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Overexpression of epoxide hydrolase in Rhodococcus ruber with high robustness for the synthesis of chiral epichlorohydrin
Process Biochemistry 79 (2019) 49–56
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Overexpression of epoxide hydrolase in Rhodococcus ruber with high robustness for the synthesis of chiral epichlorohydrin
T
⁎
Youxiang Lianga,b, Song Jiaoa,b, Miaomiao Wanga,b, Huimin Yua,b,c, , Zhongyao Shena,b a
Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China Key Laboratory of Industrial Biocatalysis (Tsinghua University), the Ministry of Education, Beijing, 100084, China c Center for Synthetic and Systems Biology, Tsinghua University, Beijing, 100084, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Rhodococcus ruber Whole-cell biocatalysts Epoxide hydrolase Stability and organic-solvent tolerance Chiral epoxides
Epoxide hydrolases (EHs) are attractive enzymes for producing enantiopure epoxides and diols, but do not display enough stability when lysates or Escherichia coli whole cells are used as biocatalysts. In this work, an organic-solvent tolerant strain Rhodococcus ruber THdAdN was utilized to overexpress an epoxide hydrolase from Agrobacterium radiobacter (ArEH), using E. coli BL21(DE3) as a control. The proportion of ArEH in all soluble proteins of R. ruber THdAdN(ArEH) reached 30.3%, which was comparable to that of E. coli BL21(DE3)(ArEH). Due to a higher cell density in flask cultivation, the maximum ArEH activity of R. ruber THdAdN(ArEH) toward epichlorohydrin (ECH) reached 5.4 U/mL, approximately 5-fold higher than that of E. coli BL21(DE3)(ArEH). More importantly, compared with E. coli BL21(DE3)(ArEH), ArEH in R. ruber THdAdN(ArEH) showed a 10-fold enhanced thermostability, better tolerance against alkali pH, and reduced substrate and product inhibition, which significantly improved its performance in the resolution of high concentration ECH. By substrate feeding, 98.5% ee (R)-ECH was obtained with a 35.5% yield from 512 mM racemic ECH hydrolyzed by R. ruber THdAdN (ArEH). Compared with using free enzyme as biocatalysts, utilization of the R. ruber cells harboring ArEH improved the final (R)-ECH concentration by 46%.
1. Introduction Optically pure epoxides and vicinal diols are valuable chiral building blocks for the preparation of bioactive compounds such as pharmaceuticals, agrochemicals and other fine chemicals [1,2]. Traditional chemical production methods, including asymmetric epoxidation or dihydroxylation of olefin substrate and resolution of racemic epoxides with metal-based catalysts [3,4], lead to safety and environmental concerns. In the past two decades, epoxide hydrolases (EHs) that catalyze the hydrolysis of racemic epoxides to corresponding vicinal diols have attracted much attention. EHs are cofactor-independent, enantioselective and ubiquitous in nature, making them highly appealing in the preparation of enantiopure epoxides and vicinal diols [5,6]. Currently, Escherichia coli cells are the most common host for the overexpression of EH [1,7–11]. However, EHs do not display enough stability under operating conditions when lysates or E. coli whole cells are used as biocatalysts [8,12]. Enzyme inhibition caused by toxic
organic substrates and products hinders the application of EHs at high substrate concentrations [8]. To overcome instability and low solubility of epoxides in the aqueous phase, organic solvents are frequently introduced as an alternative reaction media or to form a two-liquid-phase system. The consequent enzyme inactivation caused by the phase interface and organic solvents is not negligible [13,14]. Therefore, it is of great importance to improve the stability of EHs. Strategies such as immobilization and directed evolution have been widely studied [10,15–19]. For example, with ethylenediamine functionalized epoxy resin as a carrier, the half-life of immobilized epoxide hydrolase from Agrobacterium radiobacter (ArEH) was 6.8-fold longer than that of the free enzyme [15]. Through iterative saturation mutagenesis (ISM), a mutant of ArEH (T247 K/I108 L/D131S) was obtained with an 8-fold longer half-life than the wide-type [16]. However, enzyme immobilization is often coupled with a great loss of activity, while directed evolution is labor-intensive and requires high-throughput screening methods. A number of microorganisms, including Pseudomonas, Bacillus and
Abbreviations: EHs, epoxide hydrolases; ArEH, epoxide hydrolase of Agrobacterium radiobacter; ECH, epichlorohydrin; 3-MCPD, 3-chloro-1,2-propanediol; ee, enantiomeric excess; IPTG, isopropyl-β-thiogalactopyranoside; g dcw/L, gram dry cell weight per liter ⁎ Corresponding author at: Yingshi Building 412, Tsinghua University, Beijing, 100084, China. E-mail address: [email protected] (H. Yu). https://doi.org/10.1016/j.procbio.2018.12.023 Received 3 October 2018; Received in revised form 19 December 2018; Accepted 21 December 2018 Available online 23 December 2018 1359-5113/ © 2018 Elsevier Ltd. All rights reserved.
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this mutant. Plasmid pET28a-ArEH (see Fig. S2) was constructed by inserting the mutated ArEH gene (amplified by primers BamH I-ArEH-sense and Hind III-ArEH-anti) into the plasmid pET28a between the BamH I and Hind III restriction endonuclease sites and transformed into E. coli BL21(DE3) competent cells to obtain E. coli BL21(DE3)(ArEH). ArEH was fused with a 6×His-tag, thrombin site and T7 tag, amplified by the Xba IArEH-sense and EcoR I-ArEH-anti primers from pET28a-ArEH, and inserted into the plasmid pNV18.1 [28] under an amidase promoter Pa2 (see Table S2) [29]. The resulting plasmid pNV18.1-Pa2-ArEH (see Fig. S3) was transformed into the competent cells of R. ruber THdAdN by electroporation [30]. Restriction enzymes, DNA polymerases, T4 DNA ligases and a sitedirected mutagenesis kit were purchased from Takara (Dalian, China) and Vazyme (Nanjing, China). DNA purification kits, gel extraction kits, BCA-protein quantification assay kits, and plasmid extraction kits were purchased from Biomega (Shanghai, China). Protein marker (MM1397500) was purchased from GenScript (Nanjing, China). ArEH gene synthesis was completed by Qinglan (Wuxi, China) and DNA sequencing was performed by GENEWIZ (Suzhou, China). DNA manipulations including PCR, agarose gel electrophoresis, restriction enzyme digestion, ligation and transformation were performed following standard procedures from the manufacturers’ protocols.
Rhodococcus, have shown excellent organic-solvent tolerance that helps improve their stability in reaction media containing organic solvents or toxic compounds [20]. Among them, Rhodococcus is a group of grampositive bacteria with a range of metabolic activities that enable them to degrade toxic organic compounds such as nitriles, halogenated and aromatic hydrocarbons [21–23]. Rhodococcus has been widely applied in bioconversion, bioremediation and biosynthesis. Production of acrylamide from acrylonitrile catalyzed by Rhodococcus harboring nitrile hydratase (NHase) is one of the most successful cases of industrial biotechnology [24,25]. Previously, NHase was cloned in E. coli but suffered from severe inactivation caused by high concentration acrylamide and high temperature [26]. In comparison, NHase in Rhodococcus showed higher tolerance against acrylamide and acrylonitrile, as well as better thermostability [25], due to the special features of Rhodococcus cells. In this work, an engineered strain Rhodococcus ruber THdAdN with high robustness was utilized for the overexpression of a mutant epoxide hydrolase ArEH. The most common host E. coli BL21(DE3) was used as a control. Characteristics of the two engineered strains were assessed, including in-cell EHs activity, kinetic parameters, and optimal temperature and pH. Moreover, their stability against temperature, pH, epoxides and diols, as well as their performance in the resolution of high concentration epichlorohydrin (ECH) were compared.
2.2. Cell cultivation and protein expression 2. Material and methods R. ruber THdAdN was cultivated in a 100 mL conical flask containing 20 mL of seed media (30 g/L glucose, 1 g/L yeast extract, 7 g/L tryptone, 0.5 g/L K2HPO4•3H2O, 0.5 g/L KH2PO4, 0.5 g/L MgSO4•7H2O, 1 g/L monosodium glutamate, pH 7.5) with 25 mg/L kanamycin, at 200 rpm and 28 °C [27]. The cell density was monitored spectrophotometrically at 460 nm to determine the growth curves. After 48 h, 5 mL of seed liquid in the exponential phase was transferred into a 500 mL shaking flask containing 50 mL of fermentation media (30 g/L glucose, 7–8 g/L yeast extract, 10 g/L urea, 2.28 g/L K2HPO4•3H2O, 0.5 g/L KH2PO4, 1 g/L MgSO4•7H2O, 1 g/L monosodium glutamate, pH 7.5), and incubated at 200 rpm and 28 °C to induce the expression of ArEH with urea as inducer for 36 h. Samples were withdrawn periodically to monitor the cell density and ArEH activity. E. coli BL21(DE3) was cultured in 10 mL Luria–Bertani (LB) media (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) containing 50 mg/L of kanamycin in a 50 mL shaking flask at 200 rpm and 37 °C for 12 h. Next, 0.5 mL of seed was transferred into 50 mL of LB media containing 50 mg/L kanamycin in a 300 mL shaking flask. The expression of ArEH was induced for 8 h at 200 rpm and 28 °C by adding 0.5 mM isopropylβ-thiogalactopyranoside (IPTG) when the OD600 reached 0.6˜0.8 [8]. Samples were withdrawn periodically to monitor the cell density and ArEH activity. The cells above were harvested by centrifugation at 10,000×g for 15 min at 4 °C, and stored at −20 °C.
2.1. Strains, plasmids and DNA manipulations The strains and plasmids used in this study are listed in Table 1, and the primers are listed in Table S1. R. ruber THdA was an amiE-negative mutant that was deposited in the China General Microbiological Culture Collection Center (CGMCC No. 2381). R. ruber THdAdN was a mutant with both amidase and NHase knocked out, serving as an efficient host for overexpression of heterologous genes [27]. E. coli DH5α was used for plasmid construction. E. coli BL21(DE3) was used as the control host for protein expression. The EH gene of Agrobacterium radiobacter AD1 (GenBank accession No. Y12804) was synthesized according to the codon preference of R. ruber and E. coli, as shown in Fig. S1. The GenBank accession numbers of the codon-optimized gene is MH480511. Four mutations (F108I, P205H, Y215H, E271 V) [11] were introduced via three rounds of sitedirected mutagenesis to obtain the mutant ArEH (GenBank accession No. MH480512). In this study, ArEH mentioned afterward indicated Table 1 Strains and plasmids used in this study. Strains/Plasmids Plasmids pET28a pMV-ArEH pET28a-ArEH pNV18.1 pNV18.1-Pa2-ArEH Strains E. coli DH5α E. coli BL21(DE3) E. coli BL21(DE3)(pET28a) E. coli BL21(DE3)(ArEH) R. ruber THdAdN R. ruber THdAdN(pNV18.1) R. ruber THdAdN(ArEH)
Relevant genotypes
Source
5.4 kb, KanR Plasmid carrying codon-optimized ArEH gene pET28a with ArEH gene E. coli-Rhodococcus shuttle vector, KanR pNV18.1 with ArEH gene under promoter Pa2
Novagen This study
Host for plasmid construction Host for protein expression E. coli BL21(DE3) harboring pET28a E. coli BL21(DE3) harboring pET28aArEH R. ruber TH with amidase and NHase deleted R. ruber THdAdN harboring pNV18.1 R. ruber THdAdN harboring pNV18.1Pa2-ArEH
2.3. Protein purification and SDS-PAGE analysis This study [28]
The cells (approximately 2 g) were resuspended with 30 mL of 0.2 M sodium phosphate buffer (pH = 8.0) and disrupted by a high-pressure homogenizer at 1500 bar in a bath of ice water. After centrifugation (10,000 × g) at 4 °C for 15 min, the supernatant was applied onto a preequilibrated Ni-NTA column (5 mL). Buffer A (20 mM Na2HPO4/ NaH2PO4, 30 mM imidazole, pH = 8.0) was then applied to remove the unbound proteins, followed by buffer B (20 mM Na2HPO4/NaH2PO4, 300 mM imidazole, pH = 8.0) to elute the bound protein. The enzyme solution was dialyzed at 4 °C for 12 h against 20 mM Na2HPO4/ NaH2PO4 (pH = 8.0) to remove imidazole and then stored at −20 °C. Cell lysate was centrifuged at 10,000×g for 15 min at 4 °C. The precipitate was suspended to equal volume. Both the supernatant and precipitate suspension were analyzed with 12% sodium dodecyl sulfate-
This study
Solarbio Salarbio This study This study [27] This study This study
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phosphate buffer with a pH ranging from 2 to 12 at 30 °C for 30 min and then centrifuged at 10,000 × g for 3 min to remove the supernatant. The cells were then resuspended with 0.2 M sodium phosphate buffer (pH = 8.0) for activity measurement. Two mol/L NaOH was used to adjust solutions with a pH higher than 9, and 50% HCl was used to adjust solutions with a pH lower than 5. The initial activity of ArEH at pH 8.0 was defined as 100%.
polyacrylamide gel electrophoresis (SDS-PAGE). For the supernatant, 0.03 mg protein was added per well in the gel. For precipitation suspension, equal volume to the supernatant was added. 2.4. Activity measurement of ArEH R. ruber or E. coli cells was suspended in 0.2 M sodium phosphate buffer (pH = 8.0) to the initial volume, and then diluted to suitable cell concentrations (R. ruber was diluted by 5-fold, and E. coli was not diluted). In a 1.5 mL centrifuge tube, 0.36 mL of diluted cells or enzymes was mixed with 0.04 mL of 2.5% (v/v) racemic ECH in 0.2 M sodium phosphate buffer (pH = 8.0). Due to high level of the spontaneous hydrolysis of ECH in aqueous phase, a control without cells or enzymes was set as a background. The reaction was conducted at 30 °C and 1000 rpm in a thermo shaker for 5 min and terminated by adding 0.8 mL of hexane containing 0.1% (v/v) 1-chlorohexane as an internal standard. Samples were mixed on a vortex shaker for 1 min and then centrifuged. The organic layer was analyzed by gas chromatography after being dehydrated with anhydrous sodium sulfate. One activity unit was defined as the amount of cells or enzymes that catalyzed 1 μmol ECH into diol per minute. ECH concentration was determined by a GC system (Thermo Fisher Scientific, America) equipped with an Astec Chiraldex G-TA column (0.25 mm ID, 30 m length, 0.12 μm film thickness) and a FID detector. The temperature of the oven, injector and detector were 60 °C, 180 °C and 180 °C, respectively, and the pressure of carrier gas (nitrogen) was 108 kPa with a split ratio of 1:50. The retention time of the 1-chlorohexane, (S)-ECH and (R)-ECH were 5.4, 6.0 and 6.7 min, respectively.
2.9. Tolerance against substrate and product Recombinant cells were incubated in 0.2 M sodium phosphate buffer (pH = 8.0) containing ECH ranging from 0 to 638 mM or 3chloro-1,2-propanediol (3-MCPD) from 0 mM to 450 mM at 30 °C for 30 min. The cells were centrifuged at 10,000 × g for 3 min and washed twice to remove the residual ECH or 3-MCPD. The remained activities were then measured at pH 8.0 and 30 °C. The initial activity of ArEH before treatment was defined as 100%. 2.10. Enantioselective resolution of racemic ECH The ECH hydrolysis was conducted in a 100 mL bottle sealed with a rubber septum containing 30 mL of cell suspension in 0.2 M sodium phosphate buffer (pH = 9.0) at 30 °C. The reaction was initiated by adding a certain concentration of ECH. The hydrolysis course was monitored by periodically taking samples to analyze the residual ECH, and the reaction was stopped when the enantiomeric excess (ee value) of (R)-ECH reached 98%. The ee value was calculated using the equation ee = (R − S )/(R + S ) , where R and S represent the concentration of (R)ECH and (S)-ECH, respectively. The yield of (R)-ECH was calculated by the equation c = R/(R 0 + S0) , where R0 and S0 represent the initial concentration of (R)-ECH and (S)-ECH. The E value was calculated by the equation E = ln[(1 − c ) × (1 − ee )]/ln[(1 − c ) × (1 + ee )], where c represented the yield of (R)-ECH [8]. To reduce the inactivation of recombinant cells caused by ECH, substrate feeding was performed with an initial ECH concentration of 192 mM and a feeding rate of 10.67 mM/min for 30 min until the total ECH added reached 512 mM. The reaction was terminated when the ee value of (R)-ECH reached 98%.
2.5. Determination of kinetic parameters Kinetic parameters of ArEH toward (S)-ECH and (R)-ECH were estimated by measuring the initial activity of the recombinant cells or free enzymes at 30 °C and pH 8.0 with a substrate concentration range of 0.1˜15 mM and 5˜150 mM, respectively. Km and Vmax were calculated using the double reciprocal Lineweaver-Burk plot method. 2.6. Optimum temperature and pH of recombinant cells and purified enzymes
3. Results
The temperature characteristics of recombinant cells or purified enzymes were measured at pH 8.0 with temperature ranging from 15 °C to 60 °C. The pH characteristics were measured at 30 °C in 0.2 M sodium phosphate buffer with a pH range of 6 to 12. Two mol/L NaOH was used to adjust pH higher than 9.
3.1. Cloning and overexpression of ArEH in R. ruber THdAdN and E. coli BL21(DE3) A novel idea was proposed to develop R. ruber with high robustness [25,31] as an excellent whole-cell biocatalyst for enantioselective resolution of racemic ECH to produce (R)-ECH with overexpressed epoxide hydrolases, as shown in Fig. 1. In presence of inducer urea, the natively overexpressed NHase in R. ruber would reduce the expression efficiency of target enzymes, so the NHase-negative mutant R. ruber THdAdN [27] was utilized as the host strain. Several epoxide hydrolases have been utilized for the enantioselective resolution of ECH, but most of them showed limited enantioselectivty or activity [11,32–37]. Among them, EH from Agrobacterium radiaobacter (ArEH) is a classical one that has been well-studied [8,9,11,13]. The wild-type of ArEH showed no enantioselectivty toward ECH, but through directed evolution, Van et al. obtained a mutant (F108I/P205 H/Y215 H/E271 V) with an E-value of 40 [11], which is better than most of the reported EHs. In this study, we chose this mutant of ArEH for the production of (R)-ECH. R. ruber THdAdN(ArEH) was constructed and cultured in flasks, in parallel with E. coli BL21(DE3)(ArEH) and the strains containing blank plasmids. The expressions of ArEH in R. ruber THdAdN and E. coli BL21(DE3) were confirmed by SDS-PAGE. After cell disruption, both supernatant and precipitate were analyzed. As shown in Fig. 2, ArEH
2.7. Effect of substrate and product concentration on the activities of recombinant cells and purified enzymes The effect of substrate concentration was studied by measuring the initial ArEH activities of recombinant cells and purified enzymes with an ECH concentration ranging from 0 mM to 512 mM. Product inhibition was studied by measuring the initial ArEH activity in the presence of 3-chloro-1,2-propanediol (3-MCPD) ranging from 45˜450 mM. 2.8. Thermostability and pH stability of ArEH in recombinant cells and purified enzymes The thermostability of ArEH in the recombinant cells and purified enzymes was assessed by incubating the cell suspension in 0.2 M sodium phosphate buffer (pH = 8.0) at 55 °C for the indicated periods and measuring the residual activities at 30 °C. The initial activity of ArEH at 30 °C was defined as 100%. The half-lives of recombinant cells were calculated by the equation t1/2 = 0.693/ki. ki is the first-order constant for enzyme inactivation. To measure pH stability, the cells were incubated in 0.2 M sodium 51
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Fig. 1. Enantioselective resolution of racemic ECH for the production of (R)-ECH with engineered R. ruber THdAdN(ArEH) cells.
3.3. Characterization of recombinant cells and purified enzymes
achieved efficient expression in both of the strains, and nearly no inclusion body was formed. Cell growth curves and ArEH activities toward ECH were measured as shown in Section 2.2, and summarized in Fig. 3. The maximum ArEH activity in E. coli BL21(DE3)(ArEH) was 1.1 U/mL at 10.5 h when the biomass reached 0.74 g dcw/L. For R. ruber THdAdN(ArEH), the cell density was 6.8 g dcw/L at 36 h, and correspondingly the maximum ArEH activity reached 5.4 U/mL, approximately 5-fold higher than that of E. coli BL21(DE3)(ArEH). To compare the expression efficiency, the proportion of ArEH in all soluble proteins was analyzed by Quantity One (Bio-Rad, USA). ArEH contributed 38.4% of the total soluble proteins in E. coli BL21(DE3) (ArEH), and 30.3% in R. ruber THdAdN(ArEH).
The optimum temperature and pH of ArEH contained or not contained in cells were measured and compared in Fig. 4a and 4b. Generally, the cell-free ArEH purified from R. ruber THdAdN and E. coli BL21(DE3), and the ArEH contained in E. coli BL21(DE3)(ArEH) cells showed no significant difference. Their optimum catalysis conditions ranged from 45 to 50 °C and pH 8-9. For the ArEH contained in R. ruber THdAdN(ArEH), however, the optimum catalysis temperature and pH were enhanced to 55 °C and pH 10, respectively. More interestingly, ArEH in R. ruber THdAdN(ArEH) still retained 75% of its activity at pH 12; under this condition, it was completely inactivated in E. coli BL21(DE3)(ArEH). In addition, the effect of substrate and product on the ArEH activity was assessed as shown in Fig. 4c and 4d. Both intracellular and free ArEH showed a maximum activity at an ECH concentration of 256 mM and a sharp decline at 384 mM owing to the inactivation of ArEH caused by toxic ECH. Surprisingly, product inhibition of ArEH was reduced significantly in R. ruber THdAdN(ArEH). R. ruber THdAdN(ArEH) showed 88% of its initial ArEH activity in presence of 360 mM 3-MCPD, while E. coli BL21(DE3)(ArEH) and free enzymes showed no more than 56%.
3.2. Kinetic parameters of ArEH Kinetic parameters of intracellular ArEH and free ArEH for both enantiomers were measured and compared in Table 2. The Vmax for (R)ECH was higher than that for (S)-ECH, but the Km for (S)-ECH was much lower than that for (R)-ECH. Both intracellular ArEH and free ArEH hydrolyzed (S)-ECH preferentially, with a higher Vmax/Km for (S)-ECH than that for (R)-ECH. The apparent Km values of both R. ruber THdAdN (ArEH) and E. coli BL21(DE3)(ArEH) for the same enantiomers of ECH were similar to that of free enzymes. This result indicated that the mass transfer resistance conferred by the cell envelopes is low probably because ECH is a relatively small molecule. We found that the Vmax/Km of R. ruber for (S)-ECH was much lower than that of E. coli. This result is probably caused by the difference of intracellular environment between R. ruber and E. coli, as well as the expression level of ArEH.
3.4. Stability of whole-cell biocatalysts Stability of biocatalysts is of great importance to industrial applications, which requires biocatalysts to resist against harsh environments such as high temperature, extreme pH, organic solvents, and high concentrations of substrate and product. Fig. 2. SDS-PAGE analyses of recombinant cells expressing ArEH. (a) Supernatant of the cell lysate. (b) Precipitate of the cell lysate. The E. coli BL21(DE3)(ArEH) cells were harvested after an 8 h induction with 0.5 mM IPTG in LB media. R. ruber THdAdN(ArEH) cells were harvested after a 36 h induction with 10 g/L of urea in fermentation media. The molecular mass of ArEH fused with a 6×His-Tag and T7-Tag was calculated to be 37.3 kDa.
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Fig. 3. Cell growth and ArEH activity curves of engineered strains. Experiments were performed in triplicate.
To reduce the enzyme inactivation caused by high concentrations of ECH, substrate feeding was conducted. With R. ruber THdAdN(ArEH) as biocatalysts, 182 mM (R)-ECH was obtained with 98.5% ee and 35.5% yield from 512 mM ECH, as shown in Fig. 6b. However, when the total ECH concentration was further increased to 576 mM, cells suffered from severe inhibition after 30 min and only 48.5% ee of (R)-ECH was obtained, as shown in Fig. S5.
Thermostability and pH stability of intracellular and/or free ArEH were assessed and compared as shown in Fig. 5a and b. After incubation at 55 °C for 30 min, the residual activity of ArEH in R. ruber THdAdN (ArEH) cells was approximately 90%, but free ArEH and ArEH in E. coli BL21(DE3)(ArEH) was almost completely inactivated. The half-life of ArEH in R. ruber THdAdN(ArEH) was 50.3 min, approximately 10-fold longer than that in E. coli BL21(DE3)(ArEH), as shown in Fig S4. Moreover, R. ruber THdAdN(ArEH) could tolerate a much wider pH range than E. coli BL21(DE3)(ArEH), as shown in Fig. 5b. Tolerance of ArEH in recombinant cells against toxic substrate and product was measured and summarized in Fig. 5c and d. Incubation in 256 mM ECH for 30 min didn’t decrease the activity of ArEH in R. ruber THdAdN. However, under the same conditions, only 25% of the initial activity remained in E. coli BL21(DE3). In addition, incubation in 3MCPD solution showed no significant impact on the activity of ArEH in R. ruber THdAdN(ArEH).
4. Discussion Microbial enzymes and cells are important tools in asymmetric biocatalysis for the preparation of value-added chemicals including pharmaceuticals, agrochemicals and flavors [38]. Compared to free or immobilized enzyme, whole-cell biocatalysts are often preferred as they are simple and convenient to operate. Moreover, microbial cells can provide a relatively mild environment for enzymes, preventing denaturation of protein under harsh reaction conditions [39]. Among various host strains, E. coli is known as a standard “workhorse”. However, the insufficient stability of biocatalysts under operating conditions is still the main limitation for their industrial application [12]. For example, epoxide hydrolases, which are important enzymes widely used in the preparation of chiral epoxides and diols, often suffer from limited thermostability, substrate inactivation and product inhibition when lysates or E. coli whole cells are used as biocatalysts. The primary development methods such as directed evolution and immobilization are widely performed to improve the stability of enzymes [15,16]. In addition to E. coli, many researchers are interested in solvent-tolerant microorganisms including Pseudomonas, Bacillus and Rhodococcus, for their adaptation to harsh conditions [20]. In this study, the engineered strain R. ruber THdAdN with high tolerance to solvents and environmental stresses was utilized for the first time to overexpress heterologous EHs for the enhancement of incell enzyme stability. With a urea-induced promoter Pa2, efficient expression of ArEH was achieved in R. ruber THdAdN. The proportion of ArEH in all soluble proteins of R. ruber THdAdN(ArEH) reached 30.3%,
3.5. Enantioselective resolution of ECH at high concentrations For practical applications of epoxide hydrolases in the production of chiral epoxides or diols, it is unavoidable to increase the concentration of ECH, thereby reducing production costs. However, when substrate concentration increases, ee value and yield usually decrease because of the inactivation effect caused by toxic epoxides or diols. To assess the performance of the recombinant strains, a gradient of racemic ECH was hydrolyzed by recombinant cells until the ee value of (R)-ECH reached 98% or no longer improved. The statistics are shown in Table 3. The E value of E. coli BL21(DE3)(ArEH) sharply declined to 1.6 when the initial ECH concentration was over 384 mM; for R. ruber THdAdN (ArEH), however, the E value still remained as high as 13.2 under the same conditions. The productivities of different cell biocatalysts at various ECH concentrations were further calculated and summarized in Fig. 6a. For R. ruber THdAdN(ArEH), it was more than 2-fold higher than that of E. coli BL21(DE3)(ArEH) at 256 mM ECH and approximately 10-fold higher at 384 mM. Table 2 Kinetic parameters for hydrolysis of ECH by recombinant cells or free ArEHa. Free ArEHb
R. ruber THdAdN(ArEH)
E. coli BL21(DE3)(ArEH) 32.3 ± 3.8
R Km (mM)
35.5 ± 1.2
31.9 ± 4.7
S Km (mM)
0.57 ± 0.13
0.18 ± 0.04
0.19 ± 0.03
R (μmol/min/mg protein or mg dcw)c Vmax
66.8 ± 12
4.7 ± 0.4
8.6 ± 2.1
S Vmax (μmol/min/mg protein or mg dcw)c
13.6 ± 0.2
0.47 ± 0.03
1.93 ± 0.16
R R (L/min/mg protein or mg dcw) Vmax / Km
1.88
0.15
0.27
S S Vmax / Km (L/min/mg protein or mg dcw)
23.8
2.61
10.16
a. Kinetic parameters were measured at pH 8.0 and 30 °C. Experiments were performed in triplicate. b. ArEH was purified from E. coli BL21(DE3)(ArEH). c. The unit of Vmax is μmol/min/mg protein for free ArEH and μmol/min/mg dcw for intracellular ArEH. 53
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Fig. 4. Effect of various parameters on ArEH activities of recombinant cells and purified enzymes. (a) Temperature. (b) pH. (c) Substrate concentration. The initial activities of intracellular ArEH were measured at various concentration of racemic ECH. (d) Product concentration. The initial activities were measured in the presence of various concentration of 3-MCPD. Experiments were performed in triplicate.
Fig. 5. Residual activities of ArEH under different conditions. (a) Incubation at 55 °C for various periods. (b) Incubation at various pH values for 30 min. (c) Incubation in various concentrations of ECH for 30 min. (d) Incubation in various concentrations of 3-MCPD for 30 min. Experiments were performed in triplicate.
bioprocesses. More importantly, ArEH in R. ruber THdAdN showed enhanced thermostability, pH stability and tolerance against toxic substrate and product, thereby resulting in a better performance during the resolution of ECH at high concentrations. The half-life of ArEH in R. ruber THdAdN (ArEH) at 55 °C was 10-fold longer than that of E. coli BL21(DE3)
comparable to that of E. coli BL21(DE3)(ArEH). The result indicated the great potential of R. ruber THdAdN as an efficient host for overexpression of heterologous enzymes. Also, R. ruber could reach a higher cell density than E. coli during flask cultivation, which resulted in a higher activity. Meanwhile, compared to the widely used but expensive inducer IPTG, the low-cost urea inducer is more suitable for industrial 54
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Table 3 Effect of substrate concentration on the resolution of ECH catalyzed by recombinant cellsa. ECH con. (mM)
26 64 128 256 384 a
Biomass con. (g dcw/L)
Reaction time (min)
ee of (R)-ECH (%)
Yield of (R)-ECH (%)
E value
E. coli
R. ruber
E. coli
R. ruber
E. coli
R. ruber
E. coli
R. ruber
E. coli
R. ruber
0.5 1 2 5 10
0.75 1.5 3 7.5 15
35 40 45 61 120
30 38 33 18 20
> 98 > 98 > 98 > 98 7.4 ± 1.2
> 98 > 98 > 98 > 98 > 98
41.5 ± 1.2 41.0 ± 0.9 38.1 ± 1.1 37.5 ± 0.6 35.0 ± 1.5
38.5 ± 0.6 37.2 ± 0.4 35.5 ± 1.3 38.3 ± 0.5 30 ± 1.2
24.6 ± 1.5 24.0 ± 1.8 17.5 ± 1.3 17.6 ± 0.9 1.6 ± 0.5
20.7 ± 2.0 18.0 ± 2.1 19.7 ± 1.5 20.4 ± 1.4 12.9 ± 1.8
Experiments were performed in triplicate.
(ArEH), as shown in Fig. S4. In this work, a simple but effective approach was developed with R. ruber as the expression host to enhance enzyme stability, which avoided the obvious loss of activity and laborintensive screening encountered with immobilization and directed evolution. We speculated that the robustness was probably due to the special intracellular microenvironment in R. ruber created by multiple factors such as molecular chaperones [40] and cell envelope structure [41]. The R. ruber cells can be regarded as capsule-like carriers for in situ immobilization, protecting the intracellular enzymes against tough environments and permitting the transfer of small molecules with a low diffusion limit. Finally, R. ruber THdAdN(ArEH) whole cell was utilized in the enantioselective resolution of high concentration ECH for the production of (R)-ECH. The results were compared with some literatures, as shown in Table 4. To be noted, Jin et al. reported that by substrate feeding, 124 mM (R)-ECH with > 98% ee and 27.7% yield was obtained from 448 mM ECH hydrolyzed by cell-free ArEH [8]. In this work, with the same enzyme, utilization of R. ruber THdAdN as host strain improved the final (R)-ECH concentration by 46%, with a higher substrate concentration and higher yield. To improve the economic feasibility, the recovery of 3-MCPD should be also considered, which is possibly enantiomerically enriched. Moreover, the performance of EHs could be further improved by protein engineering. For example, through iterative saturation mutagenesis, Zou et al. obtained a mutant based on the ArEH utilized in this study with enhanced enantioselectivity, activity and stability, and 99.9% ee (R)-ECH could be obtained from 660 mM racemic ECH with a yield of 40.2% [16]. In summary, this study proposed to develop R. ruber as an efficient and robust platform for the overexpression of epoxide hydrolases to utilize as a high-performance whole-cell biocatalyst, which showed promise in the biotransformation of high-value chiral chemicals. It is expected that the combination of protein engineering and cell engineering will not only accelerate but also expand the industrial applications of whole-cell biocatalysts. Evolved enzymes with enhanced enantioselectivity, activity and stability can be obtained through directed evolution, then overexpressed in the engineered R. ruber to further improve their performance under operating conditions.
Table 4 Resolution of racemic ECH for the preparation of chiral ECH. Source of EHs
Catalytic form
CECH (mM)
ee (%) (enantiomer)
Yield (%)
Reference
A. niger R. glutinis
Cells Pichia pastoris cells Enzymes E. coli cells Enzymes R. ruber cells
60 50
100 (S) 100 (R)
20 26
[37] [34]
500 450 448 512
99.99 (R) > 99 (S) > 98 (R) 98.5 (R)
20.7 40.5 27.7 35.5
[33] [36] [8] This study
N. aromaticivorans A. mediolanus A. radiobacter A. radiobacter
Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements We thank professor Jun Ishikawa (Japan) for supplying plasmid pNV18.1. This work was supported by the National Natural Science Foundation (No. 21476126; No. 21776157) of China and the 973 National Key Basic Research Project (2013CB733600) of China.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.procbio.2018.12.023. Fig. 6. Enantioselective resolution of ECH at high concentrations. (a) Biocatalyst productivities of recombinant cells during the resolution of ECH at various concentrations. (b) Resolution of ECH with R. ruber THdAdN (ArEH) by substrate feeding. Initial concentration of ECH was 192 mM with a feeding rate of 10.67 mM/min for 30 min, resulting in a total concentration of 512 mM added into the system. The concentration of R. ruber THdAdN (ArEH) added was 7.5 g dcw/L.
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Overexpression of epoxide hydrolase in Rhodococcus ruber with high robustness for the synthesis of chiral epichlorohydrin
T
⁎
Youxiang Lianga,b, Song Jiaoa,b, Miaomiao Wanga,b, Huimin Yua,b,c, , Zhongyao Shena,b a
Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China Key Laboratory of Industrial Biocatalysis (Tsinghua University), the Ministry of Education, Beijing, 100084, China c Center for Synthetic and Systems Biology, Tsinghua University, Beijing, 100084, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Rhodococcus ruber Whole-cell biocatalysts Epoxide hydrolase Stability and organic-solvent tolerance Chiral epoxides
Epoxide hydrolases (EHs) are attractive enzymes for producing enantiopure epoxides and diols, but do not display enough stability when lysates or Escherichia coli whole cells are used as biocatalysts. In this work, an organic-solvent tolerant strain Rhodococcus ruber THdAdN was utilized to overexpress an epoxide hydrolase from Agrobacterium radiobacter (ArEH), using E. coli BL21(DE3) as a control. The proportion of ArEH in all soluble proteins of R. ruber THdAdN(ArEH) reached 30.3%, which was comparable to that of E. coli BL21(DE3)(ArEH). Due to a higher cell density in flask cultivation, the maximum ArEH activity of R. ruber THdAdN(ArEH) toward epichlorohydrin (ECH) reached 5.4 U/mL, approximately 5-fold higher than that of E. coli BL21(DE3)(ArEH). More importantly, compared with E. coli BL21(DE3)(ArEH), ArEH in R. ruber THdAdN(ArEH) showed a 10-fold enhanced thermostability, better tolerance against alkali pH, and reduced substrate and product inhibition, which significantly improved its performance in the resolution of high concentration ECH. By substrate feeding, 98.5% ee (R)-ECH was obtained with a 35.5% yield from 512 mM racemic ECH hydrolyzed by R. ruber THdAdN (ArEH). Compared with using free enzyme as biocatalysts, utilization of the R. ruber cells harboring ArEH improved the final (R)-ECH concentration by 46%.
1. Introduction Optically pure epoxides and vicinal diols are valuable chiral building blocks for the preparation of bioactive compounds such as pharmaceuticals, agrochemicals and other fine chemicals [1,2]. Traditional chemical production methods, including asymmetric epoxidation or dihydroxylation of olefin substrate and resolution of racemic epoxides with metal-based catalysts [3,4], lead to safety and environmental concerns. In the past two decades, epoxide hydrolases (EHs) that catalyze the hydrolysis of racemic epoxides to corresponding vicinal diols have attracted much attention. EHs are cofactor-independent, enantioselective and ubiquitous in nature, making them highly appealing in the preparation of enantiopure epoxides and vicinal diols [5,6]. Currently, Escherichia coli cells are the most common host for the overexpression of EH [1,7–11]. However, EHs do not display enough stability under operating conditions when lysates or E. coli whole cells are used as biocatalysts [8,12]. Enzyme inhibition caused by toxic
organic substrates and products hinders the application of EHs at high substrate concentrations [8]. To overcome instability and low solubility of epoxides in the aqueous phase, organic solvents are frequently introduced as an alternative reaction media or to form a two-liquid-phase system. The consequent enzyme inactivation caused by the phase interface and organic solvents is not negligible [13,14]. Therefore, it is of great importance to improve the stability of EHs. Strategies such as immobilization and directed evolution have been widely studied [10,15–19]. For example, with ethylenediamine functionalized epoxy resin as a carrier, the half-life of immobilized epoxide hydrolase from Agrobacterium radiobacter (ArEH) was 6.8-fold longer than that of the free enzyme [15]. Through iterative saturation mutagenesis (ISM), a mutant of ArEH (T247 K/I108 L/D131S) was obtained with an 8-fold longer half-life than the wide-type [16]. However, enzyme immobilization is often coupled with a great loss of activity, while directed evolution is labor-intensive and requires high-throughput screening methods. A number of microorganisms, including Pseudomonas, Bacillus and
Abbreviations: EHs, epoxide hydrolases; ArEH, epoxide hydrolase of Agrobacterium radiobacter; ECH, epichlorohydrin; 3-MCPD, 3-chloro-1,2-propanediol; ee, enantiomeric excess; IPTG, isopropyl-β-thiogalactopyranoside; g dcw/L, gram dry cell weight per liter ⁎ Corresponding author at: Yingshi Building 412, Tsinghua University, Beijing, 100084, China. E-mail address: [email protected] (H. Yu). https://doi.org/10.1016/j.procbio.2018.12.023 Received 3 October 2018; Received in revised form 19 December 2018; Accepted 21 December 2018 Available online 23 December 2018 1359-5113/ © 2018 Elsevier Ltd. All rights reserved.
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this mutant. Plasmid pET28a-ArEH (see Fig. S2) was constructed by inserting the mutated ArEH gene (amplified by primers BamH I-ArEH-sense and Hind III-ArEH-anti) into the plasmid pET28a between the BamH I and Hind III restriction endonuclease sites and transformed into E. coli BL21(DE3) competent cells to obtain E. coli BL21(DE3)(ArEH). ArEH was fused with a 6×His-tag, thrombin site and T7 tag, amplified by the Xba IArEH-sense and EcoR I-ArEH-anti primers from pET28a-ArEH, and inserted into the plasmid pNV18.1 [28] under an amidase promoter Pa2 (see Table S2) [29]. The resulting plasmid pNV18.1-Pa2-ArEH (see Fig. S3) was transformed into the competent cells of R. ruber THdAdN by electroporation [30]. Restriction enzymes, DNA polymerases, T4 DNA ligases and a sitedirected mutagenesis kit were purchased from Takara (Dalian, China) and Vazyme (Nanjing, China). DNA purification kits, gel extraction kits, BCA-protein quantification assay kits, and plasmid extraction kits were purchased from Biomega (Shanghai, China). Protein marker (MM1397500) was purchased from GenScript (Nanjing, China). ArEH gene synthesis was completed by Qinglan (Wuxi, China) and DNA sequencing was performed by GENEWIZ (Suzhou, China). DNA manipulations including PCR, agarose gel electrophoresis, restriction enzyme digestion, ligation and transformation were performed following standard procedures from the manufacturers’ protocols.
Rhodococcus, have shown excellent organic-solvent tolerance that helps improve their stability in reaction media containing organic solvents or toxic compounds [20]. Among them, Rhodococcus is a group of grampositive bacteria with a range of metabolic activities that enable them to degrade toxic organic compounds such as nitriles, halogenated and aromatic hydrocarbons [21–23]. Rhodococcus has been widely applied in bioconversion, bioremediation and biosynthesis. Production of acrylamide from acrylonitrile catalyzed by Rhodococcus harboring nitrile hydratase (NHase) is one of the most successful cases of industrial biotechnology [24,25]. Previously, NHase was cloned in E. coli but suffered from severe inactivation caused by high concentration acrylamide and high temperature [26]. In comparison, NHase in Rhodococcus showed higher tolerance against acrylamide and acrylonitrile, as well as better thermostability [25], due to the special features of Rhodococcus cells. In this work, an engineered strain Rhodococcus ruber THdAdN with high robustness was utilized for the overexpression of a mutant epoxide hydrolase ArEH. The most common host E. coli BL21(DE3) was used as a control. Characteristics of the two engineered strains were assessed, including in-cell EHs activity, kinetic parameters, and optimal temperature and pH. Moreover, their stability against temperature, pH, epoxides and diols, as well as their performance in the resolution of high concentration epichlorohydrin (ECH) were compared.
2.2. Cell cultivation and protein expression 2. Material and methods R. ruber THdAdN was cultivated in a 100 mL conical flask containing 20 mL of seed media (30 g/L glucose, 1 g/L yeast extract, 7 g/L tryptone, 0.5 g/L K2HPO4•3H2O, 0.5 g/L KH2PO4, 0.5 g/L MgSO4•7H2O, 1 g/L monosodium glutamate, pH 7.5) with 25 mg/L kanamycin, at 200 rpm and 28 °C [27]. The cell density was monitored spectrophotometrically at 460 nm to determine the growth curves. After 48 h, 5 mL of seed liquid in the exponential phase was transferred into a 500 mL shaking flask containing 50 mL of fermentation media (30 g/L glucose, 7–8 g/L yeast extract, 10 g/L urea, 2.28 g/L K2HPO4•3H2O, 0.5 g/L KH2PO4, 1 g/L MgSO4•7H2O, 1 g/L monosodium glutamate, pH 7.5), and incubated at 200 rpm and 28 °C to induce the expression of ArEH with urea as inducer for 36 h. Samples were withdrawn periodically to monitor the cell density and ArEH activity. E. coli BL21(DE3) was cultured in 10 mL Luria–Bertani (LB) media (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) containing 50 mg/L of kanamycin in a 50 mL shaking flask at 200 rpm and 37 °C for 12 h. Next, 0.5 mL of seed was transferred into 50 mL of LB media containing 50 mg/L kanamycin in a 300 mL shaking flask. The expression of ArEH was induced for 8 h at 200 rpm and 28 °C by adding 0.5 mM isopropylβ-thiogalactopyranoside (IPTG) when the OD600 reached 0.6˜0.8 [8]. Samples were withdrawn periodically to monitor the cell density and ArEH activity. The cells above were harvested by centrifugation at 10,000×g for 15 min at 4 °C, and stored at −20 °C.
2.1. Strains, plasmids and DNA manipulations The strains and plasmids used in this study are listed in Table 1, and the primers are listed in Table S1. R. ruber THdA was an amiE-negative mutant that was deposited in the China General Microbiological Culture Collection Center (CGMCC No. 2381). R. ruber THdAdN was a mutant with both amidase and NHase knocked out, serving as an efficient host for overexpression of heterologous genes [27]. E. coli DH5α was used for plasmid construction. E. coli BL21(DE3) was used as the control host for protein expression. The EH gene of Agrobacterium radiobacter AD1 (GenBank accession No. Y12804) was synthesized according to the codon preference of R. ruber and E. coli, as shown in Fig. S1. The GenBank accession numbers of the codon-optimized gene is MH480511. Four mutations (F108I, P205H, Y215H, E271 V) [11] were introduced via three rounds of sitedirected mutagenesis to obtain the mutant ArEH (GenBank accession No. MH480512). In this study, ArEH mentioned afterward indicated Table 1 Strains and plasmids used in this study. Strains/Plasmids Plasmids pET28a pMV-ArEH pET28a-ArEH pNV18.1 pNV18.1-Pa2-ArEH Strains E. coli DH5α E. coli BL21(DE3) E. coli BL21(DE3)(pET28a) E. coli BL21(DE3)(ArEH) R. ruber THdAdN R. ruber THdAdN(pNV18.1) R. ruber THdAdN(ArEH)
Relevant genotypes
Source
5.4 kb, KanR Plasmid carrying codon-optimized ArEH gene pET28a with ArEH gene E. coli-Rhodococcus shuttle vector, KanR pNV18.1 with ArEH gene under promoter Pa2
Novagen This study
Host for plasmid construction Host for protein expression E. coli BL21(DE3) harboring pET28a E. coli BL21(DE3) harboring pET28aArEH R. ruber TH with amidase and NHase deleted R. ruber THdAdN harboring pNV18.1 R. ruber THdAdN harboring pNV18.1Pa2-ArEH
2.3. Protein purification and SDS-PAGE analysis This study [28]
The cells (approximately 2 g) were resuspended with 30 mL of 0.2 M sodium phosphate buffer (pH = 8.0) and disrupted by a high-pressure homogenizer at 1500 bar in a bath of ice water. After centrifugation (10,000 × g) at 4 °C for 15 min, the supernatant was applied onto a preequilibrated Ni-NTA column (5 mL). Buffer A (20 mM Na2HPO4/ NaH2PO4, 30 mM imidazole, pH = 8.0) was then applied to remove the unbound proteins, followed by buffer B (20 mM Na2HPO4/NaH2PO4, 300 mM imidazole, pH = 8.0) to elute the bound protein. The enzyme solution was dialyzed at 4 °C for 12 h against 20 mM Na2HPO4/ NaH2PO4 (pH = 8.0) to remove imidazole and then stored at −20 °C. Cell lysate was centrifuged at 10,000×g for 15 min at 4 °C. The precipitate was suspended to equal volume. Both the supernatant and precipitate suspension were analyzed with 12% sodium dodecyl sulfate-
This study
Solarbio Salarbio This study This study [27] This study This study
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phosphate buffer with a pH ranging from 2 to 12 at 30 °C for 30 min and then centrifuged at 10,000 × g for 3 min to remove the supernatant. The cells were then resuspended with 0.2 M sodium phosphate buffer (pH = 8.0) for activity measurement. Two mol/L NaOH was used to adjust solutions with a pH higher than 9, and 50% HCl was used to adjust solutions with a pH lower than 5. The initial activity of ArEH at pH 8.0 was defined as 100%.
polyacrylamide gel electrophoresis (SDS-PAGE). For the supernatant, 0.03 mg protein was added per well in the gel. For precipitation suspension, equal volume to the supernatant was added. 2.4. Activity measurement of ArEH R. ruber or E. coli cells was suspended in 0.2 M sodium phosphate buffer (pH = 8.0) to the initial volume, and then diluted to suitable cell concentrations (R. ruber was diluted by 5-fold, and E. coli was not diluted). In a 1.5 mL centrifuge tube, 0.36 mL of diluted cells or enzymes was mixed with 0.04 mL of 2.5% (v/v) racemic ECH in 0.2 M sodium phosphate buffer (pH = 8.0). Due to high level of the spontaneous hydrolysis of ECH in aqueous phase, a control without cells or enzymes was set as a background. The reaction was conducted at 30 °C and 1000 rpm in a thermo shaker for 5 min and terminated by adding 0.8 mL of hexane containing 0.1% (v/v) 1-chlorohexane as an internal standard. Samples were mixed on a vortex shaker for 1 min and then centrifuged. The organic layer was analyzed by gas chromatography after being dehydrated with anhydrous sodium sulfate. One activity unit was defined as the amount of cells or enzymes that catalyzed 1 μmol ECH into diol per minute. ECH concentration was determined by a GC system (Thermo Fisher Scientific, America) equipped with an Astec Chiraldex G-TA column (0.25 mm ID, 30 m length, 0.12 μm film thickness) and a FID detector. The temperature of the oven, injector and detector were 60 °C, 180 °C and 180 °C, respectively, and the pressure of carrier gas (nitrogen) was 108 kPa with a split ratio of 1:50. The retention time of the 1-chlorohexane, (S)-ECH and (R)-ECH were 5.4, 6.0 and 6.7 min, respectively.
2.9. Tolerance against substrate and product Recombinant cells were incubated in 0.2 M sodium phosphate buffer (pH = 8.0) containing ECH ranging from 0 to 638 mM or 3chloro-1,2-propanediol (3-MCPD) from 0 mM to 450 mM at 30 °C for 30 min. The cells were centrifuged at 10,000 × g for 3 min and washed twice to remove the residual ECH or 3-MCPD. The remained activities were then measured at pH 8.0 and 30 °C. The initial activity of ArEH before treatment was defined as 100%. 2.10. Enantioselective resolution of racemic ECH The ECH hydrolysis was conducted in a 100 mL bottle sealed with a rubber septum containing 30 mL of cell suspension in 0.2 M sodium phosphate buffer (pH = 9.0) at 30 °C. The reaction was initiated by adding a certain concentration of ECH. The hydrolysis course was monitored by periodically taking samples to analyze the residual ECH, and the reaction was stopped when the enantiomeric excess (ee value) of (R)-ECH reached 98%. The ee value was calculated using the equation ee = (R − S )/(R + S ) , where R and S represent the concentration of (R)ECH and (S)-ECH, respectively. The yield of (R)-ECH was calculated by the equation c = R/(R 0 + S0) , where R0 and S0 represent the initial concentration of (R)-ECH and (S)-ECH. The E value was calculated by the equation E = ln[(1 − c ) × (1 − ee )]/ln[(1 − c ) × (1 + ee )], where c represented the yield of (R)-ECH [8]. To reduce the inactivation of recombinant cells caused by ECH, substrate feeding was performed with an initial ECH concentration of 192 mM and a feeding rate of 10.67 mM/min for 30 min until the total ECH added reached 512 mM. The reaction was terminated when the ee value of (R)-ECH reached 98%.
2.5. Determination of kinetic parameters Kinetic parameters of ArEH toward (S)-ECH and (R)-ECH were estimated by measuring the initial activity of the recombinant cells or free enzymes at 30 °C and pH 8.0 with a substrate concentration range of 0.1˜15 mM and 5˜150 mM, respectively. Km and Vmax were calculated using the double reciprocal Lineweaver-Burk plot method. 2.6. Optimum temperature and pH of recombinant cells and purified enzymes
3. Results
The temperature characteristics of recombinant cells or purified enzymes were measured at pH 8.0 with temperature ranging from 15 °C to 60 °C. The pH characteristics were measured at 30 °C in 0.2 M sodium phosphate buffer with a pH range of 6 to 12. Two mol/L NaOH was used to adjust pH higher than 9.
3.1. Cloning and overexpression of ArEH in R. ruber THdAdN and E. coli BL21(DE3) A novel idea was proposed to develop R. ruber with high robustness [25,31] as an excellent whole-cell biocatalyst for enantioselective resolution of racemic ECH to produce (R)-ECH with overexpressed epoxide hydrolases, as shown in Fig. 1. In presence of inducer urea, the natively overexpressed NHase in R. ruber would reduce the expression efficiency of target enzymes, so the NHase-negative mutant R. ruber THdAdN [27] was utilized as the host strain. Several epoxide hydrolases have been utilized for the enantioselective resolution of ECH, but most of them showed limited enantioselectivty or activity [11,32–37]. Among them, EH from Agrobacterium radiaobacter (ArEH) is a classical one that has been well-studied [8,9,11,13]. The wild-type of ArEH showed no enantioselectivty toward ECH, but through directed evolution, Van et al. obtained a mutant (F108I/P205 H/Y215 H/E271 V) with an E-value of 40 [11], which is better than most of the reported EHs. In this study, we chose this mutant of ArEH for the production of (R)-ECH. R. ruber THdAdN(ArEH) was constructed and cultured in flasks, in parallel with E. coli BL21(DE3)(ArEH) and the strains containing blank plasmids. The expressions of ArEH in R. ruber THdAdN and E. coli BL21(DE3) were confirmed by SDS-PAGE. After cell disruption, both supernatant and precipitate were analyzed. As shown in Fig. 2, ArEH
2.7. Effect of substrate and product concentration on the activities of recombinant cells and purified enzymes The effect of substrate concentration was studied by measuring the initial ArEH activities of recombinant cells and purified enzymes with an ECH concentration ranging from 0 mM to 512 mM. Product inhibition was studied by measuring the initial ArEH activity in the presence of 3-chloro-1,2-propanediol (3-MCPD) ranging from 45˜450 mM. 2.8. Thermostability and pH stability of ArEH in recombinant cells and purified enzymes The thermostability of ArEH in the recombinant cells and purified enzymes was assessed by incubating the cell suspension in 0.2 M sodium phosphate buffer (pH = 8.0) at 55 °C for the indicated periods and measuring the residual activities at 30 °C. The initial activity of ArEH at 30 °C was defined as 100%. The half-lives of recombinant cells were calculated by the equation t1/2 = 0.693/ki. ki is the first-order constant for enzyme inactivation. To measure pH stability, the cells were incubated in 0.2 M sodium 51
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Fig. 1. Enantioselective resolution of racemic ECH for the production of (R)-ECH with engineered R. ruber THdAdN(ArEH) cells.
3.3. Characterization of recombinant cells and purified enzymes
achieved efficient expression in both of the strains, and nearly no inclusion body was formed. Cell growth curves and ArEH activities toward ECH were measured as shown in Section 2.2, and summarized in Fig. 3. The maximum ArEH activity in E. coli BL21(DE3)(ArEH) was 1.1 U/mL at 10.5 h when the biomass reached 0.74 g dcw/L. For R. ruber THdAdN(ArEH), the cell density was 6.8 g dcw/L at 36 h, and correspondingly the maximum ArEH activity reached 5.4 U/mL, approximately 5-fold higher than that of E. coli BL21(DE3)(ArEH). To compare the expression efficiency, the proportion of ArEH in all soluble proteins was analyzed by Quantity One (Bio-Rad, USA). ArEH contributed 38.4% of the total soluble proteins in E. coli BL21(DE3) (ArEH), and 30.3% in R. ruber THdAdN(ArEH).
The optimum temperature and pH of ArEH contained or not contained in cells were measured and compared in Fig. 4a and 4b. Generally, the cell-free ArEH purified from R. ruber THdAdN and E. coli BL21(DE3), and the ArEH contained in E. coli BL21(DE3)(ArEH) cells showed no significant difference. Their optimum catalysis conditions ranged from 45 to 50 °C and pH 8-9. For the ArEH contained in R. ruber THdAdN(ArEH), however, the optimum catalysis temperature and pH were enhanced to 55 °C and pH 10, respectively. More interestingly, ArEH in R. ruber THdAdN(ArEH) still retained 75% of its activity at pH 12; under this condition, it was completely inactivated in E. coli BL21(DE3)(ArEH). In addition, the effect of substrate and product on the ArEH activity was assessed as shown in Fig. 4c and 4d. Both intracellular and free ArEH showed a maximum activity at an ECH concentration of 256 mM and a sharp decline at 384 mM owing to the inactivation of ArEH caused by toxic ECH. Surprisingly, product inhibition of ArEH was reduced significantly in R. ruber THdAdN(ArEH). R. ruber THdAdN(ArEH) showed 88% of its initial ArEH activity in presence of 360 mM 3-MCPD, while E. coli BL21(DE3)(ArEH) and free enzymes showed no more than 56%.
3.2. Kinetic parameters of ArEH Kinetic parameters of intracellular ArEH and free ArEH for both enantiomers were measured and compared in Table 2. The Vmax for (R)ECH was higher than that for (S)-ECH, but the Km for (S)-ECH was much lower than that for (R)-ECH. Both intracellular ArEH and free ArEH hydrolyzed (S)-ECH preferentially, with a higher Vmax/Km for (S)-ECH than that for (R)-ECH. The apparent Km values of both R. ruber THdAdN (ArEH) and E. coli BL21(DE3)(ArEH) for the same enantiomers of ECH were similar to that of free enzymes. This result indicated that the mass transfer resistance conferred by the cell envelopes is low probably because ECH is a relatively small molecule. We found that the Vmax/Km of R. ruber for (S)-ECH was much lower than that of E. coli. This result is probably caused by the difference of intracellular environment between R. ruber and E. coli, as well as the expression level of ArEH.
3.4. Stability of whole-cell biocatalysts Stability of biocatalysts is of great importance to industrial applications, which requires biocatalysts to resist against harsh environments such as high temperature, extreme pH, organic solvents, and high concentrations of substrate and product. Fig. 2. SDS-PAGE analyses of recombinant cells expressing ArEH. (a) Supernatant of the cell lysate. (b) Precipitate of the cell lysate. The E. coli BL21(DE3)(ArEH) cells were harvested after an 8 h induction with 0.5 mM IPTG in LB media. R. ruber THdAdN(ArEH) cells were harvested after a 36 h induction with 10 g/L of urea in fermentation media. The molecular mass of ArEH fused with a 6×His-Tag and T7-Tag was calculated to be 37.3 kDa.
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Fig. 3. Cell growth and ArEH activity curves of engineered strains. Experiments were performed in triplicate.
To reduce the enzyme inactivation caused by high concentrations of ECH, substrate feeding was conducted. With R. ruber THdAdN(ArEH) as biocatalysts, 182 mM (R)-ECH was obtained with 98.5% ee and 35.5% yield from 512 mM ECH, as shown in Fig. 6b. However, when the total ECH concentration was further increased to 576 mM, cells suffered from severe inhibition after 30 min and only 48.5% ee of (R)-ECH was obtained, as shown in Fig. S5.
Thermostability and pH stability of intracellular and/or free ArEH were assessed and compared as shown in Fig. 5a and b. After incubation at 55 °C for 30 min, the residual activity of ArEH in R. ruber THdAdN (ArEH) cells was approximately 90%, but free ArEH and ArEH in E. coli BL21(DE3)(ArEH) was almost completely inactivated. The half-life of ArEH in R. ruber THdAdN(ArEH) was 50.3 min, approximately 10-fold longer than that in E. coli BL21(DE3)(ArEH), as shown in Fig S4. Moreover, R. ruber THdAdN(ArEH) could tolerate a much wider pH range than E. coli BL21(DE3)(ArEH), as shown in Fig. 5b. Tolerance of ArEH in recombinant cells against toxic substrate and product was measured and summarized in Fig. 5c and d. Incubation in 256 mM ECH for 30 min didn’t decrease the activity of ArEH in R. ruber THdAdN. However, under the same conditions, only 25% of the initial activity remained in E. coli BL21(DE3). In addition, incubation in 3MCPD solution showed no significant impact on the activity of ArEH in R. ruber THdAdN(ArEH).
4. Discussion Microbial enzymes and cells are important tools in asymmetric biocatalysis for the preparation of value-added chemicals including pharmaceuticals, agrochemicals and flavors [38]. Compared to free or immobilized enzyme, whole-cell biocatalysts are often preferred as they are simple and convenient to operate. Moreover, microbial cells can provide a relatively mild environment for enzymes, preventing denaturation of protein under harsh reaction conditions [39]. Among various host strains, E. coli is known as a standard “workhorse”. However, the insufficient stability of biocatalysts under operating conditions is still the main limitation for their industrial application [12]. For example, epoxide hydrolases, which are important enzymes widely used in the preparation of chiral epoxides and diols, often suffer from limited thermostability, substrate inactivation and product inhibition when lysates or E. coli whole cells are used as biocatalysts. The primary development methods such as directed evolution and immobilization are widely performed to improve the stability of enzymes [15,16]. In addition to E. coli, many researchers are interested in solvent-tolerant microorganisms including Pseudomonas, Bacillus and Rhodococcus, for their adaptation to harsh conditions [20]. In this study, the engineered strain R. ruber THdAdN with high tolerance to solvents and environmental stresses was utilized for the first time to overexpress heterologous EHs for the enhancement of incell enzyme stability. With a urea-induced promoter Pa2, efficient expression of ArEH was achieved in R. ruber THdAdN. The proportion of ArEH in all soluble proteins of R. ruber THdAdN(ArEH) reached 30.3%,
3.5. Enantioselective resolution of ECH at high concentrations For practical applications of epoxide hydrolases in the production of chiral epoxides or diols, it is unavoidable to increase the concentration of ECH, thereby reducing production costs. However, when substrate concentration increases, ee value and yield usually decrease because of the inactivation effect caused by toxic epoxides or diols. To assess the performance of the recombinant strains, a gradient of racemic ECH was hydrolyzed by recombinant cells until the ee value of (R)-ECH reached 98% or no longer improved. The statistics are shown in Table 3. The E value of E. coli BL21(DE3)(ArEH) sharply declined to 1.6 when the initial ECH concentration was over 384 mM; for R. ruber THdAdN (ArEH), however, the E value still remained as high as 13.2 under the same conditions. The productivities of different cell biocatalysts at various ECH concentrations were further calculated and summarized in Fig. 6a. For R. ruber THdAdN(ArEH), it was more than 2-fold higher than that of E. coli BL21(DE3)(ArEH) at 256 mM ECH and approximately 10-fold higher at 384 mM. Table 2 Kinetic parameters for hydrolysis of ECH by recombinant cells or free ArEHa. Free ArEHb
R. ruber THdAdN(ArEH)
E. coli BL21(DE3)(ArEH) 32.3 ± 3.8
R Km (mM)
35.5 ± 1.2
31.9 ± 4.7
S Km (mM)
0.57 ± 0.13
0.18 ± 0.04
0.19 ± 0.03
R (μmol/min/mg protein or mg dcw)c Vmax
66.8 ± 12
4.7 ± 0.4
8.6 ± 2.1
S Vmax (μmol/min/mg protein or mg dcw)c
13.6 ± 0.2
0.47 ± 0.03
1.93 ± 0.16
R R (L/min/mg protein or mg dcw) Vmax / Km
1.88
0.15
0.27
S S Vmax / Km (L/min/mg protein or mg dcw)
23.8
2.61
10.16
a. Kinetic parameters were measured at pH 8.0 and 30 °C. Experiments were performed in triplicate. b. ArEH was purified from E. coli BL21(DE3)(ArEH). c. The unit of Vmax is μmol/min/mg protein for free ArEH and μmol/min/mg dcw for intracellular ArEH. 53
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Fig. 4. Effect of various parameters on ArEH activities of recombinant cells and purified enzymes. (a) Temperature. (b) pH. (c) Substrate concentration. The initial activities of intracellular ArEH were measured at various concentration of racemic ECH. (d) Product concentration. The initial activities were measured in the presence of various concentration of 3-MCPD. Experiments were performed in triplicate.
Fig. 5. Residual activities of ArEH under different conditions. (a) Incubation at 55 °C for various periods. (b) Incubation at various pH values for 30 min. (c) Incubation in various concentrations of ECH for 30 min. (d) Incubation in various concentrations of 3-MCPD for 30 min. Experiments were performed in triplicate.
bioprocesses. More importantly, ArEH in R. ruber THdAdN showed enhanced thermostability, pH stability and tolerance against toxic substrate and product, thereby resulting in a better performance during the resolution of ECH at high concentrations. The half-life of ArEH in R. ruber THdAdN (ArEH) at 55 °C was 10-fold longer than that of E. coli BL21(DE3)
comparable to that of E. coli BL21(DE3)(ArEH). The result indicated the great potential of R. ruber THdAdN as an efficient host for overexpression of heterologous enzymes. Also, R. ruber could reach a higher cell density than E. coli during flask cultivation, which resulted in a higher activity. Meanwhile, compared to the widely used but expensive inducer IPTG, the low-cost urea inducer is more suitable for industrial 54
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Table 3 Effect of substrate concentration on the resolution of ECH catalyzed by recombinant cellsa. ECH con. (mM)
26 64 128 256 384 a
Biomass con. (g dcw/L)
Reaction time (min)
ee of (R)-ECH (%)
Yield of (R)-ECH (%)
E value
E. coli
R. ruber
E. coli
R. ruber
E. coli
R. ruber
E. coli
R. ruber
E. coli
R. ruber
0.5 1 2 5 10
0.75 1.5 3 7.5 15
35 40 45 61 120
30 38 33 18 20
> 98 > 98 > 98 > 98 7.4 ± 1.2
> 98 > 98 > 98 > 98 > 98
41.5 ± 1.2 41.0 ± 0.9 38.1 ± 1.1 37.5 ± 0.6 35.0 ± 1.5
38.5 ± 0.6 37.2 ± 0.4 35.5 ± 1.3 38.3 ± 0.5 30 ± 1.2
24.6 ± 1.5 24.0 ± 1.8 17.5 ± 1.3 17.6 ± 0.9 1.6 ± 0.5
20.7 ± 2.0 18.0 ± 2.1 19.7 ± 1.5 20.4 ± 1.4 12.9 ± 1.8
Experiments were performed in triplicate.
(ArEH), as shown in Fig. S4. In this work, a simple but effective approach was developed with R. ruber as the expression host to enhance enzyme stability, which avoided the obvious loss of activity and laborintensive screening encountered with immobilization and directed evolution. We speculated that the robustness was probably due to the special intracellular microenvironment in R. ruber created by multiple factors such as molecular chaperones [40] and cell envelope structure [41]. The R. ruber cells can be regarded as capsule-like carriers for in situ immobilization, protecting the intracellular enzymes against tough environments and permitting the transfer of small molecules with a low diffusion limit. Finally, R. ruber THdAdN(ArEH) whole cell was utilized in the enantioselective resolution of high concentration ECH for the production of (R)-ECH. The results were compared with some literatures, as shown in Table 4. To be noted, Jin et al. reported that by substrate feeding, 124 mM (R)-ECH with > 98% ee and 27.7% yield was obtained from 448 mM ECH hydrolyzed by cell-free ArEH [8]. In this work, with the same enzyme, utilization of R. ruber THdAdN as host strain improved the final (R)-ECH concentration by 46%, with a higher substrate concentration and higher yield. To improve the economic feasibility, the recovery of 3-MCPD should be also considered, which is possibly enantiomerically enriched. Moreover, the performance of EHs could be further improved by protein engineering. For example, through iterative saturation mutagenesis, Zou et al. obtained a mutant based on the ArEH utilized in this study with enhanced enantioselectivity, activity and stability, and 99.9% ee (R)-ECH could be obtained from 660 mM racemic ECH with a yield of 40.2% [16]. In summary, this study proposed to develop R. ruber as an efficient and robust platform for the overexpression of epoxide hydrolases to utilize as a high-performance whole-cell biocatalyst, which showed promise in the biotransformation of high-value chiral chemicals. It is expected that the combination of protein engineering and cell engineering will not only accelerate but also expand the industrial applications of whole-cell biocatalysts. Evolved enzymes with enhanced enantioselectivity, activity and stability can be obtained through directed evolution, then overexpressed in the engineered R. ruber to further improve their performance under operating conditions.
Table 4 Resolution of racemic ECH for the preparation of chiral ECH. Source of EHs
Catalytic form
CECH (mM)
ee (%) (enantiomer)
Yield (%)
Reference
A. niger R. glutinis
Cells Pichia pastoris cells Enzymes E. coli cells Enzymes R. ruber cells
60 50
100 (S) 100 (R)
20 26
[37] [34]
500 450 448 512
99.99 (R) > 99 (S) > 98 (R) 98.5 (R)
20.7 40.5 27.7 35.5
[33] [36] [8] This study
N. aromaticivorans A. mediolanus A. radiobacter A. radiobacter
Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements We thank professor Jun Ishikawa (Japan) for supplying plasmid pNV18.1. This work was supported by the National Natural Science Foundation (No. 21476126; No. 21776157) of China and the 973 National Key Basic Research Project (2013CB733600) of China.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.procbio.2018.12.023. Fig. 6. Enantioselective resolution of ECH at high concentrations. (a) Biocatalyst productivities of recombinant cells during the resolution of ECH at various concentrations. (b) Resolution of ECH with R. ruber THdAdN (ArEH) by substrate feeding. Initial concentration of ECH was 192 mM with a feeding rate of 10.67 mM/min for 30 min, resulting in a total concentration of 512 mM added into the system. The concentration of R. ruber THdAdN (ArEH) added was 7.5 g dcw/L.
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