Genome Data Mining Of Quinuclidinone Reductases For The Asymmetric Synthesis Of (R)-3-Quinuclidinol

Optically pure (R)-3-quinuclidinol is an important chiral building block of many anti-muscarinic agents for the treatment of Alzheimer’s disease and COPD (chronic obstructive pulmonary disease), such as talsaclidine, revatropate, and aclidinium bromide. This research was focused on the screening of robust carbonyl reductases for the synthesis of (R)-3-quinuclidinol by asymmetric reduction.Firstly, genome mining was performed in NCBI and Uniprot database according to the target reaction. Two enzymes ArQR and SaQR from Agrobacterium radiobacter ECU2556and Sulfobacillus acidophilus respectively were selected further as their higher abilities of reducing3-quinuclidinone to (R)-3-quinuclidinol after the recombinant expression in E. coli.Secondly,ArQR and SaQR with N-terminal His-tag were purified to electrophoretic homogeneity by nickel affinity chromatography. The specific activities of ArQR and SaQR were198U/mg and42U/mg and both of them were homodimers. ArQR exhibited its highest activity at pH7.0,40℃and its half time at30℃,35℃,40℃were89h,8.3h,1.2h. While SaQR performed its best at pH7.0,50℃and its half-lives at30℃,40℃,50℃were130h,24h,2.5h. Metal ion Zn2+showed serious deactivation to ArQR while Ni2+showed deactivation to SaQR. Otherwise, EDTA decreased the activity of ArQR, but didn’t have obvious influence to SaQR. The Km and kcat values of ArQR and SaQR towards3-quinuclidinone were0.4mM,116s-1and1.67mM,92.8s-1respectively.Finally, the coexpression plasmids of ArQR and BmGDH (glucose dehydrogenase from Bacillus megaterium) were constructed in a tandem mode for cofactor regeneration. The fusion sequence of ArQR and BmGDH was studied and the induction conditions of the coexpressed strain were optimized. Then all the important factors for3-quinuclidinone reduction including the biocatalyst dosage, substrate loading, and external NAD+concentration were assessed systematically. To our satisfactory, with the addition of242g L-13-quinuclidinone,0.1mM NAD+,1.5equivalence glucose and0.1g lyophilized cells of E. coli (pET28a-ArQR-BmGDH) into the reaction system, the conversion reached100%in4.5h and the space time yield was916g L-1d-1with ee>99%. So far among all the quinuclidinone reductases reported, ArQR was the only one with the substrate loading more than200g/L, demonstrating its great potential in industrial manufacturing.