**1. Introduction**

Many natural products and active pharmaceutical ingredients share a common piperidine core, and the introduction of a chiral hydroxyl group on the C3-position of the piperidine ring may alter the bioactivity of the molecule [1–3]. (*S*)-*N*-Boc-3-hydroxypiperidine ((*S*)-NBHP) is a key chiral intermediate in the synthesis of ibrutinib as the inhibitor of Bruton's tyrosine kinase [4]. In the chemical synthesis of (*S*)-NBHP, employed strategies include the synthesis of racemic 3-hydroxypiperidine followed by chiral resolution and the enantiospecific synthesis of (*S*)-NBHP from chiral precursors. The former only achieves a maximum yield of 50%, making the process economically unviable, while the latter appears to be limited because of the lengthy procedure, rather poor yields of the products, and the use of potentially hazardous reagents [1,5,6]. Alternatively, the carbonyl-reductase-catalyzed asymmetric reduction of *N*-Boc-3-piperidone (NBPO) has gained increasing focus due to its mild reaction conditions, high yield, and remarkable enantioselectivity [4,7–9].

Coenzymes are required in carbonyl reductase-catalyzed reactions, and well-established approaches for coenzyme regeneration include the use of a second enzyme and a second substrate (i.e., glucose dehydrogenase and glucose), and the use of the second substrate catalyzed by the same enzyme (i.e., 2-propanol) [10]. Recently, an NADPH-dependent carbonyl reductase from *Saccharomyces cerevisiae* (YDR541C) was employed for the efficient synthesis of (*S*)-NBHP from NBPO by adopting a biphasic system to alleviate product inhibition and using glucose/glucose dehydrogenase to achieve coenzyme regeneration [8]. The glucose/glucose dehydrogenase system yields to the continuous production of gluconic acid; thus, pH adjustment is needed during the reaction, eventually making the process more complex and forming a large quantity of solid waste salt. Alternatively, the 2-propanol oxidation catalyzed by the same carbonyl reductase was widely used for coenzyme regeneration in order to simplify the operating process and increase the solubility of the substrates [11]. An efficient process catalyzed by the commercially-available ketoreductase KR-110 has been demonstrated to reduce 0.5 M NBPO to render the (*S*)-NBHP yield of 97.6% after a 24-h reaction [4]. The enzyme KR-110 was heat-sensitive and the substrate inhibition was obviously observed at a substrate concentration of 0.5 M. In addition, the 2-propanol concentration is usually required in excess to increase the product yield. Thus, high concentrations of the co-substrate together with the substrate further aggravate the inhibition of the enzyme activity in the 2-propanol-coupled strategy [4,11].

To overcome the inhibition from the high load of substrate/co-substrate, protein engineering is one of the promising approaches expanding the upper limit of the substrate/co-substrate concentration on a larger preparative scale [12,13]. Variants of the phenylacetaldehyde reductase from *Rhodococcus* sp. ST-10 (PAR) have been constructed through directed evolution, fully converting 200 g/L ethyl 4-chloro-3-oxobutanoate into ethyl (*S*)-4-chloro-3-hydroxybutyrate in the presence of 15% (*v*/*v*) 2-propanol [14,15]. Furthermore, attempts with biphasic catalysis in the presence of water-immiscible organic solvents have demonstrated an intriguing potential for overcoming the inhibition from substrate/co-substrate, increasing the solubility of substrates, easy product removal, decreasing the spontaneous hydrolysis of substrate/product, and avoiding unfavorable equilibria [16–19]. In an aqueous/octanol biphasic system, the biosynthesis process of ethyl (*R*)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from *Burkholderia gladioli* was established, in which 1.2 M ethyl 4-chloro-3-oxobutanoate was completely converted to afford ethyl (*R*)-4-chloro-3-hydroxybutyrate through the substrate fed-batch strategy [20]. In addition, the integration of protein engineering and medium engineering can further improve the effectiveness of asymmetric reduction at a high substrate load [20–22].

Although several processes for the efficient biosynthesis of (*S*)-NBHP have been developed, the pivot carbonyl reductases as biocatalysts still lack an in-depth characterization. Our previous genome mining enabled the discovery of chiral ketoreductases from *Rhodococcus erythropolis* WZ010 and the exploration of its application in the synthesis of chiral alcohols [23,24]. Here, a strictly (*S*)-enantioselective carbonyl reductase from *R. erythropolis* WZ010 (ReCR) and its variant Y54F were characterized for the efficient bioreduction of NBPO to (*S*)-NBHP, providing a basis for process development with an efficient coenzyme regeneration employing (*R*/*S*)-2-octanol or 2-propanol as the co-substrate (Scheme 1).

**Scheme 1.** Asymmetric bioreduction of *N*-Boc-3-piperidone (NBPO) using (*R*/*S*)-2-octanol or 2-propanol as co-substrate for NADH regeneration.

#### **2. Results and Discussion**
