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Multi-Parameter Optimization in Directed Evolution: Engineering Thermostability, Enantioselectivity and Activity of an Epoxide Hydrolase Guangyue Li, Hui Zhang, Zhoutong Sun, Xinqi Liu, and Manfred T. Reetz ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b01113 • Publication Date (Web): 02 May 2016 Downloaded from http://pubs.acs.org on May 7, 2016
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Multi-Parameter Optimization in Directed Evolution: Engineering Thermostability, Enantioselectivity and Activity of an Epoxide Hydrolase Guangyue Li1,2,§, Hui Zhang3,§, Zhoutong Sun1,2, Xinqi Liu3,* and Manfred T. Reetz1,2,* 1
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany 2
Fachbereich Chemie der Philipps-Universität, Hans-Meerwein-Strasse, 35032 Marburg, Germany
3
State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, 300071 Tianjin, China
ABSTRACT: The challenge of optimizing several parameters in directed evolution of enzymes remains a central issue. In this study we address thermostability, enantioselectivity and activity of limonene epoxide hydrolase (LEH) as the catalyst in the hydrolytic desymmetrization of cyclohexene oxide with formation of (R,R)- and (S,S)-cyclohexane-1,2-diol. Wildtype LEH shows a thermostability of T5030= 41ο C and an enanioselectivity of 2% ee (S,S). Two approaches are described herein. In one strategy, the mutations generated previously by Janssen, Baker and coworkers for notably increased thermostability are combined with mutations evolved earlier for enhanced enantioselectivity. Although highly enantioselective (R,R)- and (S,S)-variants (92-93% ee) with increases in T5030 by 10-11ο C were obtained, relative to Wildtype LEH the tradeoff in activity was significant. The second strategy based on the simultaneous optimization of both parameters using iterative saturation mutagenesis (ISM) with minimized tradeoff in activity proved to be superior. Several notably improved variants were observed, a reasonable “compromise” being (R,R)- and (S,S)-selective LEH variants (80-94% ee) showing enhanced thermostability by 5-10ο C and still reasonable levels of activity. Analysis of the X-ray structure of the (S,S)-variant (94% ee) with and without diol product sheds light on the origin of altered stereoselectivity. KEYWORDS: directed evolution, enantioselectivity, thermostability, activity, saturation mutagenesis
3,4
especially in industrial processes. It has been 5 pointed out that stability promotes evolvability. Many different strategies for thermostabilization have been reported, the common feature being the predominance of surface mutations.3,4 In most cases the tradeoff in terms of catalytic properties such as activity remained small or negligible, which does not surprise since the mutations are remote from the active site. However, these additional properties are generally not improved in the process of thermostabilization. Various approaches to the optimization of two parameters in specific cases have been reported, but general guidelines remain to be issued.1-4,6 In most cases substantial experimental efforts had to be invested. Two general options are possible, sequential or simultaneous optimization of two or more parameters.
INTRODUCTION Directed evolution is a versatile tool for engineering the catalytic profiles of enzymes as catalysts in organic chemistry and biotechnology,1 the control of stereo- and regioselectivity being of particular interest.2 It constitutes a prolific source of selective biocatalysts which complement chiral man-made transition metal catalysts and 2 organocatalysts. The degree of success depends upon the right choice of the mutagenesis method and the amount of experimental work that the researcher is willing to invest. Since the screening step is the bottleneck, much effort has been invested in order to maximize the efficacy of directed evolution.1,2 Directed evolution has also been applied in order to enhance protein thermostability, 1
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Here we describe the directed evolution of thermostability and enantioselectivity of an enzyme, while attempting to maintain activity as much as possible. Limonene epoxide hydrolase from Rhodococcus erythropolis (LEH)7 was chosen as the catalyst in the hydrolytic desymmetrization of cyclohexene oxide (1) with formation of (R,R)-2 and (S,S)-2 (Scheme 1). The goal was to evolve robust and active (R,R)- and (S,S)-selective variants on an optional basis. O 1
OH
OH
LEH H2O
OH
OH (R, R)-2
source. In a recent study we evolved (R,R)- and (S,S)-selective LEH variants for the model reaction 9 (Scheme 1). Structure-guided saturation mutagenesis based on three amino acids (in addition to WT) as building blocks was employed at large CAST 2 sites lining the binding pocket in a process dubbed triple code saturation mutagenesis (TCSM).9 The initial libraries harbored both (R,R)- and (S,S)-selective variants, 2 making iterative saturation mutagenesis (ISM) 9 superfluous. The variants were shown to be distinctly better than those generated in earlier directed evolution study utilizing other saturation 10 mutagenesis strategies. Two of the best variants were identified as: SZ529 (4 mutations; (R,R)-selective; 97% ee): M32V/M78V/I80V/L114F and SZ348 (3 mutations; (S,S)-selective; 99% ee): I80Y/L114V/I116V Combining the mutations of the variants evolved for thermostability and enantioselectivity provided four variants: RR-8 (LEH-P + SZ529), RR-11 (LEH-F1b + SZ529), SS-8 (LEH-P + SZ348) and SS-11 (LEH-F1b + SZ348). The catalytic profiles of the four new variants in terms of enantioselectivity and thermostability are listed in Table 1. Thermostability was assessed by measuring T5030 values, the temperature at which 50% of enzyme activity is lost following a heat 3 treatment for 30 minutes. The results of kinetic studies are summarized in Table 2. It can be seen that tradeoffs of some sort occur in all of the four new variants. If only thermostability and enantioselectivity are considered, the (R,R)-selective variant RR-11 and the (S,S)-selective variant SS-8 appear to be acceptable compromises (Table 1, entries 4 and 8, respectively). However, inspection of Table 2 shows that catalytic efficiency relative to WT LEH as measured by kcat/Km is diminished considerably (Table 2, entries 4 and 8, respectively). Evolving three parameters simultaneously. In view of the limited success of the above approach, a different approach was needed. In the interest of efficacy in directed evolution, we intended to keep the screening effort at a minimum. As in the previous LEH 9 study, we employed Reymond’s adrenaline 11 on-plate assay. It is a convenient on-plate pre-test which rapidly identifies active mutants on the basis of a color change by visual inspection or 11 semi-quantitatively by a UV/Vis plate reader.
(S, S)-2
Scheme 1. Hydrolytic desymmetrization catalyzed by the limonene epoxide hydrolase LEH. 8
In previous studies, thermostability and 9 enantioselectivity were evolved separately. It was therefore logical to combine the respective point 8,9 mutations of the best variants, which forms the first part of the present study. In the second part, we present a different approach in which these two parameters in addition to activity are optimized simultaneously. RESULT AND DISCUSSION Combining mutations previously evolved for enhanced thermostability and enantioselectivity. Janssen, Baker and coworkers have shown that the thermostability of LEH can be notably increased by applying a technique dubbed FRESCO (Framework for Rapid Enzyme Stabilization by Computational libraries).8 It is a promising computationally guided approach to protein thermostabilization in which Rosettaddg, FoldX and DD (Disulfide Discovery) software packages are employed. In the case of LEH, several multi-site mutants resulted, showing an impressive increase in apparent melting ο ο temperature relative to WT LEH from 50 to 85 C and a more than 250-fold longer half-life. We chose two of the best variants for our present study: LEH-P (8 mutations): S15P/A19K/E45K/T76K/T85V/N92K/Y96F/E124D LEH-F1b (11 mutations): I5C/S15P/A19K/T76K/E84C/T85V/G89C/S91C/N92 K/Y96F/E124D Mutations for enhanced and inverted enantioselectivity were taken from a different
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Table 1. Catalytic profiles of the mutants formed by combining mutations of variants previously evolved 8 9 a) for thermostabilization and enantioselectivity, respectively. Data refers to the model reaction 1 → (R,R)-2 + (S,S)-2 (Scheme 1). The bold faced enzymes are the designations of the new variants formed by combining mutations. 30
Entry
Enzyme
T50
1 2 3
WT LEH SZ529 RR-8
41 C ο 40 C ο 51 C
4
RR-11
5
Mutations
2 97 93
Preferred enantiomer (S,S) (R,R) (R,R)
58ο C
89
(R,R)
I5C/S15P/A19K/M32V/T76K/M78V/I8 0V/E84C/T85V/G89C/S91C/N92K/Y9 6F/L114F/E124D
LEH-P
54ο C
3
(S,S)
S15P/A19K/E45K/T76K/T85V/N92K/Y 96F/E124D
6
LEH-F1b
62ο C
3
(S,S)
I5C/S15P/A19K/T76K/E84C/T85V/G89 C/S91C/N92K/Y96F/E124D
7
SZ348
38ο C
99
(S,S)
I80Y/ L114V/ I116V
8
SS-8
52ο C
92
(S,S)
ο
ee (%)
M32V/M78V/I80V/L114F S15P/A19K/M32V/E45K/T76K/M78V/I 80V/T85V/N92K/Y96F/L114F/E124D
S15P/A19K/E45K/T76K/T85V/N92K/Y 96F/E124D/ I80Y/L114V/I116V (S,S) I5C/S15P/A19K/T76K/I80Y/E84C/T85 9 SS-11 63ο C 87 V/G89C/S91C/N92K/Y96F/L114V/I116 V/E124D a) 30 Explanatory notes: ee = enantiomeric excess; T50 : the temperature at which 50% of enzyme activity is lost following a heat treatment for 30 minutes. The enzyme was incubated at different temperatures (30–70°C) for 30 min followed by measuring the residual activity by monitoring the conversion of substrate by GC using 1- heptanol as the internal standard. Table 2. Results of kinetic experimentsa) using the model reaction (Scheme 1). Entry
Enzyme
Km (mM)
-1
kcat (S )
kcat/Km -1 -1 (S M ) 1 WT LEH 6.70 ±0.45 0.82 ±0.017 122.39 2 SZ529 10.12 ±0.81 0.18 ±0.0051 17.78 3 RR-8 16.26 ±1.28 0.41 ±0.013 25.21 4 RR-11 17.70 ±1.66 0.51 ±0.02 28.81 5 LEH-P 7.74 ±0.42 1.03 ±0.018 133.07 6 LEH-F1b 8.79 ±0.65 1.31 ±0.032 149.03 7 SZ348 19.11 ±1.93 0.11 ±0.0048 5.75 8 SS-8 15.30 ±1.63 0.13 ±0.0057 8.50 9 SS-11 19.32 ±1.18 0.19 ±0.0049 9.84 a) The kinetic parameters were measured by monitoring the conversion of substrate by GC using 1-heptanol as the internal standard. About 30-40 96-format plates can be assayed per day, the active hits then being analyzed for enantioselectivity by automated GC (Supplementary Table S8). Previous experience9,10 has shown that about 10-30% of the LEH
transformants are active, depending upon the 9 particular randomization site. In the present project, we decided to limit the screening effort to about 8,000 transformants. 3
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We planned to apply saturation mutagenesis at two types of LEH residues (Fig. 1): Those that can be expected to be sensitive to changes in thermostability on the basis of the previous study [S15, A19, E45, T76, T85, N92, Y96, E124],8 and those that can be expected to be sensitive to changes in enantioselectivity and/or activity on the basis of the other previous study [M32, L74, M78, I80, L114, I116, F139, L147].9 For clarity, these are mapped in Figure 1 which is based on the 12 crystal structure of WT LEH. The 16 positions were grouped into four potential randomization sites, A, B, C and D, each containing representatives from each of the two types of residues (Table 3). The plan was to apply ISM in the expectation that all three parameters could be positively influenced, thermostability, enantioselectivity and activity. Randomization of a 4-residue site using NNK codon degeneracy encoding all 20 canonical amino acids would entail for 95% library coverage excessive screening 6 2,13 (3 x 10 transformants). Therefore, triple code 9 saturation mutagenesis (TCSM) based on reduced amino acid alphabets was considered, which would require the screening of about 770 transformants for 95% library coverage. When applying saturation mutagenesis at a randomization site, two different triple codes were chosen in one and the same experiment: One to control designed mutagenesis at the “thermostability” residues, and the other at the “enantioselectivity” residues. For this purpose triple codes Lys-Pro-Asp (K-P-D) and Val-Phe-Tyr (V-F-Y) were chosen, respectively. These choices were made on the basis of structural information and previous studies in which thermostability and stereoselectivity were evolved separately.8,9
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enantioselectivity and/or activity. CAST is a convenient acronym for randomization at sites lining the binding pocket (Combinatorial Active-site Saturation Test), thereby distinguishing it from saturation mutagenesis at other sites for different purposes. Table 3. Grouping of the 16 chosen LEH residues into four randomization sites A, B, C and D and the respective triple codes used in saturation mutagenesis. The green numbers denote “thermostability” positions, the red ones “enantioselectivity” positions. Site A: S15, A19, L74, M78 B: T76, T85, L114, I116 C: N92, Y96, F139, L147 D: E45, E124, M32, I80
Triple code K-P-D / V-F-Y K-P-D / V-F-Y K-P-D / V-F-Y K-P-D / V-F-Y
Following saturation mutagenesis, the supernatants of the lysates were placed in the wells of 96-format micro-titer plates, which were ο subjected to a heat treat in an oven at 70 C for 20 minutes. Under these conditions WT LEH loses about 90% of its activity. Thus, this procedure was designed so that less stable LEH variants would be eliminated, although possibly having high enantioselectivity. A three-phase screening procedure was applied: On-plate pre-test for activity,10 automated GC analysis for enantioselectivity followed by automated GC analysis for conversion. The latter requires a longer elution time. The best variants were characterized by sequence determination and kinetics. Whereas saturation mutagenesis at sites C and D did not lead to notably improved variants, the initial libraries at sites A and B harbored a number of hits. Thereafter, ISM was applied using the best variants as templates. All theoretically possible pathways were explored, the process terminating whenever a given library failed to harbor improved variants. In these cases local minima on the fitness pathway landscape 14 were encountered. Although it is possible to escape from such “dead ends” by utilizing a non-improved or even inferior mutant as the template in the subsequent ISM step,14 this procedure was not attempted in the present case. Scheme 2 summarizes all results. During the entire directed evolution process, 8448 transformants were screened in the first phase, to be followed by 676 and 106 transformants in the second and third phase, respectively.
Figure 1. LEH residues chosen for saturation mutagenesis marked in the crystal structure of WT LEH.12 The green-colored residues indicate positions at which potential mutations may enhance thermostability, while the red-colored residues mark CAST positions lining the binding pocket where potential mutations may influence 4
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Tables 4 and 5 display data concerning thermostability and enantioselectivity of the best variants as well as the results of kinetic experiments, respectively. The assignment of “best” hit was made on the basis of improved enantioselectivity as the main requirement, thermostabilization being the second parameter that was considered. In all of the variants some sort of tradeoff occurs, which is not unexpected. The best “compromises” are variants H-2-H5 showing a ο 30 T50 value of 51 C and an enantioselectivity of 30 ο 94% ee (S,S), and J-7-A12 (T50 = 46 C; 80% ee (R,R)). This is not ideal, but significantly better than previous variants.8,9 Variant H-2-H5 shows a 13ο C improvement in the T5030 value and an 8-fold
increase in catalytic efficiency relative to the 9 previous (S,S)-selective variant SZ348. When comparing variant J-7-A12 with the previous 30 (R,R)-selective variant SZ529, an increase in T50 ο by about 6 C and a 5-fold enhanced catalytic efficiency are observed. In order to get additional insight concerning the improved T5030, the melting points of WT LEH (50.3 ℃ ) and LEH mutant ο H-2-H5 (92.4 C) were measured by differential scanning calorimetry (DSC) (Fig. 2). Variant H-2-H5 shows a significant increase in Tm (~42.1℃ ) compared to WT. These results are consistent 30 with the improvement of the T50 value, and indicates that the mutational changes do not primarily induce faster refolding.
Scheme 2. ISM exploration of LEH as catalyst in the model reaction (Scheme 1) focusing on enantioselectivity. A, B, C and D denote 4-residue randomization sites as defined in Table 3. Table 4. Profiles of LEH variants as catalysts in the model reaction (Scheme 1). 30
Entry
Enzyme
T50
1 2 3 4
WT LEH B-1-F12 F-6-A9 G-7-H2
41 C 44ο C ο 45 C ο 51 C
ο
ee (%) 2 69 80 92
Preferred enantiomer (S,S) (S,S) (S,S) (S,S)
a)
Mutations
T76K/L114V/I116V T76K/L114V/I116V/N92K/F139V/L147F T76K/L114V/I116V/N92K/F139V/L147F/S 15D/A19K/L74F/M78F ο 5 H-2-H5 51 C 94 (S,S) T76K/L114V/I116V/N92K/F139V/L147F/S 15D/A19K /L74F/M78F/ E45D 6 A-5-C1 47ο C 34 (R,R) S15P/M78F 39 (R,R) S15P/M78F/N92K/F139V 7 E-5-A8 48ο C 8 I-4-H10 44ο C 45 (R,R) S15P/M78F/N92K/F139V/T76K/T85K 9 J-7-A12 46ο C 80 S15P/M78F/N92K/F139V/T76K/T85K/E (R,R) 45D/I80V/E124D 30 Explanatory notes: ee = enantiomeric excess; T50 : the temperature at which 50% of enzyme activity is lost following a heat treatment for 30 minutes. The enzyme was incubated at different temperatures (30–70°C) for 30 min followed by measuring the residual activity by monitoring the conversion of substrate by GC using 1- heptanol as the internal standard. 5
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a)
Table 5. Kinetic characterization of best LEH variants as catalysts in the model reaction (Scheme 1).
Entry Enzyme Km (mM) kcat (S-1) kcat/Km (S-1M-1) 1 WT LEH 6.70 ±0.45 0.82 ±0.017 122.39 2 B-1-F12 14.41 ±1.40 1.55 ±0.06 107.56 3 F-6-A9 15.44 ±1.01 0.76 ±0.02 49.22 4 G-7-H2 19.41 ±1.18 0.82 ±0.02 42.25 5 H-2-H5 16.10 ±1.08 0.84 ±0.02 52.17 6 A-5-C1 16.97 ±0.81 1.25 ±0.02 73.66 7 E-5-A8 16.06 ±0.50 0.86 ±0.01 53.54 8 I-4-H10 8.23 ±0.43 0.59 ±0.01 71.69 9 J-7-A12 6.39 ±0.36 0.64 ±0.01 100.15 b) 10 13.09±1.76 0.93±0.05 71.05 H-2-H5 (37ºC) b) 11 6.96±0.39 0.84±0.01 120.68 J-7-A12 (37ºC) a) The kinetic parameters in entries 1-9 were measured by monitoring the conversion of substrate at 30°C by GC using 1-heptanol as the internal standard. b) The kinetic parameters were measured at 37 ºC.
enzyme holds two water molecules by residues R99, D101 and D132, which are located on one side of the binding pocket. In contrast, the two water molecules are absent in the enzyme/product complex, and the binding pocket is occupied by the product diol (S,S)-2. In order to shed light on the reaction process, substrate cyclohexene oxide (1) was docked into the position of (S,S)-2, the structure then being refined for three cycles by PHENIX in order to minimize the steric clashes. It was found that the substrate fits well into the product position. Moreover, the orientation of oxygen atom is consistent with the oxygen in (S,S)-2 diol. With this composed structure, we proceeded to analyze the possible reaction process catalyzed by the H-2-H5 mutant. When comparing the structure of the WT LEH/1-complex obtained by previous docking experiments,10a the substrate is now located in a new pose because the phenyl ring of residue F134 has “turned away” (Fig. 3C and 3E). Additionally, the sidechain of residue R99 occupies a new position, thereby also providing more space for two water molecules to be bound in this region of the binding pocket (Fig. 3C and 3F). With the relocation of the sidechains of the two residues, these new water molecules are tightly coordinated by R99, D101, D132 and positioned close to the substrate’s prochiral C1-atom so that the (S,S)-diol is formed preferentially. In this model, it makes no difference which one of the two water molecules participates in the SN2 reaction, since both of them are closer to C1 leading to (S,S)-2. It is also noteworthy that the O-atom of the substrate still forms the usual hydrogen bond with protonated aspartate D101 which is necessary for activation. We propose that the combination of hydrogen
We also performed kinetic experiments and ee-determinations using variants H-2-H5 and ο J-7-A12 at a higher temperature (37 C). The result was a 37% and 20% increase in catalytic efficiency relative to the measurements at 30ο C, respectively (Table 5), while maintaining high stereoselectivity; H-2-H5: 92% ee (S,S), and J-7-A12 (79% ee (R,R) (Table S3). Relative to the good performance of the previously reported epoxide hydrolases from 15 Sphingomonas sp. HXN-200 and Rhodotorula 16 glutinis, variants H-2-H5 and J-7-A12 show comparable or higher enantioselectivities.
Figure 2. DSC curves of WT LEH (blue) and LEH mutant H-2-H5 (red). In order to gain some insight regarding the source of enantioselectivity improvement, the crystal structures of the (S,S)-selective variant H-2-H5 with and without complexed product (S,S)-2 were solved at 1.89 Å and 2.30 Å resolution, respectively (Fig. 3A and 3B). The two structures are very similar and can be superimposed well, showing negligible conformational differences at most residues. At the active site, the substrate-free 6
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bond network helping to bind and activate substrate 1, two well positioned water molecules, and certain LEH sidechains accounts for the origin of the observed (S,S)-selectivity of variant H-2-H5 (Fig. 3D). This model implies a catalytic machinery that is somewhat different from WT
LEH, in which the nucleophilic water molecule is 10a positioned and activated by D132, N55, and Y53. Unfortunately, some residues in the N- and C- termini are missing in our crystal structures. These residues are cleaved off during
Figure 3. Analysis of active site of LEH variant H-2-H5 in comparison with WT LEH, based on X-ray structures. A) Active site of variant H-2-H5. B) Structure of active site of variant H-2-H5 complexed with (S,S)-cyclohexane-1,2-diol. C) Model of substrate 1 and water molecule in active site of WT LEH; WO represents water molecule. D) Model of substrate 1 and two water molecules in active site of variant H-2-H5. W1 and W2 represent two separate water molecules. The black and blue dashed lines represent the hydrogen bond network and the distances from W1, W2 to C1 and C2, respectively. The distances are labeled in angstrom (Å). E) Model of the alteration of the F134 rotamer conformation resulting in the change of the location of substrate 1 and water molecules. Orange and light blue sticks represent WT and H-2-H5, respectively. F) Model of the alteration of the R99 rotamer conformation resulting in the change of the location of substrate 1 and water molecules. Orange and light blue sticks represent WT and H-2-H5, respectively. The critical residues are shown as sticks in all of these pictures. crystallization as judged by SDS-PAGE analysis using protein crystal samples (Supplementary Fig. S10). During the ISM process, both of the two improved variants B-1-F12 (T76K/L114V/I116V) and B-4-B11 (T76D/L114V/I116V), which lack the Nand Cterminal mutations, maintain enantioselectivity of 71% ee in favor of (S,S)-2 (Table S2). Similarly, other positive mutants F-1-E4 (T76K/L114V/I116V/N92D/F139V/L147F), F-2-H10 (T76K/L114V/I116V /F139V/L147F) and F-6-A9 (T76K/L114V/I116V/N92K/F139V/L147F) lacking the N-terminal mutations, show enantioselectivity of 82%, 82% and 83% ee, respectively, in favor of (S,S)-2 (Table S2).
Additionally, we constructed a new mutant H-2-H5-1(E45D/L74F/T76K/M78F/N92K/L114V/I11 6V) lacking the N- and C- terminal mutations, and this mutant displays 86% ee in favor of (S,S)-2. Based on these control experiments, it can be concluded that the N- and C- terminal mutations show minimal effect on the enantioselectivity. In order to gain some insight regarding the source of enhanced thermostability, we analyzed the stability-related mutations on the crystal WT structure (Fig. 4). Accordingly, mutations E45D, T76K and N92K are located on or near the surface of LEH. It is likely that these mutations stabilize the protein by optimizing the distribution of
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Figure 4. The evolved thermostability-related mutations shown as spheres in WT (left) and variant H-2-H5 (right). Gray: uncharged amino acids; purple: negatively charged amino acid; red: positively charged amino acids. charges on the enzyme surface, which is an established method of protein stabilization.3, 17 Furthermore, S15D may form an ionic bond with A19K, thereby stabilizing a flexible N-terminal loop. However, since mutation S15D and A19K in the N-terminus are missing in our crystal structure, the respective interaction cannot be visualized. CONCLUSION
mutagenesis (ISM) and reduced amino acid alphabets based on triple code saturation 9 mutagenesis (TCSM), better results were obtained. Insight regarding the origin of enantioselectivity was gained by analyzing the crystal structures of the (S,S)-selective variant with and without complexed product. Due to the mutations introduced by TCSM, the substrate and the nucleophilic water were re-positioned for smooth and stereoselective ring-opening. This insight was gained by the analyzing the X-ray structure of variant H-2-H5, which reveals a hydrogen bond network instrumental in binding and activating the substrate. The comparison of the traditional with the present approach in one and the same enzyme system may be of help when performing future multi-parameter optimization by directed evolution while minimizing the screening effort. Sequential optimization, e.g., directed evolution of thermostability followed by protein engineering of stereoselectivity and 1,3 activity also needs increased attention. Finally, protein engineering of stereoselectivity and activity followed by stabilization for industrial 19 applications by means of immobilization also deserves mention as a possible alternative. MATERIAL AND METHODS
This study demonstrates once more the challenge of optimizing several enzyme parameters when performing directed evolution.1-5 It is one of the remaining problems in directed evolution for which a reliable and general strategy still needs to be developed, especially when considering industrial applications. In the present study we attempted to evolve thermostability and enantioselectivity, while also considering activity. Two approaches were explored using the epoxide hydrolase LEH as the catalyst in the hydrolytic desymmetrization of cyclohexene oxide (1), the goal being the evolution of both (R,R)- and (S,S)-selective variants. In one strategy, mutations from two previous studies reporting enhanced thermostability and increased as well as inverted enantioselectivity, respectively, were combined in a “conventional” approach. This traditional strategy proved to be only partially successful, because activity was somewhat reduced. This finding may have different reasons, including the possibility that mutational effects on several enzyme properties are not necessarily additive, as 18 already demonstrated in one-parameter systems. Upon applying an alternative strategy in which the parameters were optimized simultaneously using iterative saturation
KOD Hot Start DNA Polymerase was obtained from Novagen. Restriction enzyme Dpn I was bought from NEB. The oligonucleotides were synthesized by Life Technologies. Plasmid preparation kit was ordered from Zymo Research, and PCR purification kit was bought from QIAGEN. DNA sequencing was conducted by GATC Biotech. All commercial chemicals were purchased from Sigma-Aldrich or Tokyo Chemical 8
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Industry (TCI) or Alfa Aesar. Lysozyme and DNase I were purchased from AppliChem. PCR based methods for site-directed mutagenesis and library construction of LEH. Libraries were constructed using the Over-lap PCR and megaprimer approach with KOD Hot Start polymerase. 50 µL reaction mixtures typically contained 30 µL water, 5 µL KOD hot start polymerase buffer (10×), 3 µL 25 mM MgSO4, 5 µL 2 mM dNTPs, 2.5 µL DMSO, 0.5 µL (50~100 ng) template DNA, 100 µM primers Mix (0.5 µL each) and 1 µL KOD hot start polymerase. The PCR conditions for short fragment: 95 °C 3 min, (95 °C 30 sec, 56 °C 30 sec, 68 °C 40 sec) × 30 cycles, 68 °C 120 sec. For mega-PCR: 95 °C 3 min, (95 °C 30 sec, 60 °C 30 sec, 68 °C 5 min 30 sec) × 24 cycles, 68 °C 10 min. The PCR products were analyzed on agarose gel by electrophoresis and purified using a Qiagen PCR purification kit. 2 µL NEB CutSmart™ Buffer and 2 µL Dpn I were added in 50 µL PCR reaction mixture and the digestion was carried out at 37 °C for 7 h. After Dpn I digestion, the PCR products 1.5 µL were directly transformed into electro-competent E. coli BL21(DE3) to create the final library for Quick Quality Control and screening. Primer design and site-directed mutagenesis of LEH. Primer design depend upon the particular amino acid residues sites and mutagenesis, and in the case of LEH mutant LEH-P creation eight residues which were divided into four groups (Supplementary Fig.S1): 1) Amplification of the short fragments of LEH using mixed primers LEH-1-F/LEH-2-R, LEH-2-F/LEH-3-R and LEH-3-F/LEH-4-R, respectively; 2) Over-lap PCR using the products of step 1 as template and mixed primers LEH-1-F/LEH-4-R; 3) Amplification of the whole plasmid pET22bLEHwt using the products of step 2 as megaprimers, leading to the final mutagenesis plasmids for library generation. Primers are listed in Supplementary Table S5. The PCR products were digested by Dpn I and transformed into electro-competent E. coliBL21(DE3) to create the library. Mutants LEH-F-b, SS-8, RR-8, SS-11, RR-11 and H-2-H5-1 were created using the same procedure as mentioned above (Supplementary Figure S2-S7). All the primers used are listed in Supplementary Table S5. Primer design and library creation of LEH. Primer design and library construction depend upon the particular amino acid chosen, and in the case of LEH this involves sixteen residues which were divided into four groups (Supplementary Fig. S8 and Fig. S9): 1)
Amplification of the short fragments of LEH using mixed primers F1/R1, F2/R2 and F3/R3 for Library A, B and C respectively. Amplification of the short fragments of LEH using mixed primers F4/R4 and F5/R5 for Library D; 2) Over-lap PCR using the PCR products of F4/R4 and F5/R5 as template and mixed primers F4/R5; 3) Amplification of the whole plasmid pET22bLEHwt using the PCR products of F1/R1, F2/R2 and F3/R3 and over-lap PCR product of step2 as megaprimers, leading to the final variety plasmids for library generation. Primers are listed in Supplementary Table S6. The PCR products were digested by Dpn I and transformed into electro-competent E. coliBL21(DE3) to create the library for screening. Other Libraries were created using the same procedure as mentioned above. All the primers used are listed in Supplementary Table S7. Screening Procedures. Colonies were picked up and inoculated into deep-well plates containing 300 µL LB medium with 50 µg/ml carbenicillin and cultured overnight at 37 °C with shaking. An aliquot of 120 µL was transferred to glycerol stock plate and stored at -80 °C. Then, 800 µL TB medium with 0.5% (m/v) lactose and 50 µg/mL carbenicillin was added directly to the culture plate for 8 h at 28 °C with shaking for protein expression. The cell pellets were harvested and washed with 400 µL 50 mM pH 7.4 potassium phosphate buffer and centrifuged for 10 min 4000 rpm at 4 °C. Then, the pellets were resuspended in 400 µL of the same buffer with 6 U/mL DNase I and 1 mg/mL lysozyme for breaking the cell at 30 °C for 1 h with shaking. The crude lysate was centrifuged for 30 min 4000 rpm at 4°C. 50 µL of the supernatant was transferred into 96-well-micrioplate and heated at 70°C for 20 minutes at oven (The 96-well-micrioplate has protection function for the inside supernatant. After heated at 70°C for 20 minutes at oven, the wt LEH remain about 10% activity, so we select this condition for the high-throughput screening of thermostability). Then the heat-treated supernatant 50 ul was used for an adrenaline assay. 110 µL potassium phosphate buffer (50 mM, pH 7.4) with 5% acetonitrile and substrate 1 (final concentration 10 mM) were added. The plates were incubated at 30 °C, 500 rpm, 1 h. Afterwards, NaIO4 solution 20 µL (77 mg in 24 mL of water) was added and the plates were further incubated at 30 °C, 500 rpm, 10 min. Subsequently, adrenaline solution 20 µL (epinephrine 132 mg, water 24 mL, concentrated HCl 5 drops for solubilizing the adrenaline) was added, which caused the immediate formation of a red color in the inactive reactions. Active variants gave lighter 9
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colour relative to WT. 300 µL rest supernatant of the active transformants was transferred into new deep-well plates for reaction with 10 mM substrate 1 and 5% acetonitrile as co-solvent for 5 h at 30 °C 800rpm, the final volume was 400 µL. The product and remaining substrate were extracted using equal volumes of ethyl acetate (EtOAc) for GC analysis by chiral column (Supplementary Table S8). The screening results were shown in Supplementary Table S2. Protein expression and purification. The WT and positive mutants were inoculated in 5 mL LB containing 50 µg/mL carbenicillin and cultured overnight at 37 °C with shaking, and then scaled up to 500 mL TB containing 50 µg/mL carbenicillin. When the OD600 reached 0.8, 0.4 mM IPTG was added to induce the protein expression. The cell cultures continued to grow for 10 hours at 25 °C before being harvested by centrifugation at 6,000 × g and resuspended in a PBS buffer (20 mM, pH 7.4) containing 500 mM NaCl, 20 mM imidazole. The cell pellets were disrupted by sonication and the cell debris was removed by centrifugation at 15,000 × g for 60 min. The soluble protein sample was loaded onto a nickel affinity column (GE Healthcare) and washed with 20~500 mM imidazole solution containing 500 mM NaCl and 20 mM PBS buffer (pH 7.4). Proteins from the flow through were pooled and concentred, and then loaded onto a HiTrap™ Q HP 5 ml column (GE Healthcare) and eluted with 0~1000 mM Nacl solution containing 500 mM Tris buffer (pH 8.0). The eluted protein sample was pooled and concentred, and then loaded onto a Hiload™ 16/600 Superdex™ 200 pg column (GE Healthcare) and eluted with 50 mM Tris (pH 8.0), 500 mM Nacl. Fractions containing highly pure LEH proteins were pooled and concentrated with Centricon filtration devices (Amicon) to 10 mg/ml. The protein concentrations were determined by Bradford. The measurement of kinetic parameters of purified enzyme. The enzymatic hydrolysis rate was measured by monitoring the conversion of substrate by GC using 1-heptanol as the internal standard. Pure enzymes were kept at 30°C or 37°C for 10min and added to potassium phosphate buffer (50 mM, pH 7.4) with a total volume of 400 μL containing epoxide of varied concentration (2–64 mM) and 2.5% (v/v) of acetonitrile, and the reaction was performed at 30°C or 37°C for 10min with shaking (800 rpm). The reaction was terminated by the addition of 400 μL EtOAc containing 2.0 mM 1-heptanol. The mixture was vigorously mixed and the organic layer was separated by centrifugation at 4,000 × rpm for 15
min. The EtOAc layer was analyzed by GC (The column is Hydrodex-β-TBDAc, 25 m x 0.25 mm ID). Effects of temperature on enzyme activity. For thermostability, the enzyme was incubated at different temperatures (30–70°C) for 30 min followed measuring the residual activity by monitoring the conversion of substrate by GC using 1- heptanol as the internal standard. Differential scanning calorimetry. The protein samples of WT LEH and LEH mutant H-2-H5 were dissolved in the 50 mM potassium phosphate buffer (pH 8.0) with a final concentration of 12mg/mL. DSC examinations of the sample were carried out using the instrument Rigaku Thermo plus EVO2 DSC8231. DSC scans were taken from 20 to 100°C at a heating rate of 5°C/min. The 50 mM potassium phosphate buffer (pH 8.0) was used as a reference. The heat flow versus temperature plot of the WT LEH and LEH mutant H-2-H5 was obtained after subtraction of the buffer signal. The peak value of the DSC plot 8 was considered as the melting point (Tm). X-ray structural analysis. The H-2-H5 crystals were grown in 0.1 M HEPES (pH 7.5), 2% v/v Polyethylene glycol 400 and 2.0 M ammonium sulfate by the sitting-drop vapor diffusion method at 20 °C. Proteins at 10 mg/mL were mixed in a 1:1 ratio with the reservoir solution in a final volume of 2 μL and equilibrated against the reservoir solution. Single crystals of H-2-H5 were obtained directly, whereas the complex structures of H-2-H5-(S,S)-cyclohexane-1,2-diol were obtained by soaking the crystals in growth buffer containing 100 mM of epoxide for 1 min. All crystals were mounted in nylon loops and flash-frozen in liquid nitrogen. Diffraction data of all crystals was collected at the wave length of 0.9792 Å using an ADSC Quantum 315r detector at beamline BL17U of Shanghai Synchrotron Radiation Facility (SSRF) at 100 K. All data sets were indexed, integrated, and scaled using the 20 HKL2000 package. The structures of H-2-H5 and H-2-H5-(S,S)-cyclohexanediol complex were solved by molecular replacement method using the program PHASER21 and the coordinate of wild-type LEH (PDB code 1NU3) as a search model. Rounds of automated refinement were 22 performed with PHENIX and the models were 23 extended and rebuilt manually with COOT. The structures of H-2-H5 and H-2-H5-(S,S)-cyclohexane-1,2-diol complex have been refined to 2.30 and 1.89 Å, respectively. The statistics for data collection and crystallographic refinement are summarized in Supplementary
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Table S4. All structural figures were prepared using Pymol (http://www.pymol.org/). ASSOCIATED CONTENT Supporting Information: The figures of primer design and creation of LEH mutants and libraries, results of libraries screening, data collection and refinement statistics of H-2-H5 crystal structure, list of primers, SDS-PAGE of H-2-H5 and analytic conditions of GC are available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Correspondence should be addressed to (M.T.R.)
[email protected] and (Xinqi Liu)
[email protected] Author Contributions § These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS Support from the Max-Planck-Society and the LOEWE Research Cluster SynChemBio is gratefully acknowledged. We thank Dr. Richard Lonsdale for constructing Fig. 1. REFERENCES (1) Recent reviews of directed evolution: (a) Bommarius, A. S. Annu. Rev. Chem. Biomol. Eng. 2015, 6, 319-345; (b) Gillam, E. M. J.; Copp, J. N.; Ackerley, D. F. Directed Evolution Library Creation. Methods in Molecular Biology, Humana Press, Totowa, 2014; (c) Denard, C. A.; Ren, H.; Zhao, H. Curr. Opin. Chem. Biol. 2015, 25, 55-64; (d) Reetz, M. T. Directed Evolution of Enzymes. Enzyme Catalysis in Organic Synthesis, Wiley-VCH, Weinheim, 2012, Vol. 1, pp.119-190; (e) Jäckel, C.; Hilvert, D. Curr. Opin. Biotechnol. 2010, 21, 753-759; (f) Brustad, E. M.; Arnold, F. H. Curr. Opin. Chem. Bio. 2011, 15, 201-210; (g) Goldsmith, M.; Tawfik, D. S. Curr. Opin. Struct. Biol. 2012, 22, 406-412; (h) Widersten, M. Curr. Opin. Chem. Biol. 2014, 21, 42-47; (i) Currin, A.; Swainston, N.; Day, P. J.; Kell, D. B. Chem. Soc. Rev. 2015, 44, 1172-1239; (j) Turner, N. J. Nat. Chem. Biol. 2009, 5, 567-573; (k) Siloto, R. M. P.; Weselake, R. J. Biocatal. Agric. Biotechnol. 2012, 1, 181-189; l) Lutz, S.; Bornscheuer, U. T. Protein Engineering Handbook. Wiley-VCH, Weinheim, 2009; (m) Sun, Z.; Wikmark, Y.; Bäckvall, J.-E., Reetz, M. T. Chem. Eur. J. 2016, 22, 5046-5054. (2) Reetz, M. T. Angew. Chem. Int. Ed. 2011, 50, 138-174. (3) (a) Eijsink, V. G.; Gåseidnes, S.; Borchert, T. V.; vandenburg, B. Biomol. Eng. 2005, 22, 21-30; (b) Polizzi, K. M.; Bommarius, A. S.; Broering, J. M.; Chaparro-Riggers, J. F. Curr. Opin. Chem. Biol. 2007, 11, 220-225; (c) Liszka, M. J.; Clark, M. E.; Schneider, E.; 11
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