- Open Access
Gene mining-based identification of aldo–keto reductases for highly stereoselective reduction of bulky ketones
© The Author(s) 2018
- Received: 10 April 2018
- Accepted: 5 July 2018
- Published: 16 July 2018
Aldo–keto reductase (AKR) or alcohol dehydrogenases (ADH)-mediated stereoselective reduction of prochiral carbonyl compounds is an efficient way of preparing single enantiomers of chiral alcohols. However, steric hindrance of substrate affects the catalytic performance of enzymes. The present study aims to discover and identify AKRs/ADHs capable of catalyzing highly stereoselective reduction of sterically hindered ketones.
Five AKRs from different microorganisms (CaCR, ScCR, KmCR, CPR-C1, and CPR-C2) were identified through gene mining, and overexpressed in recombinant Escherichia coli BL21(DE3). The specific activity and stereoselectivity of the AKRs were further evaluated towards various ketoesters and heterocyclic ketones, which are sterically bulky and are valuable in industrial applications. Each purified enzyme exhibited catalytic activity to one or more of the tested substrates. Among the enzymes, ScCR showed a broader substrate spectrum compared to the others. Regarding Km values related to substrate association, we also provided insights into the specificity and preference of certain enzymes. Consequently, enantiopure (R)-methyl mandelate, ethyl (R)-mandelate, ethyl (R)-2-hydroxy-4-phenylbutyrate, and (S)-N-benzyl-3-pyrrolidinol (> 99%e.e.) were obtained through the identified AKRs.
- Aldo–keto reductase
- Carbonyl compound
- Gene mining
- Substrate specificity
Optically active alcohols have been recognized as key chiral intermediates for synthesizing fine chemicals and pharmaceuticals (Schoemaker et al. 2003; Zhao et al. 2017). Aldo–keto reductase (AKR) or alcohol dehydrogenase (ADH)-mediated stereoselective reduction of prochiral carbonyl compounds is an efficient way of preparing single enantiomers of chiral alcohols due to the high chemo-, enantio-, and regioselectivity of the enzymes (Ma et al. 2013). However, the majority of prochiral ketones used in industrial applications are structurally complex. These include ketoesters and heterocyclic ketones bearing bulky substituents, such as ethyl (R)-2-hydroxy-4-phenylbutyrate [(R)-HPBE], a key precursor for the preparation of an anti-hypertension drug, the angiotensin-converting enzyme (ACE) inhibitor (de Lacerda et al. 2006), and (S)-N-benzyl-3-pyrrolidinol, an important intermediate for the preparation of antitumor and anesthetic drugs (Kizaki et al. 2008). Substrate size-induced deficiency of enzyme activity is still a challenge for the enzymatic reduction of ketones. Therefore, it is necessary to develop suitable enzymes for substrates with bulky groups, for the production of corresponding chiral alcohols with high optical purity (Athanasiou et al. 2001).
For the discovery and identification of useful enzymes, bioinformatics analysis-based search from databases can be helpful in increasing the possibility and efficiency of finding candidate ideal biocatalysts (Kaluzna et al. 2004). Gene mining, a method through which homologous proteins are searched from a gene database using known enzymes as the template, is a promising approach (Wang et al. 2011). Through this method, various carbonyl reductases and AKRs exhibiting catalytic activities towards various carbonyl substrates have been discovered from the genome of Candida parapsilosis (Guo et al. 2014; Nie et al. 2011).
Substrates and applications of the corresponding alcohols
Product configuration and application
(R)-1b, a precursor of semi-synthetic penicillins, cephalosporins, vasodilators, fungicides, and sedatives
Guo et al. (2010)
(R)-2b, a precursor of cyclandelate, hydrobenzole, or pemoline
Kasprzak et al. (2016)
(R)-3b, a precursor of enalapril and lisinopril
de Lacerda et al. (2006)
(S)-4b, a precursor of fluoxetine
Engelking et al. (2006)
(S)-5b, a precursor of antitumor, anesthetic, antispasmodic, hepatotoxic, anti-inflammatory, and anti-HIV products
Kizaki et al. (2008)
Chemicals and strains
Ketone substrates such as, methyl benzoylformate (1a), ethyl benzoylformate (2a), ethyl 2-oxo-4-phenylbutyrate (3a), ethyl benzoylacetate (4a), 1-benzyl-3-pyrrolidinone (5a), and the enantiomers of corresponding chiral alcohols, methyl 2-hydroxy-2-phenylacetate (1b), ethyl mandelate (2b), ethyl 2-hydroxy-4-phenylbutyrate (3b), 3-hydroxy-3-phenylpropionate (4b), and N-benzyl-3-pyrrolidinol (5b), were obtained from J&K Chemical (Beijing, China). The cofactors (NADH and NADPH) were purchased from Solarbio (Beijing, China). The organic solvents used for product analysis by high-performance liquid chromatography (HPLC) were purchased from Tedia (Anhui, China).
Candida albicans CICC 31283, S. cerevisiae CICC1002, and K. marxianus CICC 1609 from the China Center of Industrial Culture Collection (Beijing, China) were used to obtain the genes encoding CaCR, ScCR, and KmCR, respectively. C. parapsilosis CCTCC M203011 from the China Center of Typical Culture Collection (Wuhan, China) was applied to obtain the genes encoding CPR-C1 and CPR-C2. Escherichia coli BL21(DE3) was adopted for expressing AKRs. The plasmid pET28a was purchased from Novagen (USA). Restriction endonucleases and PrimeSTAR® DNA polymerase (Premix) were obtained from Takara (Dalian, China).
Homologous protein-searching analysis
The BLAST tool at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to discover potential AKRs for bulky substrates, with the CaAKR amino acid sequence (GenBank Accession No. XP_711680.1) as the searching template, which has been identified to reduce bulky ketones (Wang et al. 2015). ClustalX software and Espript 3.0 were used for multiple sequence alignment.
Expression of AKRs in recombinant E. coli
The AKR genes were amplified from extracted genomic DNAs of C. albicans, S. cerevisiae, K. marxianus, and C. parapsilosis using the restriction sites-involved primer pairs synthesized according to the gene sequences of potential AKRs, respectively (Additional file 1: Table S1). By digestion with the corresponding restriction endonucleases, the resulting DNA fragments were inserted into the vector pET28a for expression. The recombinant plasmids, pET-28a-CaCR, pET-28a-ScCR, pET-28a-KmCR, pET-28a-CPR-C1, and pET-28a-CPR-C2, were transformed individually into the expression host cells of E. coli BL21(DE3). The transformants were incubated in the LB medium comprising 50 µg mL−1 kanamycin at 37 °C with shaking at 200 rpm.
The expression of target recombinant proteins was induced with 1 mM IPTG when the biomass of the culture reached approximately 0.6–0.8 in OD600, followed by further incubation at 17 °C for 14–16 h.
Purification of recombinant enzymes
Through centrifugation at 7104×g and 4 °C for 20 min, the harvested cells were washed twice with saline. After resuspension in the binding buffer (500 mM NaCl, 20 mM imidazole, 20 mM Tris/HCl, and pH 8.0), the cells were disrupted by an Vibra-Cell™ Ultrasonic Liquid Processor VCX750 (Sonic, USA). Through centrifugation at 26,000×g and 4 °C for 40 min, the supernatant was collected and then purified by Ni–NTA affinity chromatography on AKTA purifier 10 (GE Healthcare, USA). The enzymes were obtained with an elution buffer (500 mM NaCl, 150 mM imidazole, 20 mM Tris/HCl, and pH 8.0). The collected protein fractions were dialyzed against the buffer (500 mM NaCl, 20 mM Tris/HCl, and pH 8.0) at 4 °C for 12 h. The molecular weight and purity of the enzymes were determined through SDS-PAGE with a 10% polyacrylamide gel.
By recording the decline of the absorbance of the cofactor at 340 nm, enzyme activity was determined in the standard reaction mixture (100 µL) consisting of sodium phosphate buffer (0.1 M, pH 6.5), 0.5 mM NAD(P)H, 5 mM substrate, and an appropriate amount of purified enzyme. Protein concentration was measured through Bradford assay with bovine serum albumin (BSA) as the standard. One unit of AKR activity was defined as the amount of enzyme demanded to catalyze the oxidation of 1 µmol cofactor (NADH or NADPH) per minute under the given conditions.
Measurement of kinetic parameters
To determine the Michaelis–Menten kinetic parameters of the enzymes, the initial velocities of enzymatic reactions were measured at different concentrations of each substrate (0.05–4 mM), with the cofactor at saturated concentrations. Tetrahydrofuran (1% v/v) was used as the cosolvent for the dissolution of carbonyl substrates. Apparent kinetic parameters of the enzymes were calculated from Lineweaver–Burk double-reciprocal plot.
Asymmetric reductions and analytical methods
Analytical methods of enantiomers of chiral alcohols
Retention time (min)
OB-H, 210 nm, hexane:2-propanol = 88:12
Guo et al. (2010)
OD-H, 210 nm, hexane:2-propanol = 97:3
Kasprzak et al. (2016)
OD-H, 210 nm, hexane:2-propanol = 19:1
Nikaido et al. (1992)
OB-H, 220 nm, hexane:2-propanol = 9:1
Ou et al. (2011)
OB-H, 220 nm, hexane:2-propanol = 19:1
Yamada-Onodera et al. (2007)
Discovery of potential AKRs
Selected proteins from GenBank by BLAST against CaAKR
Theoretical molecular weight (Da)
Protein expression and purification
Stereospecific AKRs were discovered through gene mining. The catalytic performance of the newly identified enzymes was evaluated for the reduction of various carbonyl compounds, including ketoesters and heterocyclic ketones, from which chiral alcohol products that are valuable intermediates for the synthesis of important pharmaceuticals are obtained. All the enzymes exhibited catalytic activity towards ethyl benzoylformate. In addition, CPR-C2 even showed activity towards 1-benzyl-3-pyrrolidinone. Moreover, the enzymes were capable of catalyzing highly stereospecific reduction of the tested substrates to their corresponding chiral alcohols in high optical purity (> 99%e.e.). Based on their catalytic performance and excellent stereoselectivity, the newly identified AKRs may be useful in preparation of important enantiopure alcohols.
YN, XM, and YX designed the experiments; CL performed the experiments; CL and YN wrote this manuscript. All authors read and approved the final manuscript.
We would like to thank Editage (http://www.editage.cn) for English language editing.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this article.
Consent for publication
All authors have read and approved to submit it to bioresources and bioprocessing. There is no conflict of interest of any author in relation to the submission.
Ethics approval and consent to participate
Financial supports from the National Natural Science Foundation of China (21336009, 21676120), the Natural Science Foundation of Jiangsu Province (BK20151124), the 111 Project (111-2-06), the High-end Foreign Experts Recruitment Program (GDT20183200136), the Program for Advanced Talents within Six Industries of Jiangsu Province (2015-NY-007), the National Program for Support of Top-Notch Young Professionals, the Fundamental Research Funds for the Central Universities (JUSRP51504), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Jiangsu province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program, and the National First-Class Discipline Program of Light Industry Technology and Engineering (LITE2018-09) are greatly appreciated.
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