Engineering of an Evolved Artificial Formolase Enzyme To Facilitate In Vivo Synthetic C1Metabolism

To accomplish a sustainable circular economy, the conversion of CO<inf>2</inf>to products through biotechnological processes is a promising approach. One method uses synthetic methylotrophic microbes that use CO<inf>2</inf>-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 K<inf>M</inf>, 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 K<inf>M</inf>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.