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

Abstract

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.

Structure‐Guided Modulation of the Catalytic Properties of [2Fe−2S]‐Dependent Dehydratases

“…[8] However, while CcXylDHT and RlArDHT are reactive toward long sugar acid substrates (e. g. D-gluconate) we recently showed that they are practically inactive toward D-glycerate. [9] In that study, several novel dehydratases (DHADs and DHTs) with Both showed up to 50-fold higher activity toward D-glycerate when compared to SsDHAD. Further substrate profiling demonstrated however that FtDHAD, in contrast to CcXylDHT and RlArDHT, is significantly less active toward longer sugar acids (such as D-gluconate), while PuDHT is not active on branchedchain substrates such as DHIV, in contrast to SsDHAD (Table 1).…”

“…Further substrate profiling demonstrated however that FtDHAD, in contrast to CcXylDHT and RlArDHT, is significantly less active toward longer sugar acids (such as D-gluconate), while PuDHT is not active on branchedchain substrates such as DHIV, in contrast to SsDHAD (Table 1). [9] DHTs and DHADs belong to the ilvD/EDD (isoleucine, leucine, valine Dehydratase/Entner-Doudoroff Dehydratase) superfamily of enzymes. [4,8,10] To date, there is no consistent naming system for enzymes from this superfamily, which complicates their comparative analysis.…”

Selective Biocatalytic Defunctionalization of Raw Materials

“…[80] A broad substrate scope has been demonstrated for the dehydratase from Paralcaligenes ureilyticus, which is not only highly active in catalyzing the hydration of d-gluconate, d-xylonate, and l-arabinonate, [81] but is also useful for the efficient conversion of d-glyceric acid to pyruvate. [82] Dihydroxyacids such as d-glyceric acid, 2,3-dihydroxyisovaleric acid, and d-gluconic acid are also substrates of a dihydroxyacid dehydratase from Sulfolobus solfataricus. [74] Scheme 3.…”

Cell-Free Biocatalysis for the Production of Platform Chemicals

“…The replacement of fossil fuels in the production of high value chemicals was examined by Sutiono et al (2020) with reference to cell-free bio-transformations of D-glucose and glycerol to pyruvate as a precursor for alcohols, amino acids and other building blocks. They concentrated on the slow dehydration step of D-glycerate to pyruvate catalyzed by Sulfolobus solfataricus previously incorporated into enzyme cascades and searched for alternative enzymes with higher activities, finding two distinct enzymes with greater activity and higher turnover numbers than the enzyme from S. solfataricus.…”