Identification of a novel ene reductase from Pichia angusta with potential application in (R)-levodione production

Abstract

Asymmetric reduction of electronically activated alkenes by ene reductases (ERs) is an attractive approach for the production of enantiopure chiral products. Herein, a novel FMN-binding ene reductase (PaER) from Pichia angusta was heterologously expressed in Escherichia coli BL21(DE3), and the recombinant enzyme was characterized for its biocatalytic properties. PaER displayed optimal activity at 40 °C and pH 7.5, respectively. The purified enzyme was quite stable below 30 °C over a broad pH range of 5.0-10.0. PaER was identified to have a good ability to reduce the C[double bond, length as m-dash]C bond of various α,β-unsaturated compounds in the presence of NADPH. In addition, PaER exhibited a high reduction rate (k _cat = 3.57 s^-1) and an excellent stereoselectivity (>99%) for ketoisophorone. Engineered E. coli cells harboring PaER and glucose dehydrogenase (for cofactor regeneration) were employed as biocatalysts for the asymmetric reduction of ketoisophorone. As a result, up to 1000 mM ketoisophorone was completely and enantioselectively converted to (R)-levodione with a >99% ee value in a space-time yield of 460.7 g L^-1 d^-1. This study provides a great potential biocatalyst for practical synthesis of (R)-levodione.

Fig. 1. Phylogenetic relationship of amino acid sequences of PaER to other OYEs with known function. Bootstrap values at the nodes were expressed as percentages of 1000 replications. The accession number of OYEs is shown in brackets.

Fig. 2. SDS-PAGE analysis of the expression and purification of PaER. Lane M, protein molecular weight marker; Lane 1, whole-cell of E. coli expressing PaER; Lane 2, crude extract of E. coli expressing PaER; Lane 3, precipitate of E. coli expressing PaER; Lane 4, purified PaER.

Fig. 3. Effect of pH (A) and temperature (B) on enzyme activity of PaER.

Fig. 4. (A) The pH stability of PaER in various pH (5.0–10.0) conditions. (B) The thermostability of PaER at various temperatures (4, 30 and 40 °C).

Fig. 5. Catalytic profile of PaER and the measured specific activities toward 1a–14a of the substrate library.

Fig. 6. (A) Proposed catalytic mechanism of PaER for the reduction of ketoisophorone. (B) 3D structure generated by docking of ketoisophorone into the active pocket of PaER.

Fig. 7. Asymmetric bioreduction of ketoisophorone. (A) PaER-mediated reduction reaction for preparation of (R)-levodione. (B) Construction of engineered E. coli strains. (C) Comparison of the (R)-levodione production in each constructed E. coli strain. The reaction system (1 mL) contained 20 mM substrate (3a), 1 g L⁻¹ lyophilized E. coli cells, 0.2 mM NADP⁺, 40 mM glucose, and 100 mM PBS (pH 7.5).

Fig. 8. Reaction progress curves of ketoisophorone by Strain 3 on a preparative scale. Reaction mixtures (20 mL): ketoisophorone (1000 mM), glucose (1050 mM), NADP⁺ (0.05–0.2 mM), 10 g L⁻¹ lyophilized E. coli cell (Strain 3), and PBS buffer (100 mM, pH 7.5).