Engineering of an Evolved Artificial Formolase Enzyme To Facilitate <i>In Vivo</i> Synthetic C₁ Metabolism

To accomplish a sustainable circular economy, the conversion of CO2 to products through biotechnological processes is a promising approach. One method uses synthetic methylotrophic microbes that use CO2-based methanol as the carbon source. However, its toxicity, as well as its byproduct formaldehyde, poses a challenge. To address this, a synthetic enzyme called formolase (FLS) has been designed to convert formaldehyde directly into dihydroxyacetone. So far, its utilization is limited due to low velocity, high KM, and the formation of the byproduct glycolaldehyde. To enable the in vivo application of formolase, the enzyme was subjected to iterative rounds of directed evolution. The key to success was a combined ″semirational” approach, which led to the identification of an advanced formolase variant exhibiting higher expression levels, enabling better formaldehyde tolerance in E. coli, and a 30% decrease in KM for the formose reaction. Furthermore, this double mutant exhibits an accelerated conversion of glycolaldehyde to dihydroxyacetone, which is particularly relevant for its future application in synthetic methylotrophy.