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Development of an Engineered Ketoreductase with Simultaneously Improved Thermostability and Activity for Making a Bulky Atorvastatin Precursor xumin gong, zhen qin, Fu-Long Li, Bu-Bing Zeng, Gao-Wei Zheng, and Jian-He Xu ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b03382 • Publication Date (Web): 21 Nov 2018 Downloaded from http://pubs.acs.org on November 22, 2018
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ACS Catalysis
Development of an Engineered Ketoreductase with Simultaneously Improved Thermostability and Activity for Making a Bulky Atorvastatin Precursor Xu-Min Gong,† Zhen Qin,‡ Fu-Long Li,† Bu-Bing Zeng,§ Gao-Wei Zheng,*,† and Jian-He Xu*,† †State
Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China ‡State Key Laboratory of Bioreactor Engineering and R&D Center of Separation and Extraction Technology in Fermentation Industry, East China University of Science and Technology, Shanghai 200237, China §Shanghai Key Laboratory of New Drug Design, East China University of Science and Technology, Shanghai 200237, China ABSTRACT: Protein engineering is a powerful strategy for enhancing the properties of enzymes for industrial applications. However, thermostabilizing an enzyme via this strategy while simultaneously improving its activity is challenging due to the wellknown stability-activity trade-off. Herein, using native ketoreductase LbCR, thermostability and activity were evolved separately by directed evolution, generating mutations V198I and M154I/A155D with increased thermostability, and mutations A201D/A202L with increased enzymatic activity. Based on cooperative mutational effects, variants LbCRM6 (M154I/A155D/A201D/A202L) and LbCRM8 (M154I/A155D/V198I/A201D/A202L) with simultaneously improved thermostability and activity were subsequently constructed by combining mutations. Analysis of variant structures demonstrated that increased thermostability was largely attributed to rigidification of flexible loops around the active site through the formation of additional hydrogen bonds and hydrophobic interactions. The best variant LbCRM8 displayed a 1944-fold increase in half-life at 40°C and a 3.2-fold improvement in catalytic efficiency compared with the wide-type enzyme. Using only 1 g L–1 of E. coli cells co-expressing this LbCRM8 and glucose dehydrogenase BmGDH as catalyst, t-butyl 6-cyano-(5R)-hydroxy-3-oxo-hexanoate up to 300 g L–1 loading was completely reduced within 6 h at 40°C, yielding the corresponding t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate (ATS-7) with >99.5% de and a space-time yield up to 1.05 kg L–1 day–1. These results demonstrated that LbCRM8 is an attractive biocatalyst for the synthesis of ATS-7, an advanced chiral intermediate for the production of the cholesterol-lowering drug atorvastatin. KEYWORDS: Biocatalysis, asymmetric reduction, ketoreductase, protein engineering, thermostabilization, atorvastatin precursor
INTRODUCTION Enzymes are exceptionally efficient catalysts conducting chemical reaction, recent advances in DNA technologies and bioinformatics have led to the discovery of numerous natural enzymes for the synthesis of fine chemicals and pharmaceutical drugs.1 However, the majority of biocatalysts are from mesophilic organisms, and cannot usually resist harsh reaction conditions such as elevated temperatures that are often utilized in industrial processes due to the advantages of increased reaction rates and improved solubility of hydrophobic substrates.2 Inherent problems can lead to rapid inactivation of enzymes under practical operation conditions, and thereby hinder their extensive industrial applications. To address these issues, efficient strategies have been developed, including the identification and use of enzymes from thermophilic organisms,3 chemical modification of enzymes,4 and enzyme immobilization.5 In addition, protein engineering is also a powerful tool for improving the properties of enzymes.6 Numerous successful examples have been reported in the past few decades. For example, several novel enzymes for reactions not catalyzed by natural biocatalysts have been created using protein engineering,7 and many improved biocatalysts have been developed for the industrial synthesis of useful molecules such as key intermediates of sitagliptin (Januvia),8 atorvastatin (Lipitor),9 and montelukast (Singulair)10. Enzyme thermostabilization is of particular interest to biochemists, and many different engineering strategies and robust enzymes have been reported.11,12 In most cases, evolution for
thermostabilization has a small or negligible effect on enzyme activity because the mutated residues are predominately located on the protein surface, distant from the active site. However, simutaneously improving enzyme activity and thermostabilization is particularly challenging due to the wellknown stability-activity trade-off,13 especially for mutations near the active site. Therefore, the development of efficient protein engineering strategies for significantly improving protein thermostability while simultaneously enhancing activity is in high demand. Herein, using the native ketoreductase (LbCR) from Lactobacillus brevis,14 we attempted to enhance its thermostability and activity at the same time using directed evolution and cooperative mutational effects, to obtain a robust and highly active variant via a simple evolutionary approach. Analysis of variant structures showed that mutated residues were mainly located on flexible loops near the active site. Using E. coli cells coexpressing LbCRM8 and BmGDH as catalyst, we successfully carried out a large-scale preparation of atorvastatin precursor t-butyl 6-cyano-(3R,5R)dihydroxyhexanoate.
RESULTS AND DISCUSSION Combining Mutations for Simultaneously Improving Thermostability and Activity. Our previous study suggested that the ketoreductase LbCR from L. brevis is a highly stereoselective biocatalyst for the synthesis of atorvastatin precursor ATS-7. However, a poor half-life (only 2.5 min at 40°C) and insufficient activity hamper its practical application
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in industry.14 Hence, concurrent improvements in thermostability and enzyme activity are of great importance for the practical application of this enzyme. Initially, we constructed a mutant library containing ~7,000 colonies by error-prone PCR with a mutation rate of 13 nucleotides per sequence due to the unknown enzyme structure. Fast screening the mutant library for enhanced thermostability in was performed in 96-well microtiter plates by incubating cell-free extracts at 37°C for 2 h, and residual activity was measured by monitoring the initial decrease in absorbance at 340 nm and at 30°C using a UV/Vis microplate reader. Three hits (LbCRM0, LbCRM1, and LbCRM2) exhibiting slightly improved thermostability relative to wild-type (WT) LbCRWT were obtained (Table 1, entries 2, 3, and 4). These mutations were subsequently combined by DNA shuffling, and screening of 200 clones identified a remarkably stable mutant LbCRM4 (M154I/A155D). The half-life of LbCRM4 at 40°C was prolonged from 2.5 min to 1070 min, representing a 430-fold increase compared with LbCRWT. However, a remarkable decrease in affinity toward substrate ATS-6 was observed for variant LbCRM4, leading to a sharp drop in catalytic efficiency (kcat/KM; Table 1, entry 6). This demonstrated the anticipated compromise between function and stability. Hence, we subsequently performed activity screening using the same mutant library to identify potential ‘hotspots’ for improving enzyme activity. After screening, variant LbCRM3 (A201D/A202L) exhibiting a 2.5-fold improvement in kcat/KM was identified. Unfortunately, a decreased thermostability was observed for this variant (Table 1, entries 1 and 3). The above results clearly indicated the trade-off between activity and stability that can occur during enzyme evolution. To solve this puzzle, it is logical to combine the ‘hotspots’. Indeed, cooperative mutational effects are often used when simplifying protein engineering because they allow simultaneous improvement of multiple properties by direct combination of beneficial mutations. This strategy has been successfully applied to simutanelously engineer uncontradicted thermostability and enantioselectivity, or activity and enantioselectivity but few examples of engineering of thermostability and activity have been reported.15 In order to assess the feasibility of this strategy for cooperatively improving thermostability and activity, we attempted to combine mutations A201D/A202L for increasing activity and mutations M154I/A155D and V198I for increasing thermostability. Excitingly, variant LbCRM6 harboring the combined M154I/A155D/A201D/A202L mutations and variant LbCRM8 harboring the combined M154I/A155D/A201D/A202L/V198I
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mutations, displayed both improved thermostability and catalytic activity compared with variants lacking these combinations. Compared with the WT enzyme, the best variant (LbCRM8) exhibited a 1944-fold increase in t1/2 at 40°C—from a few minutes to more than 3 days, and a 3.2-fold increase in kcat/KM. Thus, although the mutations V198I and A201D/A202L themselves did not obviously improve thermostability, their combination with the M154I/A155D mutations resulted in clear additivity and synergistic mutational effects for thermostability and activity. These results demonstrated that, despite the known stability-activity trade-off, the use of additivity and cooperative mutational effects for the concurrent improvement of enzyme thermostability and activity is possible.
Figure 1. Thermal inactivation/unfolding analysis of purified wild-type LbCRWT and its variants. A, T15 50 of WT and engineered mutants. B, Thermal unfolding curves of WT and variant enzymes measured using circular dichroism spectroscopy.
Table 1. Kinetic Parameters and Half-lives of purified LbCRWT and Engineered Proteins toward Substrate ATS-6. mutation
KM (mM)
kcat (s−1)
kcat/KM (s−1 mM−1)
t1/2 (min)
folda
LbCRWT
WT
0.582 ± 0.13
24.6 ± 6.7
42.4
2.5
1.0
LbCRM0
M154I
0.326 ± 0.07
17.4 ± 0.9
53.4
6.8
2.7
epPCR
LbCRM1
A155D
1.02 ± 0.15
38.4 ± 1.5
37.7
3.0
1.2
4
epPCR
LbCRM2
V198I
0.792 ± 0.05
24.4 ± 1.3
30.8
7.0
2.8
5
epPCR
LbCRM3
A201D/A202L
0.232 ± 0.02
24.9 ± 1.4
108
2.0
0.8
6
DNA shuffling
LbCRM4
M154I/A155D
1.06 ± 0.02
29.9 ± 0.6
28.3
1070
428
7
combined mutation
LbCRM6
M154I/A155D/A201D/A202L
0.188 ± 0.02
36.5 ± 1.0
194
2820
1128
0.397 ± 0.05
53.6 ± 4.3
135
4860
1944
entry
evolutionary strategy
enzyme
1
starting enzyme
2
epPCR
3
8 a Fold
combined mutation LbCRM8 M154I/A155D/A201D/A202L/V198I change enhancement in half-life at 40°C compared to the WT enzyme.
In order to further confirm the improved thermostability, two additional parameters for assessing thermal stability,
melting temperature Tm and T15 50, representing the thermodynamic stability and kinetic stability respectively,
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ACS Catalysis were also characterized. Both the thermodynamic stability and kinetic stability of LbCRM4, LbCRM6, and LbCRM8 were significantly improved compared to LbCRWT or LbCRM2 and LbCRM3 (Figure 1). The T15 50 values of LbCRM6 and LbCRM8 were 18°C and 20°C, respectively, higher than the WT enzyme (Table S2). Circular dichroism spectroscopy analysis showed that the Tm values of LbCRM6 and LbCRM8 were enhanced by 7.8°C and 7.0°C, respectively, compared to LbCRWT (Table S2). Elucidating the Mechanism Underpinning the Enhanced Thermostability. To probe the mechanism of the remarkably enhanced thermostability, we attempted to crystallize LbCRWT, LbCRM6, and LbCRM8 proteins, and determined the apo crystal structures of LbCR M6 and LbCRM8 at 2.0 Å and 2.3 Å resolutions, respectively (Table S3). Unfortuantely, we failed to obtain the crystals of the WT enzyme, which might be related to its instability. Therefore, we constructed a structure model for LbCRWT using homology modeling based on the LbCRM8 crystal structure. LbCRM6 and LbCRM8 proteins are homotetramers in the crystal structures (Figure S3). Figure 2 shows the positions of the mutations in the monomer structure of LbCRM8. We noted that most of the residues are located on loops near the active site. Mutations M154I/A155D are located on loop 9, which is positioned between sheet β5 and helix α5 and is adjacent to the catalytic residue Tyr158. Meanwhile, mutation V198I is located on the flexible loop 11 that connects sheet β6 with helix α6, and mutations A201D/A202L are on helix α6. Mutations V198I/A201D/A202L are in the substrate binding pocket.16 Comparison between the modelled structure of LbCRWT and the experimentally determined structure of LbCRM8 revealed three new hydrogen bonds formed by Asp155 in LbCRM8, with Val96 on loop 7, and with Ala157 and Tyr158 on helix α5 (Figure 3A and 3B). Together, these additional H-bonds in
crease the stability of flexible loops 7 and 9 surrounding the active site residues. Additionally, Li and co-workers also found that the activity of a thermophilic enzyme at ambient temperatures was dramatically improved by directed evolution. Theoretical analysis showed that the relatively rigid loop covering the active site became highly flexible following mutagenesis. Indeed, the two phenomena are related, since both indicate the importance of stabilizing the active site loop for enzyme stability.13d
Figure 2. Locations of mutations (ball and stick representations) in the monomer structure of LbCRM8 (M154I/A155D/A201D/A202L/V198I). Active site residues are shown as orange sticks.
Figure 3. Structural comparison of mutations in LbCRWT (A and C) and LbCRM8 (B and D). A and B, Analysis of the mutation of residues 154 and 155. Mutated residues are shwn as magenta stick, mutation site, and dotted line indicate hydrogen bonds. C and D, Analysis of the mutation of residues 198, 201, and 202. Hydrophobic interactions are shown as a light pink surface, and dotted line depict hydrogen bonds.
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We also noted that, compared to the WT enzyme (Figure 3C), mutation A201D (situated on the solvent-exposed surface) generates extra hydrogen bond(s) with solvent water molecules (Figure 3D). Meanwhile, mutation A202L forms a tighter hydrophobic interaction with the internal residue V150 located on loop 9. Using B-FITTER software17, we were able to calculate the B factors of the enzymes LbCRM6 and LbCRM8 based on their measured structures, and found that variant LbCRM8 has lower B factor values in two regions (residues 147152 and 197209) compared with those of variant LbCRM6 (Figure S4B), suggesting the possible source of the increased thermostability of LbCRM8. Indeed, the additional V198I mutation in variant LbCRM8 enhances its hydrophobic interactions with A202L, generating a longer helix α6 and an increased rigidity of loop 11 (Figure S4A). Elucidating the Mechanism of the Enhanced Activity. To gain insight into the influence of the mutations A201D/A202L on enzyme activity, the substrate ATS-6 was docked into the binding pocket of the LbCRWT, LbCRM3, and LbCRM8 using AutoDock program.18 In view of the conformational changes induced by NADPH-binding, structures of LbCRWT, LbCRM3, and LbCRM8 in complex with NADPH were reconstructed based upon the crystal structures of LkADH in complex with NADP+ (PDB ID 4RF2)19 and apo-LbCRM8. According to the elucidated catalytic mechanism of the short-chain dehydrogenase20, the carbonyl oxygen atom of ATS-6 is hydrogen bonded to the hydroxyl groups of Ser145 and Tyr158, and the carbonyl carbon atom of ATS-6 is attacked by a hydrogen from the C4 atom of NADPH at the si-face, affording the (R)-alcohol, consistent with the experimental results. Figure S5A5C shows that the binding orientations of the substrate in the active site of LbCRWT, LbCRM3, and LbCRM8 are similar. Although the mutations A201D/A202L have no directed interaction with the substrate, these mutations lead to additional hydrogen bonds formed between the residues S145, A147, and G148 and ATS-6 in the variants compared with LbCRWT (Figure S5D5F). These additional interactions result in much stronger binding affinity than in LbCRWT, consistent with the observed decrease in Km (Table 1, entries 1, 5, and 8).21 Therefore, the A201D/A202L mutations are beneficial for enzyme activity because they stabilize protein-substrate binding interactions. In addition, A201D/A202L mutations improving activity and M154/A155D mutations increasing thermostability were additive; when combined, variant LbCRM6 exhibited simultaneous improvements in thermostability and activity. This may be because these mutations are located in two separate regions (Figure 2, >10 Å), with no obvious interaction effects between them, as demonstrated by the simultaneous increase in the stability and DNA binding affinity of the gene V protein (GVP) following combinational mutagenesis.22 Moreover, the cooperative effects between mutation V198I and its adjacent mutations A201D/A202L resulted in further improvement in thermostability of LbCRM8, but a decrease in activity. Asymmetric bioreduction of bulky β-ketoester ATS-6. Asymmetric reduction of the ketoester ATS-6 to ATS-7 was performed by variant LbCRM8 in combination with glucose dehydrogenase variant BmGDHQ252L23 from Bacillus megaterium for regeneration of the NADPH cofactor. However, we found that the top two variants LbCRM6 and LbCRM8 were mainly expressed in inclusion bodies in
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recombinant E. coli cells, unlike LbCRWT and other mutants (Figure S6), which may be attributed to the large changes introduced into the enzyme. To enhance soluble expression of these two variants, several soluble fusion tags and alternative expression hosts were tested. The results revealed enhanced soluble expression levels of LbCRM6 and LbCRM8 in E. coli Rosetta–gami 2 (DE3)/pET28a that were comparable with the wild-type enzyme (Table S1 and Figure S6). Therefore, E. coli Rosetta–gami 2 (DE3)/pET28a was subsequently used for expression of LbCRM8. Given that the half-life of LbCRM8 at 40°C is more than 3 days we carried out the reaction at 40°C, instead of the previously reported 30°C, in order to shorten the reaction times. At 100 g L−1, ATS-6 could be completely reduced within 4 h by 3.0 g L−1 of lyophilized E. coli cells expressing LbCRM8 and 6 g L−1 of lyophilized E. coli cells expressing BmGDH (Table 2, entry 2). By contrast, only 26% of substrate was converted by LbCRWT, even after 13 h under the same conditions (Table 2, entry 1). Given the known poor thermostability of LbCRWT, it was not surprising that the WT enzyme lost almost all activity, while the thermostable LbCRM8 maintained its initial activity throughout the reaction period (Figure S7B). Even at a higher substrate loading of 300 g L−1, the reaction was accomplished completely within 5 h using 5 g L−1 of lyophilized E. coli cells expressing LbCRM8 and 6 g L−1 of lyophilized E. coli cells expressing BmGDH (Table 2, entry 3), highlighting the excellent robustness of the LbCRM8 enzyme for synthetic applications with extremely high substrate/product loading. Table 2. Asymmetric Reduction of ATS-6 Catalyzed by LbCRWT and LbCRM8. entry
biocatalyst
substrate loads (g L−1)
S/C ratio (g g−1) c
time (h)
conv. (%)
1
LbCRWTa
100
11
24
26
2
LbCRM8a
100
11
4
>99
3
LbCRM8a
300
27
5
>99
4
E. coli cellsb
300
150
5
>99
5 E. coli cellsb 300 300 8 >99 Reaction conditions: substrate (1 g or 3 g), glucose (1.2 equivalent), lyophilized cells expressing LbCRWT (0.03 g) or LbCRM8 (0.03 g or 0.05 g), lyophilized cells expressing GDH (0.06 g), 5% (v/v) dimethyl sulfoxide (DMSO) and NADP+ (0.1 mM), potassium phosphate buffer (100 mM, pH 6.0), 10 mL total volume, 40°C, pH was maintained at 6.0 with K2CO3. b Reaction conditions: substrate (3 g), glucose (1.2 equivalent), lyophilized E. coli cells coexpressing LbCRM8 and BmGDH (0.01 g or 0.02 g), DMSO (5%, v/v) and NADP+ (0.1 mM), potassium phosphate buffer (100 mM, pH 6.0), 10 mL total volume, 40°C, pH was maintained at 6.0 with K2CO3. c S/C: substrate/catalyst ratio. a
Although the engineered LbCRM8 displayed excellent thermostability and activity, the observed substrate/catalyst (S/C) ratio was still low due to the addition of BmGDH for regeneration of NADPH. This is clearly not economical and is unfavorable for the separation of downstream products due to emulsification formed by high biocatalyst loading. To solve this problem, we first constructed E. coli whole cell biocatalysts by coexpression of LbCRM8 and BmGDHQ252L using pET Duet-1 as vector. We also significantly improved the expression levels of LbCRM8 and BmGDHQ252L by simple optimization (Table S4). These operations significantly decreased the catalyst loading, resulting in a dramatic increase from 27 g/g to 300 g/g in terms of S/C ratio. After simple optimization, 300 g L−1 of ATS-6 could be completely
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ACS Catalysis converted using only 1 g L−1 lyophilized cells at 40°C within 8 h, affording ATS-7 with >99% ee (Table 2, entry 5). Finally, we tested the feasibility of the bioreduction reaction at 100-mL scale. With just 1 g L−1 of lyophilized cells as catalyst, 300 g L−1 of ATS-6 was completely transformed into ATS-7 with >99.5% de within 6 h (Figure S7C). After extraction and normal work-up (Figure 4), the structurally complicated and usually difficult-to-make atorvastatin precursor ATS-7 was afforded in 87% isolated yield, and with an STY up to 1.05 kg L−1 d−1 space-time yield (STY), which represents the highest productivity reported to date for biosynthesis of ATS-7 (Table S5).14,24 Compared with the process using LbCRWT developed previously,14 the STY was increased to 1050 g L−1 d−1 from 350 g L−1 d−1, while the catalyst loading was reduced to 1 g L−1 from 16 g L−1, and these values are also far superior to those reported previously in the literature (Table S5).24 These impressive quantities and yields are far beyond typical chemical process goals during large-scale manufacture,25 demonstrating that the bioprocess is technically competitive and economically viable for production of the bulky chiral atorvastatin precursor ATS-7. OH O NC
E. coli cells coexpressing LbCRM8 and BmGDH
O O
OH OH O NC
Gluconic acid
NADPH
Glucose
Ca2+ OH OH O O
This material is available free of charge via the Internet at http://pubs.acs.org. Chemicals, mutagenesis, protein expression, and purification, activity assays and kinetic parameters, thermostability analysis, crystallization, asymmetric reduction, supporting tables and figures, and NMR analysis.
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected]. * E-mail:
[email protected]. Notes The authors declare no competing financial interest.
ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (grant nos. 21472045, 21878085, and 21536004) and the Fundamental Research Funds for the Central Universities (22221818014).
REFERENCES
87% yield, >99.5% de 1.05 kg/L/day STY
NADP+
Supporting Information
O ATS-7
ATS-6 300 g/L of substrate 1 g/Lof biocatalyst
ASSOCIATED CONTENT
O-
N
NH
F Atorvastatin
2
Figure 4. Asymmetric reduction of ATS-6 by E. coli cells coexpressing LbCRM8 and BmGDH to synthesize atorvastatin intermediate ATS-7 at 100 mL scale.
CONCLUSIONS In this study, we successfully engineered several LbCR variants that exhibit simultaneous enhancement in thermostability and activity compared with the WT enzyme using directed evolution and cooperative mutational effects. This simultaneous improvement is noteworthy due to the stability-activity trade-off that is frequently encountered when attempting protein stabilization. Analysis of crystal structures of the engineered proteins demonstrated that flexible loops around the active site were rigidified through the formation of additional hydrogen bonds and hydrophobic interactions, leading to a nearly 2000-fold improvement in half-life at 40°C. Most importantly, variant LbCRM8 allows for asymmetric bioreduction of ATS-6 to ATS-7 at an elevated temperature (e.g., 40°C), dramatically shortening the reaction times and enhancing the productivity significantly. Finally, an STY of up to 1.05 kg L−1 d−1 was achieved along with a 300 g/g S/C ratio, during the preparative-scale reduction of ATS-6 using E. coli cells coexpressing LbCRM8 and BmGDH, demonstrating that LbCRM8 is a very promising biocatalyst for practical production of the advanced atorvastatin chiral intermediate ATS-7.
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