Recombinant expression and characterisation of the oxygen-sensitive 2-enoate reductase from Clostridium sporogenes

Abstract

‘Ene’-reductases have attracted significant attention for the preparation of chemical intermediates and biologically active products. To date, research has been focussed primarily on Old Yellow Enzyme-like proteins, due to their ease of handling, whereas 2-enoate reductases from clostridia have received much less attention, because of their oxygen sensitivity and a lack of suitable expression systems. A hypothetical 2-enoate reductase gene, fldZ, was identified in Clostridium sporogenes DSM 795. The encoded protein shares a high degree of homology to clostridial FMN- and FAD-dependent 2-enoate reductases, including the cinnamic acid reductase proposed to be involved in amino acid metabolism in proteolytic clostridia. The gene was cloned and overexpressed in Escherichia coli. Successful expression depended on the use of strictly anaerobic conditions for both growth and enzyme preparation, since FldZ was oxygen-sensitive. The enzyme reduced aromatic enoates, such as cinnamic acid or p-coumaric acid, but not short chain unsaturated aliphatic acids. The β,β-disubstituted nitroalkene, (E)-1-nitro-2-phenylpropene, was reduced to enantiopure (R)-1-nitro-2-phenylpropane with a yield of 90 %. By contrast, the α,β-disubstituted nitroalkene, (E)-2-nitro-1-phenylpropene, was reduced with a moderate yield of 56 % and poor enantioselectivity (16 % ee for (S)-2-nitro-1-phenylpropane). The availability of an expression system for this recombinant clostridial 2-enoate reductase will facilitate future characterisation of this unusual class of ‘ene’-reductases, and expand the biocatalytic toolbox available for enantioselective hydrogenation of carbon-carbon double bonds.

Genetic Determinants of Hydroxycinnamic Acid Metabolism in Heterofermentative Lactobacilli

“…There is also evidence of other unidentified phenolic acid decarboxylases (33), suggesting differing genotypes among strains and/or a differential regulation. The Clostridium 2-enoate reductases also show strain-specific substrate specificity for reduction of hydroxycinnamic acids (18,19). In lactobacilli, hydroxycinnamic acids are reduced by three different enzymes, HcrB, HcrF, and Par1, with different strains and species having different substrate specificities and/or regulation explaining their differential hydroxycinnamic acid metabolism (this study).…”

“…Phenolic acid decarboxylases were characterized only in L. plantarum (16,17). Until recently, Clostridium was the closest relative to Lactobacillaceae for which enzymes capable of reducing hydroxycinnamates were characterized (18,19). In 2018, phenolic acid reductases reducing hydroxycinnamic acids to substituted phenylpropionic acids (HcrB) or reducing the decarboxylated vinyl derivatives to ethyl derivatives (VrpA) were characterized in L. plantarum (20,21).…”

An NADH-Dependent Reductase from Eubacterium ramulus Catalyzes the Stereospecific Heteroring Cleavage of Flavanones and Flavanonols

“…4). Clostridial ERED enzymes have been shown to reduce cinnamic and 4hydroxycinnamic acids to 3-phenylpropionic and 3-(4-hydroxyphenyl)propionic acids, respectively (20,21).…”

Discovery, Characterization, Engineering, and Applications of Ene-Reductases for Industrial Biocatalysis

“…Unlike most bacterial genera, Clostridia showed the presence of FAD and [4Fe-4S]-containing EnoR’s, which have not been exploited industrially due to their oxygen sensitivity 17. Recently the EnoR from Clostridium sporogenes DSM 795 (FldZ) was expressed in E. coli under anaerobic conditions, and it displayed C=C reduction towards a variety of aromatic enoates, such as α-cinnamic acid derivatives (Scheme 1D), but not short chain unsaturated aliphatic acids 38. This is in contrast to other ERs, as the single carboxyl functionality is thought to be too weak an activating group for OYEs 22.…”

“…Recently the EnoR from Clostridium sporogenes DSM 795 (FldZ) was expressed in E. coli under anaerobic conditions, and it displayed CC reduction activity toward a variety of aromatic enoates, such as α-cinnamic acid derivatives (Scheme D), but not short-chain unsaturated aliphatic acids . This is in contrast to other ERs, as the single carboxyl functionality is thought to be too weak an activating group for OYEs .…”