Enabling the direct enzymatic dehydration of D-glycerate to pyruvate as the key step in synthetic enzyme cascades used in the cell-free production of fine chemicals

A high degree of dependency on fossil fuels is one of the major problems faced by modern societies. D-glucose and glycerol have emerged in recent years as sustainable replacements for fossil fuels used in the production of high-value chemicals, and cell free bioproduction routes are expected to play a crucial role in such processes. Recently, several synthetic cascades used for the cell-free biotransformations of D-glucose and glycerol to pyruvate and beyond have been described. However, these were limited by the very slow dehydration step of D-glycerate to pyruvate catalyzed by a dehydratase from Sulfolobus solfataricus (SsDHAD), making this step by far the major bottleneck. By combining the vast number of available genomes with a sequence-based discovery approach, we have identified signature sequences leading to the discovery of two novel classes of dehydratases which exhibit promising activity and total turnover number (TTN) toward D-glycerate. In particular, the dehydratase from Paralcaligenes ureily...