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

Abstract

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.

Biocatalytic Production of 1,2,4-Butanetriol beyond a Titer of 100 g/L: Boosting by Intermediates

“…KDX was produced from d -xylonate using Pu DHT. d -Xylonate and KDX were quantified using HPLC as described previously. , (S)-Dihydroxybutanal (DHB) was produced from KDX after incubating with Ll KdcA in 250 mM HEPES pH 7.25 containing 0.1 mM thiamine diphosphate (ThDP) and 5 mM MgCl 2 . The production of DHB was followed using HPLC measuring the decrease of KDX.…”

“…Pu DHT and Ll KdcA were measured using a coupled assay. For Pu DHT, D-KdpD and KgsaDH from Pseudomonas putida were used as the coupling enzymes detecting the reduction of NAD + to NADH . For Ll KdcA, Ec AdhZ3 (LND) was used as the coupling enzyme detecting the oxidation of NADH to NAD + .…”

“…d -Xylonate was produced by oxidation of d -xylose using a gold catalyst as described previously . KDX was produced from d -xylonate using Pu DHT.…”

Selective Biocatalytic Defunctionalization of Raw Materials

“…The fact that the structures of 2-keto-3-deoxy-d-xylonate, obtained from d-xylonate, and 2-keto-3-deoxy-d-arabinonate, obtained from d-arabinonate, are identical can be emphasized by the common name 2-keto-3-deoxy-d-pentonate. [83] Further defunctionalizations can be achieved by a second dehydration reaction catalyzed by 2-keto-3-deoxyaldonic acid dehydratases, such as 2-keto-3-deoxy-d-xylonate dehydratase [76,85] and 2-keto-3-deoxy-l-arabinonate dehydratase, [84,85] with both reactions resulting in the same product 2-ketoglutarate semi-aldehyde. The three sugar acid dehydratases 2-keto-3deoxy-d-xylonate dehydratase, 2-keto-3-deoxy-l-arabinonate dehydratase, and the dehydratase from Paralcaligenes ureilyticus have been key to the convergent biocatalytic transformation of d-xylose as well as l-arabinose to 2-ketoglutarate semialdehyde, which has been converted further to 1,4-butanediol and 2-ketoglutarate.…”

“…Regarding a deoxydehydration-hydrogenation reaction sequence for vicinal diols in sugar acids, the sequence of two dehydratase-catalyzed reactions in aqueous solution has been demonstrated (see Scheme 9) to provide a sustainable biocatalytic version, [76,81,143] which is attractive for O-defunctionalization of bio-based sugars and promising for further development. The combination of the versatile dehydratase from Paralcaligenes ureilyticus, which accepts both d-xylonate as well as l-arabinonate as substrates, [82] with the 2-keto-3-l-arabinonate dehydratase from Cupriavidus necator [85] and 2-keto-3-dxylonate dehydratase from Pseudomonas putida [85] has been successfully employed as part of a convergent biocatalytic synthesis of 1,4-butanediol and 2-ketoglutarate with rates of 3 and 4.2 g h À 1 L À 1 , respectively, and excellent yields. [81]…”