초록
<P>A high degree of dependence on fossil fuels is one of the major problems faced by modern societies. <SMALL>D</SMALL>-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 <SMALL>D</SMALL>-glucose and glycerol to pyruvate and beyond have been described. However, these were limited by the very slow dehydration step of <SMALL>D</SMALL>-glycerate to pyruvate catalyzed by a dehydratase from <I>Sulfolobus solfataricus</I> (<I>Ss</I>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 <SMALL>D</SMALL>-glycerate. In particular, the dehydratase from <I>Paralcaligenes ureilyticus</I> (<I>Pu</I>DHT) demonstrated >100-fold higher activity and TTN for <SMALL>D</SMALL>-glycerate in comparison to <I>Ss</I>DHAD. In addition, <I>Pu</I>DHT showed exceptionally high activity and TTN toward <SMALL>D</SMALL>-gluconate. The replacement of <I>Ss</I>DHAD by <I>Pu</I>DHT in our model cascade from <SMALL>D</SMALL>-glucose to ethanol enhanced the production rate 10-fold, reaching a 92% theoretical yield at 50 °C. <I>Pu</I>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 <I>Ss</I>DHAD at 50 °C and attaining 97% of the theoretical yield. <I>Pu</I>DHT was also compatible when crude glycerol was used as the substrate, and it no longer caused a bottleneck in the enzymatic cascade.</P><BR>[FIG OMISSION]</BR>