Engineering of a novel carbonyl reductase with coenzyme regeneration in E. coli for efficient biosynthesis of enantiopure chiral alcohols

• The novel carbonyl reductase (AcCR) from our isolated Acetobacter sp. CCTCC M209061 was, for the first time, successfully expressed in E. coli. with markedly enhanced enzymatic activity.
• A co-expression system harboring AcCR and GDH was effectively constructed to obtain an efficient whole-cell biocatalyst with in situ coenzyme regeneration.

The novel anti-Prelog stereospecific carbonyl reductase from Acetobacter sp. CCTCC M209061 was successfully expressed in E. coli combined with glucose dehydrogenase (GDH) to construct an efficient whole-cell biocatalyst with coenzyme NADH regeneration. The enzymatic activity of GAcCR (AcCR with a GST tag) reached 304.9 U/g-dcw, even 9 folds higher than that of wild strain, and the activity of GDH for NADH regeneration recorded 46.0 U/mg-protein in the recombinant E. coli. As a whole-cell biocatalyst, the recombinant E. coli BL21(DE3)pLysS (pETDuet-gaccr-gdh) possessed a broad substrate spectrum for kinds of carbonyl compounds with encouraging yield and stereoselectivity. Besides, the asymmetric reduction of ethyl 4-chloroacetoacetate (COBE) to optically pure ethyl 4-chloro-3-hydroxybutyrate (CHBE) catalyzed by the whole-cell biocatalyst was systematically investigated. Under the optimal reaction conditions, the optical purity of CHBE was over 99% e.e. for (S)-enantiomer, and the initial rate and product yield reached 8.04 μmol/min and 99.4%, respectively. Moreover, the space-time yield was almost 20 folds higher than that catalyzed by the wild strain. Therefore, a new, high efficiency biocatalyst for asymmetric reductions was constructed successfully, and the enantioselective reduction of prochiral compounds using the biocatalyst was a promising approach for obtaining enantiopure chiral alcohols.

The general process of the asymmetric reduction of carbonyl compounds to chiral alcohols with the recombinant E. coli BL21 (DE3) pLysS (pETDuet-accr-gdh).Figure optionsDownload as PowerPoint slide