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 (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 distinct classes of dehydratases which exhibit promising activity and total turnover number (TTN) toward D-glycerate. In particular, the dehydratase from Paralcaligenes ureilyticus (PuDHT) demonstrated >100-fold higher activity and TTN for D-glycerate in comparison to SsDHAD. In addition, PuDHT showed exceptionally high activity and TTN toward D-gluconate. The replacement of SsDHAD by PuDHT in our model cascade from Dglucose to ethanol enhanced the production rate 10-fold, reaching a 92% theoretical yield at 50 °C. PuDHT 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 SsDHAD at 50 °C and attaining 97% of the theoretical yield. PuDHT was also compatible when crude glycerol was used as the substrate, and it no longer caused a bottleneck in the enzymatic cascade.

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