Rhodococcus strains as source for ene-reductase activity

Abstract

Rhodococcus strains are ubiquitous in nature and known to metabolise a wide variety of compounds. At the same time, asymmetric reduction of C=C bonds is important in the production of high-valued chiral building blocks. In order to evaluate if Rhodococci can be used for this task, we have probed several Rhodococcus rhodochrous and R. erythropolis strains for ene-reductase activity. A series of substrates including activated ketones, an aldehyde, an imide and nitro-compound were screened using whole cells of seven Rhodococcus strains. This revealed that whole cells of all Rhodococcus strains showed apparent (S)-selectivity towards ketoisophorone, while most other organisms show (R)-selectivity for this compound. Three putative ene-reductases from R. rhodochrous ATCC 17895 were heterologously expressed in Escherichia coli. One protein was purified and its biocatalytic and biochemical properties were characterised, showing typical (enantioselective) properties for class 3 ene-reductases of the old yellow enzyme family.

Keywords: Asymmetric reduction; Enantioselectivity; Ene-reductase; Rhodococcus.

Fig. 1

Substrate screening for ene-reductase activity of R. rhodochrous ATCC 17895. Activity is based on product yield: substrates in red boxes were converted to the expected product and blue boxes indicate no conversion at all or conversion to unwanted products

Fig. 2

Phylogenetic relationship of RhrERs to other OYEs with known function from different classes (Scholtissek et al. 2017). The tree was constructed using the “One-Click” Mode on Phylogeny.fr (Dereeper et al. 2008), using the same OYEs as in the sequence alignment (Fig. S1). The class of known OYEs is written behind the name. Branch support values are indicated in red

Fig. 3

SDS-PAGE gel analysis of the purified RhrER 2718 from R. rhodochrous ATCC 17895. Lane 1, purified ene-reductase; lane 2, crude extract from E. coli expressing the gene; lane 3, molecular weight marker (BioRad Precision Plus Protein Standard)

Fig. 4

Temperature optima (a), pH optima (b), thermostability (c) and Michaelis–Menten kinetics (d) of the purified RhrER 2718. The activity was measured using the standard UV assay towards 2-methyl-2-cyclopentenone 2a. a All the reaction mixtures were kept at given temperatures for 5 min before NADH and enzyme solution were added to initiate the reaction. b The activity was assayed in the following 50 mM buffers: (i) sodium citrate (pH 5.0–6.0) (□), (ii) potassium phosphate (pH 6.0–9.0) (●), and (iii) glycine-NaOH (pH 9.0–11.0) (◊); reaction mixtures were incubated at 30 °C for 5 min before NADH and enzyme solution were added to initiate the reaction. c The enzyme solutions were kept for 1 h at each temperature before the samples were withdrawn to measure the residual activity in the soluble protein content. d V_max = 1.1 μmol∙min⁻¹∙mg⁻¹ and K_m = 1.6 mM