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.
Graphical abstractThe 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 full-size imageDownload as PowerPoint slide
Journal: Journal of Biotechnology - Volume 230, 20 July 2016, Pages 54–62