An ancestral carbonyl reductase: mining and application in the synthesis of (S)-acetoin

Abstract:

[Background] (S)-Acetoin is a class of important fine chemicals with wide applications. Efficient and sustainable synthesis of (S)-acetoin is of special interest. [Objective] Due to the high thermostability, ancestral carbonyl reductases are promising in industrial biocatalysis. This study aims to mine an ancestral carbonyl reductase with high activity and thermostability, characterize its enzymatic properties, and establish an efficient enzymatic catalysis system for the synthesis of (S)-acetoin. [Methods] The gene mining strategy based on ancestral sequence reconstruction was adopted to identify ancestral carbonyl reductases. The enzyme properties were characterized, and the purified ancestral carbonyl reductase was used for the establishment of the (S)-acetoin synthesis system. [Results] An ancestral carbonyl reductase AncBDH1 was identified and heterogeneously expressed in Escherichia coli in the soluble form, and it displayed a high activity in the asymmetric reduction of diethyl into (S)-acetoin. The sequence of AncBDH1 contained the conserved motifs of short-chain dehydrogenase/reductase superfamily and exhibited low similarity with the sequences of other reported carbonyl reductases. AncBDH1 was purified by nickel-affinity chromatography for enzyme characterization. It displayed high dependence on NADH instead of NADPH as a cofactor, with the optimum performance at pH 7.0 and 60℃. The activity of AncBDH1 was not dependent on metal ions and AncBDH1 could tolerate organic solvents such as dimethyl sulfoxide. AncBDH1 completely reduced 200 mmol/L diethyl into (S)-acetoin within 12 h. [Conclusion] This study provides a novel ancestral carbonyl reductase AncBDH1 with tolerance to high temperature and organic solvents and capable of catalyzing the synthesis of (S)-acetoin, laying a certain research foundation for the biocatalytic synthesis of (S)-acetoin.