To beat the heat – engineering of the most thermostable pyruvate decarboxylase to date

Sutiono, Samuel; Satzinger, Katharina; Pick, André; Carsten, J.; Sieber, Volker

doi:10.1039/c9ra06251c

Cited by 6 publications

(6 citation statements)

References 29 publications

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“…Surprisingly, DHADs from Cupriavidus ( Cn H16-, Cn N1-, and Cm DHAD), although having relatively high activities toward d -glycerate and high T m values, exhibited relatively modest TTNs (∼20000). This result is in contrast to our previous studies with α-keto acid decarboxylases, in which we observed a direct relationship between T m and kinetic stability. , The melting temperature ( T m ) may indicate the general stability (thermodynamic stability) of an enzyme . Thus, in the case of proteins with similar initial activities and T m values, a lower TTN could be linked to rapid loss of activity over time rather than to thermal denaturation.…”

Section: Resultscontrasting

confidence: 99%

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

et al. 2020

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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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Section: Resultscontrasting

confidence: 99%

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

et al. 2020

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“…No significant difference was observed when comparing the thermodynamic stability of Cn L-KdpD and Hs L-KdpD, although the kinetic stability suggested otherwise. Apparently, the relationships between the kinetic and thermodynamic stabilities were not able to be observed for L-KdpD, which stands in contrast to our previous studies of two α-keto acid decarboxylases (Sutiono et al 2018 ; Sutiono et al 2019 ).…”

Section: Discussioncontrasting

confidence: 99%

Characterization of highly active 2-keto-3-deoxy-L-arabinonate and 2-keto-3-deoxy-D-xylonate dehydratases in terms of the biotransformation of hemicellulose sugars to chemicals

Sutiono

Siebers

Sieber

2020

Appl Microbiol Biotechnol

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2-keto-3-L-arabinonate dehydratase (L-KdpD) and 2-keto-3-D-xylonate dehydratase (D-KdpD) are the third enzymes in the Weimberg pathway catalyzing the dehydration of respective 2-keto-3-deoxy sugar acids (KDP) to α-ketoglutaric semialdehyde (KGSA). The Weimberg pathway has been explored recently with respect to the synthesis of chemicals from L-arabinose and D-xylose. However, only limited work has been done toward characterizing these two enzymes. In this work, several new L-KdpDs and D-KdpDs were cloned and heterologously expressed in Escherichia coli . Following kinetic characterizations and kinetic stability studies, the L-KdpD from Cupriavidus necator ( Cn L-KdpD) and D-KdpD from Pseudomonas putida ( Pp D-KdpD) appeared to be the most promising variants from each enzyme class. Magnesium had no effect on Cn L-KdpD, whereas increased activity and stability were observed for Pp D-KdpD in the presence of Mg 2+ . Furthermore, Cn L-KdpD was not inhibited in the presence of L-arabinose and L-arabinonate, whereas Pp D-KdpD was inhibited with D-xylonate (I 50 of 75 mM), but not with D-xylose. Both enzymes were shown to be highly active in the one-step conversions of L-KDP and D-KDP. Cn L-KdpD converted > 95% of 500 mM L-KDP to KGSA in the first 2 h while Pp D-KdpD converted > 90% of 500 mM D-KDP after 4 h. Both enzymes in combination were able to convert 83% of a racemic mixture of D,L-KDP (500 mM) after 4 h, with both enzymes being specific toward the respective stereoisomer. Key points • L-KdpDs and D-KdpDs are specific toward L- and D-KDP, respectively. • Mg 2+ affected activity and stabilities of D-KdpDs, but not of L-KdpDs. • CnL-KdpD and PpD-KdpD converted 0.5 M of each KDP isomer reaching 95 and 90% yield. • Both enzymes in combination converted 0.5 M racemic D,L-KDP reaching 83% yield. Electronic supplementary material The online version of this article (10.1007/s00253-020-10742-5) contains supplementary material, which is available to authorized users.

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“…One current example is the development of minimized biocatalytic reaction cascades in cell‐free processes for the conversion of glucose to ethanol or isobutanol using only six or eight enzymes, respectively. Enzymes required for such routes are being developed to work at elevated temperatures [32] . The production of alcohols working at high temperatures can be useful to enable easy removal of the volatile product and reduce the risk of contamination.…”

Section: Resultsmentioning

confidence: 99%

carba Nicotinamide Adenine Dinucleotide Phosphate: Robust Cofactor for Redox Biocatalysis

et al. 2021

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Here we report a new robust nicotinamide dinucleotide phosphate cofactor analog (carba‐NADP+) and its acceptance by many enzymes in the class of oxidoreductases. Replacing one ribose oxygen with a methylene group of the natural NADP+ was found to enhance stability dramatically. Decomposition experiments at moderate and high temperatures with the cofactors showed a drastic increase in half‐life time at elevated temperatures since it significantly disfavors hydrolysis of the pyridinium‐N−glycoside bond. Overall, more than 27 different oxidoreductases were successfully tested, and a thorough analytical characterization and comparison is given. The cofactor carba‐NADP+ opens up the field of redox‐biocatalysis under harsh conditions.

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To beat the heat – engineering of the most thermostable pyruvate decarboxylase to date

Abstract: Pyruvate decarboxylase (PDC) is a key enzyme for the production of ethanol at high temperatures and for cell-free butanol synthesis.

Cited by 6 publications

References 29 publications

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

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

Characterization of highly active 2-keto-3-deoxy-L-arabinonate and 2-keto-3-deoxy-D-xylonate dehydratases in terms of the biotransformation of hemicellulose sugars to chemicals

carba Nicotinamide Adenine Dinucleotide Phosphate: Robust Cofactor for Redox Biocatalysis

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