Discovery, Characterisation, Engineering and Applications of Ene Reductases for Industrial Biocatalysis

Abstract

Recent studies of multiple enzyme families collectively referred to as ene-reductases (ERs) have highlighted potential industrial application of these biocatalysts in the production of fine and speciality chemicals. Processes have been developed whereby ERs contribute to synthetic routes as isolated enzymes, components of multi-enzyme cascades, and more recently in metabolic engineering and synthetic biology programmes using microbial cell factories to support chemicals production. The discovery of ERs from previously untapped sources and the expansion of directed evolution screening programmes, coupled to deeper mechanistic understanding of ER reactions, have driven their use in natural product and chemicals synthesis. Here we review developments, challenges and opportunities for the use of ERs in fine and speciality chemicals manufacture. The ER research field is rapidly expanding and the focus of this review is on developments that have emerged predominantly over the last 4 years.

Keywords: Ene-reductases; Old Yellow Enzymes; asymmetric alkene reduction; biocatalysis; synthetic biology.

Figure 1

Asymmetric activated alkene reduction catalysed by ene-reductases (ER). Enzyme classes: OYE = Old Yellow Enzyme; EnoR = oxygen-sensitive enoate reductases; SDR = short chain dehydrogenase/reductase salutaridine/menthone reductase-like subfamily; MDR = medium chain dehydrogenase/reductase leukotriene B4 dehydrogenase subfamily; QnoR = quinone reductase-like ene-reductase.

Figure 2

Comparison of ERs belonging to OYE and quinone reductase-like families. A) Overall crystal structures of PETNR (PDB: 1GVQ) and PhENR (PDB: 3ZOG). The structures are shown as rainbow cartoons, from blue to red for the N- to C-termini, respectively. The FMN and cyclohexen-2-one are shown as atom coloured sticks with yellow and magenta carbons, respectively, throughout the Figure. B) Comparative active site arrangement between cyclohexen-2-one-bound PETNR and PhENR (2 molecules bound). Residues are shown as atom coloured sticks with green carbons. The figure was generated using Pymol.

Scheme 1

Biocatalytic α,β-unsaturated alkene reduction catalysed by novel A) thermophilic, B) fungal, C) plant ERs and D) clostridial EnoR.

Scheme 2

Whole cell catalysed α,β-unsaturated alkene reduction for the production of A) α-benzyl-β-ketoesters and B) chiral γ-oxo esters.

Scheme 3

Whole cell fungal biotransformations for the generation of A) Latanoprost precursor and B) α,β- and α, β, γ, δ-unsaturated ketones and derivatives.

Scheme 4

Routes to polymer precursors A) (+)-β-methyl-δ-valerolactones, B) lactones, C) adipic acid and D) 4-aminohydrocinnamic acid via chemoenzymatic and multienzyme cascade reactions.

Scheme 5

Generation of stereo-complementary pairs of products by engineering of ERs using A) scaffold sampling and B) site-saturated mutagenesis approaches. OYE sources: NCR = Zymomonas mobilis ; DrER = Deinococcus radiodurans ; TsER = Thermus scotoductus SA-01; RmER = Ralstonia metallidurans; OYE2.6 = P. stipitis.

Scheme 6

Multi-step enzymatic bioconversions of A) ketoisophorone to (4R,6R)-actinol and B) cinnamyl alcohol to 3-phenylpropan-1-ol.

Scheme 7

Alternative ER-FMN reduction strategies using A) co-substrates + enzymes, B) sacrificial substrates and C) nicotinamide biomimetics.

Scheme 8

Strategies of cofactor-independent photoactivation of FMN using photosensitisers A) Rose bengal and B) ruthenium complexes.

Scheme 9

Introduced pathways into host microorganisms for the production of the industrially-useful compounds A) hydrocinnamic acids from amino acids, B) 2-methyl succinic acid from pyruvate and C) (2R,5S)-carvolactone from (R)-limonene. pheA = chorismate mutase/prephenate dehydratase; tyrA = chorismate mutase/prephenate dehydrogenase; TAL = tyrosine ammonia lyase; CimA = citramalate synthase; LeuCD = 3-isopropylmalate dehydratase; CHMO = cyclohexanone monooxygenase.

Scheme 10

Insights into the catalytic mechanisms of different ER families. A) Proposed mechanism of ER-catalysed radical dehalogenation. B) Hydride-independent C=C migration vs ene-reduction catalysed by OYE2.

Scheme 11

Comparison of the proposed ene-reduction and ketoreduction mechanisms catalysed by the highly conserved SDR enzymes IPR and MMR, respectively.