Cloning and expression of Kluyveromyce marxianus gene encoding carbonyl reductase in Escherichia coli

Abstract:

[Objective] The gene encoding carbonyl reductase was cloned and expressed for the synthesis of the chiral drug intermediates. [Methods] Based on the N-terminal amino acid sequence of carbonyl reductase from GenBank, cmcr was cloned from Kluyveromyce marxianus CGMCC 2.1977 and sequenced, an expression vector, pET28a-cMCR containing the full length of cmcr was constructed and introduced into Escherichia coli BL21(DE3) and Rosetta(DE3) to express the enzyme separately. [Results] This gene showing 100% similarity to reported mer contains an open reading frame of 1 038 bp encoding 345 amino acid residues. CMCR was overexpressed in Rosetta(DE3) with a protein molecular weight of 42 kD. The optimal temperature was 40 °C and pH 8. The enzyme has low thermal and pH stability. Ca2+ activates the enzyme activity, especially when its concentration is 0.5 mmol/L. The asymmetric reduction of 4-chloro-3-oxobutanoate ethyl ester to (S)-4-chloro-3-hydroxyl butanoate ethyl ester (CHBE) with Rosetta(pET28a-cMCR) cells, in which cmcr was expressed, as a catalyst was investigated (100% e.e., 81.0% yield). The application of the cells to the asymmetric reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-prapanamine(DKTP) to (S)-N,N-dimethyl-3- hydroxy-(2-thienyl)-1-propanamine [(S)-DHTP] was also attempted. [Conclusion] The gene encoding carbonyl reductase was cloned and expressed successfully in E. coli form Kluyveromyce marxianus CGMCC 2.1977 and can be applied to asymmetric reduction.