Zhoutong Sun

Reshaping an Enzyme Binding Pocket for Enhanced and Inverted Stereoselectivity: Use of Smallest Amino Acid Alphabets in Directed Evolution

Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R,R- and S,S-selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme.

Metabolic engineering of the L-phenylalanine pathway in Escherichia coli for the production of S- or R-mandelic acid

BackgroundMandelic acid (MA), an important component in pharmaceutical syntheses, is currently produced exclusively via petrochemical processes. Growing concerns over the environment and fossil energy costs have inspired a quest to develop alternative routes to MA using renewable resources. Herein we report the first direct route to optically pure MA from glucose via genetic modification of the L-phenylalanine pathway in E. coli.ResultsThe introduction of hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis into E. coli led to a yield of 0.092 g/L S-MA. By combined deletion of competing pathways, further optimization of S-MA production was achieved, and the yield reached 0.74 g/L within 24 h. To produce R-MA, hydroxymandelate oxidase (Hmo) from Streptomyces coelicolor and D-mandelate dehydrogenase (DMD) from Rhodotorula graminis were co-expressed in an S-MA-producing strain, and the resulting strain was capable of producing 0.68 g/L R-MA. Finally, phenylpyruvate feeding experiments suggest that HmaS is a potential bottleneck to further improvement in yields.ConclusionsWe have constructed E. coli strains that successfully accomplished the production of S- and R-MA directly from glucose. Our work provides the first example of the completely fermentative production of S- and R-MA from renewable feedstock.

Whole-Cell-Catalyzed Multiple Regio- and Stereoselective Functionalizations in Cascade Reactions Enabled by Directed Evolution

Biocatalytic cascade reactions using isolated stereoselective enzymes or whole cells in one-pot processes lead to value-added chiral products in a single workup. The concept has been restricted mainly to starting materials and intermediate products that are accepted by the respective wild-type enzymes. In the present study, we exploited directed evolution as a means to create E. coli whole cells for regio- and stereoselective cascade sequences that are not possible using man-made catalysts. The approach is illustrated using P450-BM3 in combination with appropriate alcohol dehydrogenases as catalysts in either two-, three-, or four-step cascade reactions starting from cyclohexane, cyclohexanol, or cyclohexanone, respectively, leading to either (R,R)-, (S,S)-, or meso-cyclohexane-1,2-diol. The one-pot conversion of cyclohexane into (R)- or (S)-2-hydroxycyclohexanone in the absence of ADH is also described.

New Concepts for Increasing the Efficiency in Directed Evolution of Stereoselective Enzymes.

Directed evolution of stereo- and regioselective enzymes constitutes a prolific source of catalysts for asymmetric transformations in organic chemistry. In this endeavor (iterative) saturation mutagenesis at sites lining the binding pocket of enzymes has emerged as the method of choice, but uncertainties regarding the question of how to group many residues into randomization sites and how to choose optimal upward pathways persist. Two new approaches promise to beat the numbers problem effectively. One utilizes a single amino acid as building block for the randomization of a 10-residue site, the other also employs only one but possibly different amino acid at each position of a 9-residue site. The small but smart libraries provide highly enantioselective epoxide hydrolase or lipase mutants, respectively.

Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single‐ versus Double‐Code Saturation Mutagenesis

Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodological efforts, enabling comparisons between single‐code saturation mutagenesis (SCSM) and triple‐code saturation mutagenesis (TCSM); these employ either only one or three amino acids, respectively, as building blocks. In this study the comparative platform is extended by exploring the efficacy of double‐code saturation mutagenesis (DCSM), in which the reduced amino acid alphabet consists of two members, chosen according to the principles of rational design on the basis of structural information. The hydrolytic desymmetrization of cyclohexene oxide is used as the model reaction, with formation of either (R,R)‐ or (S,S)‐cyclohexane‐1,2‐diol. DCSM proves to be clearly superior to the likewise tested SCSM, affording both R,R‐ and S,S‐selective mutants. These variants are also good catalysts in reactions of further substrates. Docking computations reveal the basis of enantioselectivity.

Structural and Computational Insight into the Catalytic Mechanism of Limonene Epoxide Hydrolase Mutants in Stereoselective Transformations.

Directed evolution of limonene epoxide hydrolase (LEH), which catalyzes the hydrolytic desymmetrization reactions of cyclopentene oxide and cyclohexene oxide, results in (R,R)- and (S,S)-selective mutants. Their crystal structures combined with extensive theoretical computations shed light on the mechanistic intricacies of this widely used enzyme. From the computed activation energies of various pathways, we discover the underlying stereochemistry for favorable reactions. Surprisingly, some of the most enantioselective mutants that rapidly convert cyclohexene oxide do not catalyze the analogous transformation of the structurally similar cyclopentene oxide, as shown by additional X-ray structures of the variants harboring this slightly smaller substrate. We explain this puzzling observation on the basis of computational calculations which reveal a disrupted alignment between nucleophilic water and cyclopentene oxide due to the pronounced flexibility of the binding pocket. In contrast, in the stereoselective reactions of cyclohexene oxide, reactive conformations are easily reached. The unique combination of structural and computational data allows insight into mechanistic details of this epoxide hydrolase and provides guidance for future protein engineering in reactions of structurally different substrates.

Beating Bias in the Directed Evolution of Proteins: Combining High-Fidelity on-Chip Solid-Phase Gene Synthesis with Efficient Gene Assembly for Combinatorial Library Construction

Saturation mutagenesis (SM) constitutes a widely used technique in the directed evolution of selective enzymes as catalysts in organic chemistry and in the manipulation of metabolic paths and genomes, but the quality of the libraries is far from optimal due to the inherent amino acid bias. Herein, it is shown how this fundamental problem can be solved by applying high‐fidelity solid‐phase chemical gene synthesis on silicon chips followed by efficient gene assembly. Limonene epoxide hydrolase was chosen as the catalyst in the model desymmetrization of cyclohexene oxide with the stereoselective formation of (R,R)‐ and (S,S)‐cyclohexane‐1,2‐diol. A traditional combinatorial PCR‐based SM library, produced by simultaneous randomization at several residues by using a reduced amino acid alphabet, and the respective synthetic library were constructed and compared. Statistical analysis at the DNA level with massive sequencing demonstrates that, in the synthetic approach, 97 % of the theoretically possible DNA mutants are formed, whereas the traditional SM library contained only about 50 %. Screening at the protein level also showed the superiority of the synthetic library; many highly (R,R)‐ and (S,S)‐selective variants being discovered are not found in the traditional SM library. With the prices of synthetic genes decreasing, this approach may point the way to future directed evolution.

Biocatalysis of carboxylic acid reductases: phylogenesis, catalytic mechanism and potential applications

In recent decades, biocatalysis has attracted more and more attention, contributing significantly to synthetic organic chemistry and biotechnology and thereby advancing the development of greener and more sustainable synthetic processes. As a versatile enzyme, carboxylic acid reductases (CARs) are widely distributed in bacteria, fungi and plants. Recent seminal studies focusing on CARs have demonstrated that they can efficiently and selectively reduce a broad range of carboxylic acid substrates to their corresponding aldehyde moieties, which constitutes a very difficult transformation using chemical methods. However, the full potential of CARs is yet to be explored. In this review, we summarize the progress of CARs from an evolutionary point of view, including the convergence/divergence between CARs and knowledge of the catalytic mechanism. Particular emphasis is placed on recent advances in practical applications as well as future developments.

Methodology Development in Directed Evolution: Exploring Options when Applying Triple-Code Saturation Mutagenesis

Directed evolution of stereo‐ or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building “small but smart” mutant libraries have been developed. Herein, we compared two different strategies regarding the application of triple‐code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino‐acid alphabets. By using the synthetically difficult‐to‐reduce prochiral ketone tetrahydrofuran‐3‐one as a substrate, highly R‐ and S‐selective variants were obtained (92–99 % ee) with minimal screening. The origin of stereoselectivity was provided by molecular dynamics analyses, which is discussed in terms of the Bürgi–Dunitz trajectory.

Solid-Phase Gene Synthesis for Mutant Library Construction: The Future of Directed Evolution?

Directed evolution of stereo‐ and regioselective enzymes as catalysts in organic chemistry and biotechnology constitutes a complementary alternative to selective transition‐metal catalysts and organocatalysts. Saturation mutagenesis at sites lining the binding pocket has emerged as a key method in this endeavor, but it suffers from amino acid bias, which reduces the quality of the library at the DNA level and, thus, at the protein level. Chemical solid‐phase gene synthesis for library construction offers a solution to this fundamental problem, and the Sloning and Twist platforms are two possible options. This concept article analyzes these approaches and compares them to traditional PCR‐based saturation mutagenesis; the superior commercial Twist technique shows almost no bias.