Optimized Reaction Conditions Enable the Hydration of Non‐natural Substrates by the Oleate Hydratase from Elizabethkingia meningoseptica

Abstract

The oleate hydratase from Elizabethkingia meningoseptica (Em‐OAH) catalyzes the hydration of oleic acid (C18) to (R)‐10‐hydroxystearic acid. In previous work, low activity of Em‐OAH towards chemically synthesized (Z)‐undec‐9‐enoic acid (C11) was observed. Product formation in the hydration of the truncated C11 substrate was improved by optimizing the reaction conditions by applying statistical experiment design. Optimized reaction conditions resulted in a 2.8‐fold increase in product formation in just one quarter of the time (64 % conversion in 28 h). The applicability has been assessed in the upscaling of the conversion of (Z)‐undec‐9‐enoic acid to (S)‐10‐hydroxyundecanoic acid (132 mg product, >95 % purity). Reaction conditions developed for the hydration of C11 facilitated the first hydration of non‐natural alkenes. By using a fatty acid dummy substrate, 1‐decene was successfully hydrated to (S)‐2‐decanol with excellent stereoselectivity and 50 % conversion after four days of incubation.

On the current role of hydratases in biocatalysis

“…In essence, these can be summarized by five consensus requirements for FAHs (Hiseni et al 2015; Demming et al 2018):A carbon-carbon double bond in cis -conformationA free carboxylate of the fatty acid substrateA chain length of at least C11 of an unsaturated fatty acid (Schmid et al 2016)A minimum distance of seven carbons between the carboxyl group and the hydrated cis -double bondAddition of a terminal OH-group is not possible (Demming et al 2017)…”

“…The inherent limitations of at least seven carbons between the carboxyl group and the to be hydrated double bond were bypassed upon addition of short-chain saturated fatty acids as reaction additives (Marliere 2011; Atsumi et al 2017; Demming et al 2017). Using hexanoic acid as the co-substrate for hydration of 1-decene, approx.…”

“…Using hexanoic acid as the co-substrate for hydration of 1-decene, approx. 50% of ( S )-2-decanol were obtained after 4 days of incubating E. coli cells overexpressing OhyA (Demming et al 2017). Similar approaches permitted (de)hydration of small alkenes (Marliere 2011) and water addition to ethylene upon addition of octanoic acid (Atsumi et al 2017).…”

“…While this fold is also observed in other proteins with similar functions (Wendt et al 1997), the active site of LDI is located at the interface of two subunits, which is unprecedented among (α,α) 6 barrel proteins. The importance of cysteines for LDI activity was established for an essential disulphide bond capping the substrate channel and for the contribution of the other two cysteines in the putative reaction mechanisms (Weidenweber et al 2016; Demming et al 2017). Both studies independently reported on acid/base catalysis for the (de)hydration and isomerization reactions of LDI, in which ( S )-(+)-linalool is protonated by a cysteine, followed by rehydration for geraniol or deprotonation for β-myrcene (Fig.…”

Weiterentwicklung der Substrattoleranz von Elizabethkingia meningoseptica Oleathydratase zur regio‐ und stereoselektiven Hydratisierung von Ölsäurederivaten

“…[35] Die derzeit am umfassendsten charakterisierte FAHY ist die Oleathydratase von Elizabethkingia meningoseptica (OhyA, EC 4.2.1.53). [38,43] Ungeachtet dessen konnten Demming und Kollegen vor kurzem erstmals einige der genannten Limitierungen überwinden. 98 %) ohne eine Kofaktor-Regeneration zu bençtigen.…”