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 dependence on fossil fuels is one of the major problems faced by modern societies. d -Glucose and glycerol have emerged in recent years as prospective 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 ( Ss DHAD), 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 distinct classes of dehydratases which exhibit promising activity and total turnover number (TTN) toward d -glycerate. In particular, the dehydratase from Paralcaligenes ureilyticus ( Pu DHT) demonstrated >100-fold higher activity and TTN for d -glycerate in comparison to Ss DHAD. In addition, Pu DHT showed exceptionally high activity and TTN toward d -gluconate. The replacement of Ss DHAD by Pu DHT in our model cascade from d -glucose to ethanol enhanced the production rate 10-fold, reaching a 92% theoretical yield at 50 °C. Pu DHT was also suitable for the conversion of glycerol to pyruvate at ambient temperature, leading to a >5-fold improvement in production rate in comparison to the system utilizing Ss DHAD at 50 °C and attaining 97% of the theoretical yield. Pu DHT was also compatible when crude glycerol was used as the substrate, and it no longer caused a bottleneck in the enzymatic cascade.