Protein Engineering Strategies Research Articles

Specific Residue Interactions Regulate the Binding of Dengue Antigens to Broadly Neutralizing EDE Antibodies.

Antibodies binding to antigens present on the dengue virus (DENV) represent the main defense mechanism of the host organism against the pathogen. Among the antibodies elicited by DENV and that bind to DII of protein E, EDE1‐C8 can bind all DENV serotypes. Our analysis reveals the key residues in this interaction as well as structurally conserved hydrogen bonds located at the binding interface. They stabilize the dengue antigen–antibody complex among the EDE1 group of antibodies (Abs). Combining structural alignments with molecular dynamics simulations in the EDE1 Abs, we identified the critical elements that provide a major energetic contribution to the association of antigens from protein E with Abs. We discuss possible molecular insights into the binding mechanism by using a surrogate molecular entity resembling the protein E that forms native salt bridges and hydrogen bonds, including inferences on the light of high‐resolution crystal structures of dengue Fab complexes. Finally, the molecular determinants, the free energy profile, and the binding mechanism provide inspiration for potential strategies in protein engineering to design novel immunogens of protein E against DENV.

Thermostability and Specific-Activity Enhancement of an Arginine Deiminase from Enterococcus faecalis SK23.001 via Semirational Design for l-Citrulline Production.

l-Citrulline is a nonessential amino acid with a variety of physiological functions and can be enzymatically produced by arginine deiminase (ADI, EC 3.5.3.6). The enzymatic-production approach is of immense interest because of its mild conditions, high yield, low cost, and environmental benignity. However, the major hindrances of l-citrulline industrialization are the poor thermostability and enzyme activity of ADI. Hence, in this work, directed evolution and site-directed mutagenesis aided with in silico screening, including the use of b-factor values and HoTMuSiC, were applied to a previously identified ADI from Enterococcus faecalis SK23.001 ( EfADI), and a triple-site variant R15K-F269Y-G292P was obtained. The triple-site variant displays a 2.5-fold higher specific enzyme activity (333 U mg-1), a lower Km value of 6.4 mM, and a 6.1-fold longer half-life ( t1/2,45°C = 86.7 min) than wild-type EfADI. This work provides a protein-engineering strategy to improve enzyme activity and thermostability, which might be transferrable to other ADIs and enzymes.

Strategies for directed and adapted evolution as part of microbial strain engineering

AbstractThe industrial production of chemicals by microorganisms usually requires improvements to the enzymes, pathways, and strain that go beyond the capacity of innate enzymes. To achieve these phenotypes and overcome our limited capacity to de novo design these parts, directed and adaptive evolutionary approaches are used to explore new functions. This review highlights the recent advances in both sequence diversity generation and selection strategies from traditional in vitro mutagenesis to novel in vivo continuous evolution applications. The focus here is on comparison of the different gene diversity methods in an attempt to distinguish the best strategy for protein or strain engineering for a given goal. Furthermore, the important role that screening and selection can play in advancing directed and adapted evolution is discussed. © 2018 Society of Chemical Industry

Xenoprotein engineering via synthetic libraries

Chemical methods have enabled the total synthesis of protein molecules of ever-increasing size and complexity. However, methods to engineer synthetic proteins comprising noncanonical amino acids have not kept pace, even though this capability would be a distinct advantage of the total synthesis approach to protein science. In this work, we report a platform for protein engineering based on the screening of synthetic one-bead one-compound protein libraries. Screening throughput approaching that of cell surface display was achieved by a combination of magnetic bead enrichment, flow cytometry analysis of on-bead screens, and high-throughput MS/MS-based sequencing of identified active compounds. Direct screening of a synthetic protein library by these methods resulted in the de novo discovery of mirror-image miniprotein-based binders to a ∼150-kDa protein target, a task that would be difficult or impossible by other means.

Computational protein design and protein-ligand interaction studies for the improvement of organophosphorus degrading potential of Deinococcus radiodurans

The organophosphorus hydrolase enzyme is involved in the catalyzing reaction that involve hydrolysis of organophosphate toxic compounds. An enzyme from Deinococcus radiodurans reported as homologous to phosphotriesterase and show activity against organophosphate. In the past activity of this enzyme is low and efforts made to improve the activity by experimental mutation study. However only very few organophosphates tested against very few catalytic site mutations. In order to improve the catalytic power of the organophosphorus hydrolase enzyme, we carried out systematic functional hotspot based protein engineering strategy. The mutants tested against 46 know organophosphate compounds using molecular docking study. Finally, we carried out an extensive molecular docking study to predict the binding of 46 organophosphate compounds to wild-type protein and mutant organophosphorus hydrolase enzyme. At the end we are able to improve the degrading potential of organophosphorus hydrolase enzyme against organophosphate toxic compounds. This preliminary study and the outcome would be useful guide for the experimental scientist involved in the bioremediation of toxic organophosphate compounds.

Development of an Escherichia coli-based biocatalytic system for the efficient synthesis of N-acetyl-D-neuraminic acid

N-acetyl-d-neuraminic acid (Neu5Ac) is a valuable resource that has seen increasing demand in both medicine and biotechnology. Although enzymatic systems and whole-cell biocatalysts have been developed for the synthesis of Neu5Ac, low yield and productivity still hamper the use of these methods on larger scales. We report the creation of an Escherichia coli biocatalyst for the efficient synthesis of Neu5Ac using a metabolic and protein engineering strategy. Expression of the two enzymes, N-acetyl-D-glucosamine 2-epimerase (AGE) and Neu5Ac lyase (NAL), was balanced using promoter engineering. Genes encoding competing pathways and GlcNAc catabolism were deleted, and then a structure-guided process was used to identify a more efficient NAL and an AGE mutant with a higher rate of Neu5Ac synthesis. The resulting biocatalyst produced 351.8 mM Neu5Ac with a yield of 58.6% from GlcNAc. This work exemplifies the use of rational design and protein engineering to construct a complex bacterial biocatalyst that can serve as a platform for the large-scale synthesis of a useful biological material.

Conformational Transition of Key Structural Features Involved in Activation of ALK Induced by Two Neuroblastoma Mutations and ATP Binding: Insight from Accelerated Molecular Dynamics Simulations.

Deregulated kinase activity of anaplastic lymphoma kinase (ALK) has been observed to be implicated in the development of tumor progression. The activation mechanism of ALK is proposed to be similar to other receptor tyrosine kinases (RTKs), but the distinct static X-ray crystal conformation of ALK suggests its unique conformational transition. Herein, we have illustrated the dynamic conformational property of wild-type ALK as well as the kinase activation equilibrium variation induced by two neuroblastoma mutations (R1275Q and Y1278S) and ATP binding by performing enhanced sampling accelerated Molecular Dynamics (aMD) simulations. The results suggest that the wild-type ALK is mostly favored in the inactive state, whereas the mutations and ATP binding promote a clear shift toward the active-like conformation. The R1275Q mutant stabilizes the active conformation by rigidifying the αC-in conformation. The Y1278S mutant promotes activation at the expense of a π-stacking hydrophobic cluster, which plays a critical role in the stabilization of the inactive conformation of native ALK. ATP produces a more compact active site and thereby facilitates the activation of ALK. Taken together, these findings not only elucidate the diverse conformations in different ALKs but can also shed light on new strategies for protein engineering and structural-based drug design for ALK.

KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding.

The functionalization of polymer surfaces by polymer-binding peptides offers tremendous opportunities for directed immobilization of enzymes, bioactive peptides, and antigens. The application of polymer-binding peptides as adhesion promoters requires reliable and stable binding under process conditions. Molecular modes of interactions between material surfaces, peptides, and solvent are often not understood to an extent that enables (semi-) rational design of polymer-binding peptides, hindering the full exploitation of their potential. Knowledge-gaining directed evolution (KnowVolution) is an efficient protein engineering strategy that facilitates tailoring protein properties to application demands through a combination of directed evolution and computational guided protein design. A single round of KnowVolution was performed to gain molecular insights into liquid chromatography peak I peptide, 47 aa (LCI)-binding to polypropylene (PP) in the presence of the competing surfactant Triton X-100. KnowVolution yielded a total of 8 key positions (D19, S27, Y29, D31, G35, I40, E42, and D45), which govern PP-binding in the presence of Triton X-100. The recombination of two of the identified amino acid substitutions (Y29R and G35R; variant KR-2) yielded a 5.4 ± 0.5-fold stronger PP-binding peptide compared to LCI WT in the presence of Triton X-100 (1 mM). The LCI variant KR-2 shows a maximum binding capacity of 8.8 ± 0.1 pmol/cm2 on PP in the presence of Triton X-100 (up to 1 mM). The KnowVolution approach enables the development of polymer-binding peptides, which efficiently coat and functionalize PP surfaces and withstand surfactant concentrations that are commonly used, such as in household detergents.

Glu56Ser mutation improves the enzymatic activity and catalytic stability of Bacillus subtilis l-aspartate α-decarboxylase for an efficient β-alanine production

Abstract l -aspartate α-decarboxylase (ADC) is responsible for the decarboxylation of l -aspartate to form β-alanine in the bacterial biosynthetic pathway. However, the low activity and stability of ADC are the primary factors restricting β-alanine biosynthesis. In this study, to improve the ADC activity and thermostability and to attenuate the degree of its inactivation in the reaction, we expressed and characterized the Bacillus substilis ADC (BsADC) in Escherichia coli and rationally designed several site-directed mutations adjacent to its catalytic site. The Glu56Ser (E56S) mutant showed a 1.6-fold higher activity than the wild-type enzyme. This mutant retained approximately 65% activity when incubated at 70 °C for 12 h. More importantly, the E56S mutant showed an approximately 1.4-fold increased residual activity compared with the wild type during 2 h reaction at 37 °C, suggesting that the E56S mutation attenuated the mechanism-based inactivation of the enzyme. The mutant enzyme also catalyzed the β-alanine synthesis with a product yield of 215.3 g per liter culture, which is the highest reported yield to date. This work proposes a protein engineering strategy that improves the enzymatic activity and thermostability and reduces the degree of enzyme inactivation in the reaction, thereby improving the efficiency of β-alanine biosynthesis.

Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen.

Structure-based approaches to antigen design utilize insights from antibody (Ab):antigen interactions and a refined understanding of protective Ab responses to engineer novel antigens presenting epitopes with conformations relevant to eliciting or discovering protective humoral responses. For human immunodeficiency virus-1 (HIV-1), one model of protection is provided by broadly neutralizing Abs (bnAbs) against epitopes present in the closed prefusion trimeric conformation of HIV-1 envelope glycoprotein, such as the variable loops 1–2 (V1V2) apex. Here, computational design and directed evolution yielded a novel V1V2 sequence variant with potential utility for inclusion in an immunogen for eliciting bnAbs, or as an epitope probe for their detection. The computational design goal was to engineer a minimal single-chain antigen with three copies of the V1V2 loops to support maintenance of closed prefusion V1V2 trimeric conformation and presentation of bnAb epitopes. Via directed evolution of this computationally designed single-chain antigen, we isolated a V1V2 sequence variant that in monomeric form exhibited preferential recognition by quaternary-preferring and conformation-dependent mAbs. Structural context and transferability of this phenotype to V1V2 sequences from all strains of HIV-1 tested suggest a conformation-stabilizing effect. This example demonstrates the potential utility of computational design and directed evolution-based protein engineering strategies to develop minimal, conformation-stabilized epitope-specific antigens.

A key residue for the substrate affinity enhancement of a thermophilic endo-polygalacturonase revealed by computational design.

Protein engineering has been a research hotspot to improve the catalytic efficiency of industrially important enzymes. In the present study, a novel computational strategy was developed to in silico screen mutants with enhanced binding interaction between enzyme and substrate as well as catalytic efficiency. Through homology modeling and molecular dynamics (MD) simulation, four key residues related to substrate binding were identified in the endo-polygalacturonase BiPG28A from Bispora sp. MEY-1. Further analyses of the conformation, hydrogen bond interactions, and binding free energy revealed that lysine at position 129 (subsite - 2) has the strongest affinity to substrate. Biochemical and calorimetry experiments confirmed the functional role of Lys129 in substrate binding through non-covalent interactions. The common role of Lys129 was also verified in another GH28 endo-polygalacturonase. Distinguished from other protein engineering strategies involving structure resolution and construction of certain enzymes, this computational strategy represents an insightful and efficient approach to develop a "designed" enzyme with significantly enhanced binding affinity and catalytic efficiency.

Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges.

Oxidoreductases are ubiquitous enzymes that catalyze an extensive range of chemical reactions with great specificity, efficiency, and selectivity. Most oxidoreductases are nicotinamide cofactor-dependent enzymes with a strong preference for NADP or NAD. Because these coenzymes differ in stability, bioavailability and costs, the enzyme preference for a specific coenzyme is an important issue for practical applications. Different approaches for the manipulation of coenzyme specificity have been reported, with different degrees of success. Here we present various attempts for the switching of nicotinamide coenzyme preference in oxidoreductases by protein engineering. This review covers 103 enzyme engineering studies from 82 articles and evaluates the accomplishments in terms of coenzyme specificity and catalytic efficiency compared to wild type enzymes of different classes. We analyzed different protein engineering strategies and related them with the degree of success in inverting the cofactor specificity. In general, catalytic activity is compromised when coenzyme specificity is reversed, however when switching from NAD to NADP, better results are obtained. In most of the cases, rational strategies were used, predominantly with loop exchange generating the best results. In general, the tendency of removing acidic residues and incorporating basic residues is the strategy of choice when trying to change specificity from NAD to NADP, and vice versa. Computational strategies and algorithms are also covered as helpful tools to guide protein engineering strategies. This mini review aims to give a general introduction to the topic, giving an overview of tools and information to work in protein engineering for the reversal of coenzyme specificity.

Computer-assisted engineering of hyperstable fibroblast growth factor 2.

Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications.

Tm-Values and Unfolded Fraction Can Predict Aggregation Rates for Granulocyte Colony Stimulating Factor Variant Formulations but Not under Predominantly Native Conditions.

Protein engineering and formulation optimization strategies can be taken to minimize protein aggregation in the biopharmaceutical industry. Short-term stability measures such as the midpoint transition temperature (Tm) for global unfolding provide convenient surrogates for longer-term (e.g., 2-year) degradation kinetics, with which to optimize formulations on practical time-scales. While successful in some cases, their limitations have not been fully evaluated or understood. Tm values are known to correlate with chemical degradation kinetics for wild-type granulocyte colony stimulating factor (GCSF) at pH 4-5.5. However, we found previously that the Tm of an antibody Fab fragment only correlated with its rate of monomer loss at temperatures close to the Tm. Here we evaluated Tm, the fraction of unfolded protein (fT) at temperature T, and two additional short-term stability measures, for their ability to predict the kinetics of monomer and bioactivity loss of wild-type GCSF and four variants, at 37 °C, and in a wide range of formulations. The GCSF variants introduced one to three mutations, giving a range of conformational stabilities spanning 7.8 kcal mol-1. We determined the extent to which the formulation rank order differs across the variants when evaluated by each of the four short-term stability measures. All correlations decreased as the difference in average Tm between each pair of GCSF variants increased. The rank order of formulations determined by Tm was the best preserved, with R2-values >0.7. Tm-values also provided a good predictor (R2 = 0.73) of the aggregation rates, extending previous findings to include GCSF variant-formulation combinations. Further analysis revealed that GCSF aggregation rates at 37 °C were dependent on the fraction unfolded at 37 °C (fT37), but transitioned smoothly to a constant baseline rate of aggregation at fT37 < 10-3. A similar function was observed previously for A33 Fab formulated by pH, ionic strength, and temperature, without excipients. For GCSF, all combinations of variants and formulations fit onto a single curve, suggesting that even single mutations destabilized by up to 4.8 kcal mol-1, are insufficient to change significantly the baseline rate of aggregation under native conditions. The baseline rate of aggregation for GCSF under native conditions was 66-fold higher than that for A33 Fab, highlighting that they are a specific feature of each native protein structure, likely to be dependent on local surface properties and dynamics.

Keratinolytic protease: a green biocatalyst for leather industry.

Depilation/unhairing is the crucial but heavy pollution process in leather industry. Traditional inorganic sulfide treatment was the most widely used depilation technique in the past decades, which was usually detrimental to leather quality and resulted in serious environmental pollution. Using biocatalysts to substitute inorganic sulfide showed great advantages in environment protection and unhairing efficiency. Keratinolytic protease is one of the excellent biocatalysts to hydrolyze disulfide bond-rich proteins of hair and has little damage to leather. Biological treatment with keratinolytic proteases could largely reduce the quantity and toxicity of wastewater effluent from the leather industry. But low thermostability and substrate specificity or specific activity of these enzymes limited their practical application. Therefore, recent progresses on protein engineering strategies (site-directed mutagenesis, protein fusion, N/C-terminus truncation, and domain swapping) used to enhance the keratinolytic enzyme performance were presented.

Evaluation of sequence variability in HIV-1 gp41 C-peptide helix-grafted proteins

Many therapeutically-relevant protein-protein interactions (PPIs) have been reported that feature a helix and helix-binding cleft at the interface. Given this, different approaches to disrupting such PPIs have been developed. While short peptides (<15 amino acids) typically do not fold into a stable helix, researchers have reported chemical approaches to constraining helix structure. However, these approaches rely on laborious, and often expensive, chemical synthesis and purification. Our premise is that protein-based solutions that stabilize a therapeutically-relevant helix offer a number of advantages. In contrast to chemically constrained helical peptides, or minimal/miniature proteins, which must be synthesized (at great expense and labor), a protein can be expressed in a cellular system (like all current protein therapeutics). If selected properly, the protein scaffold can stabilize the therapeutically-relevant helix. We recently reported a protein engineering strategy, which we call “helix-grafted display”, and applied it to the challenge of suppressing HIV entry. We have reported helix-grafted display proteins that inhibit formation of an intramolecular PPI involving HIV gp41 C-peptide helix, and HIV gp41 N-peptide trimer, which contain C-peptide helix-binding clefts. Here, we used yeast display to screen a library of grafted C-peptide helices for N-peptide trimer recognition. Using ‘hits’ from yeast display library screening, we evaluated the effect helix mutations have on structure, expression, stability, function (target recognition), and suppression of HIV entry.

Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant targets. Biotechnol. Bioeng. 2017;114: 2348-2359. © 2017 Wiley Periodicals, Inc.

Generation and Validation of Intracellular Ubiquitin Variant Inhibitors for USP7 and USP10

Post-translational modification of the p53 signaling pathway plays an important role in cell cycle progression and stress-induced apoptosis. Indeed, a large body of work has shown that dysregulation of p53 and its E3 ligase MDM2 by the ubiquitin-proteasome system (UPS) promotes carcinogenesis and malignant transformation. Thus, drug discovery efforts have focused on the restoration of wild-type p53 activity or inactivation of oncogenic mutant p53 by targeted inhibition of UPS components, particularly key deubiquitinases (DUBs) of the ubiquitin-specific protease (USP) class. However, development of selective small-molecule USP inhibitors has been challenging, partly due to the highly conserved structural features of the catalytic sites across the class. To tackle this problem, we devised a protein engineering strategy for rational design of inhibitors for DUBs and other UPS proteins. We employed a phage-displayed ubiquitin variant (UbV) library to develop inhibitors targeting the DUBs USP7 and USP10, which are involved in regulating levels of p53 and MDM2. We were able to identify UbVs that bound USP7 or USP10 with high affinity and inhibited deubiquitination activity. We solved the crystal structure of UbV.7.2 and rationalized the molecular basis for enhanced affinity and specificity for USP7. Finally, cell death was increased significantly by UbV.7.2 expression in a colon cancer cell line that was treated with the chemotherapy drug cisplatin, demonstrating the therapeutic potential of inhibiting USP7 by this approach.

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α1A-adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three α2 adrenoceptor subtypes. Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α1 and α2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.

Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis.

Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1I158V/P168S) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1I158V/P168S as a template, affording a triple mutant, ScCR1A60T/I158V/P168S, whose thermostability (T5015, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C5015, defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of >99% and a space-time yield of 255 g liter-1 day-1 These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor.IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis.