose. The combination of EcGCL(_R484M/N283Q/L478M/M488L/R284K) and VvSucA(_K228L) led to the cascade production of erythrulose to 8 mM from 90 mM formaldehyde via glycolaldehyde without byproduct formation. This study will contribute to valorization of C1 gases into industrially relevant multi-carbon products in an environment-friendly way.

Automated summary

This study focuses on the engineering of C1 and C2 carboligases for the selective condensation of formaldehyde into erythrulose. By designing mutants and combining different variants, the catalytic activity and stability of the carboligases were significantly improved. This allowed for the efficient production of erythrulose from formaldehyde via glycolaldehyde, without the formation of byproducts.