Directed evolution of mandelate racemase by a novel high-throughput screening method

Optically pure methyl (R)-o-chloromandelate and (R)-acetyl-o-mandelic acid are key intermediates for the synthesis of (S)-clopidogrel, which could be prepared with 100 % theoretical yield by sequential hydrolysis and racemization. At the moment, efficient sequential hydrolysis and racemization are hindered by the low catalytic activity of mandelate racemase (MR) toward (S)-o-chloromandelic acid ((S)-2-CMA). In the present work, we proposed to improve the catalytic performance of MR toward (S)-2-CMA by directed evolution and developed an enantioselective oxidation system for high-throughput screening (HTS) of MR libraries. Based on this HTS method, a triple mutant V22I/V29I/Y54F (MRDE1) with 3.5-fold greater relative activity as compared to the native MR was obtained. Kinetic analysis indicated that the enhanced catalytic efficiency mainly arose from the elevated k _cat. Further insight into the source of improved catalytic activity was gained by molecular simulations, finding that substrate binding and product release were possibly made easier by decreased steric bulk and increased hydrophobicity of substrate binding sites. In addition, the substrate (S)-2-CMA in the enzyme-substrate complex of MRDE1 seemed to have a lower binding free energy comparing with the complex of wild-type MR. The HTS method developed in this work and the successful directed evolution of MR based on this method provide an example for racemase engineering and may inspire directed evolution of other racemases toward enhanced catalytic performance on non-natural substrates.

     

Free-Energy Perturbation Simulation on Transition States and Redesign of Butyrylcholinesterase

It is recognized that an ideal anti-cocaine treatment is to accelerate cocaine metabolism by producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., butyrylcholinesterase (BChE)-catalyzed hydrolysis of cocaine. BChE mutants with a higher catalytic activity against (-)-cocaine are highly desired for use as an exogenous enzyme in humans. To develop a rational design for high-activity mutants, we carried out free-energy perturbation (FEP) simulations on various mutations of the transition-state structures in addition to the corresponding free-enzyme structures by using an extended FEP procedure. The FEP simulations on the mutations of both the free-enzyme and transition-state structures allowed us to calculate the mutation-caused shift of the free-energy change from the free enzyme (BChE) to the transition state, and thus to theoretically predict the mutation-caused shift of the catalytic efficiency (k_cat/K_M). The computational predictions are supported by the kinetic data obtained from the wet experiments, demonstrating that the FEP-based computational design approach is promising for rational design of high-activity mutants of an enzyme. One of the BChE mutants designed and discovered in this study has an ∼1800-fold improved catalytic efficiency against (-)-cocaine compared to wild-type BChE. The high-activity mutant may be therapeutically valuable.     

Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and β-substituted alcohols. The wild-type (WT) enzyme displays a high R-enantiopreference toward most aromatic substrates, whereas no S-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement in R-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from an E_R of 65 [WT] to an E_S of 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of its R-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.     

Modeling of Transition State by Molecular Dynamics. Prediction of Catalytic Efficiency of the Mutants of Mandelate Racemase

Abstract

Modeling of transition state by molecular dynamic method often requires modification of the force field parameters to describe energy profile accurately. In this work, we avoided the modification by modeling a series of mutants at binding-related site. In predicting the catalytic efficiency (k _cat /K _m ) of the mutants of mandelate racemase (MR), the prediction performance of three energy subsets was investigated. It was indicated that the interaction-energy subset exhibited better prediction performance than whole-system subset and binding-site subset in both quantity and trend. When prediction error (PE) criterion was equal to 5%, 10 out of 12 samples were predicted correctly within interaction-energy subset, which demonstrated a great application potential of this method in prediction of enzyme catalytic efficiency and enzyme rational design.     

Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity

d-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous d-amino acids, making it an attractive biocatalyst for the production of enantiopure d-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity toward non-native substrates are desired. Here, we report the development of a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of d-amino acid oxidase (DAAO). In this assay, the d-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and o-dianisidine. Using this assay, we measured apparent K_M and k_cat values for DAAT and identified mutants displaying altered substrate specificity via the screening of cell lysates in 96-well plates. The DAAO coupled assay is sensitive in that it allowed the detection of a DAAT mutant displaying an approximately 2000-fold decrease in k_cat/K_M relative to wild type. In addition, the DAAO assay enabled the identification of two DAAT mutants (V33Y and V33G) that are more efficient than wild type at transaminating the non-native acceptor phenylpyruvate. We expect that this assay will be useful for the engineering of additional mutants displaying increased activity toward non-native substrates.     

Improved enantioselective conversion of styrene epoxides and meso-epoxides through epoxide hydrolases with a mutated nucleophile-flanking residue

In epoxide hydrolase from Agrobacterium radiobacter (EchA), phenylalanine 108 flanks the nucleophilic aspartate and forms part of the substrate-binding pocket. The influence of mutations at this position on the activity and enantioselectivity of the enzyme was investigated. Screening for improved enantioselectivity towards para-nitrophenyl glycidyl ether (pNPGE) using spectrophotometric progress curve analysis yielded five different mutants with 3- to 7-fold improved enantioselectivity. The increase in enantioselectivity was in most cases the result of an enhanced catalytic efficiency toward the preferred enantiomer. Several mutations at position F108 resulted in a higher activity toward cis-disubstituted meso-epoxides, which were converted to a single product enantiomer. Mutant F108C converted cis-2,3-epoxybutane to (2R,3R)-2,3-butanediol of >99% ee with a 7-fold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1R,2R)-1,2-cyclohexanediol of >99% ee with a more than 150-fold higher activity than wild-type enzyme. It is concluded that single amino acid substitutions in the active site of epoxide hydrolase can result in enzyme variants with catalytic properties that are suitable for preparative scale production of (S)-epoxides and chiral vicinal diols in high yield and with excellent ee.     

Directed Evolution of Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Enhancement of Penicillin G Expansion

The deacetoxycephalosporin C synthase from Streptomyces clavuligerus was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the k_cat/K_m ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a k_cat/K_m ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering.     

Mutations in two amino acids in phyI1s from <Emphasis Type="Italic">Aspergillus niger</Emphasis> 113 improve its phytase activity

We applied in vitro mutagenesis and colony screening, using the wild type phyI1s gene from Aspergillus niger 113 as the template, and obtained two mutant phyI1s (gene products) after one round of screening. The two mutants had mutations at two nucleic acid sites, resulting in changes in two amino acids: K41E, E121F. None of the amino acid substitutions in the two mutants was in a position reported to be important for catalysis or substrate binding. Kinetic analysis of the phytase activity of the two mutants indicated that the substitutions gave rise to 2.5- and 3.1-fold increased specific activity, and a 1.78- and 3.24-fold reduced affinity for sodium phytate. In addition, the overall catalytic efficiency (k _cat/K _m) of the two mutants was changed by 0.52-fold and 0.68-fold compared to that of the wild type. Such mutants will be instrumental for the structure–function study of the enzyme and for industrial application.     

Directed evolution of poly[(R)-3-hydroxybutyrate] depolymerase using cell surface display system: functional importance of asparagine at position 285

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZ_RpiT1) consists of three functional domains to effectively degrade solid PHB materials, and its catalytic domain catalyzes the ester bond cleavage of the substrate. We performed the directed evolution of PhaZ_RpiT1 targeted at the catalytic domain in combination with the cell surface display method to effectively screen for mutants with improved p-nitrophenyl butyrate (pNPC4) activity. Mutated PhaZ_RpiT1 genes generated by error-prone PCR were fused to the oprI gene to display them as fusion proteins on Escherichia coli cell surface. Some cells displaying the mutant enzymes showed a two- to fourfold increase in pNPC4 hydrolysis activity relative to cells displaying wild-type enzyme. These mutant genes were recombined by a staggered extension process and the recombined enzymes were displayed to result in a five- to eightfold higher pNPC4 hydrolysis activity than the wild type. To further evaluate the mutation effects, unfused and undisplayed enzymes were prepared and applied to the hydrolysis of p-nitrophenyl esters having different chain lengths (pNPCn; n?=?2–6) and PHB degradation. One specific second-generation mutant showed an approximately tenfold increase in maximum rate for pNPC3 hydrolysis, although its PHB degradation efficiency at 1 μg/mL of enzyme concentration was approximately 3.5-fold lower than that of the wild type. Gene analysis showed that N285D or N285Y mutations were found in six of the seven improved second-generation mutants, indicating that Asn285 probably participates in the regulation of substrate recognition and may be more favorable for PHB degradation process than other amino acid residues.     

Engineering Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Optimal Ring Expansion Activity toward Penicillin G

The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k_cat/K_m ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k_cat/K_m values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k_cat/K_m ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.     

Improving catalytic efficiency of endo-β-1, 4-xylanase from Geobacillus stearothermophilus by directed evolution and H179 saturation mutagenesis

Endo-β-1, 4-xylanase was cloned from Geobacillus stearothermophilus 1A05583 by PCR. Enzymes with improved catalytic efficiency were obtained using error-prone PCR and a 96-well plate high-throughout screening system. Two variants 1-B8 and 2-H6 were screened from the mutant library containing 9000 colonies, which, when compared with the wild-type enzyme increased the catalytic efficiency (k_cat/K_m) by 25% and 89%, respectively, acting on beechwood xylan. By sequencing 1-B8 and 2-H6, an identical mutation point H179Y was detected and found to overlap in the active site cleft. Following the introduction of the remaining 19 amino acids into position 179 by site-saturation mutagenesis, the catalytic efficiency of H179F was found to be 3.46-fold that of the wild-type. When Whistidine was substituted by tryptophan, arginine, methionine or proline, the enzyme lost activity. Therefore, the position 179 site may play an important role in regulating the catalytic efficiency.     

Structure-based engineering of alkaline α-amylase from alkaliphilic Alkalimonas amylolytica for improved thermostability

This study aimed to improve the thermostability of alkaline α-amylase from Alkalimonas amylolytica through structure-based rational design and systems engineering of its catalytic domain. Separate engineering strategies were used to increase alkaline α-amylase thermostability: (1) replace histidine residues with leucine to stabilize the least similar region in domain B, (2) change residues (glycine, proline, and glutamine) to stabilize the highly conserved α-helices in domain A, and (3) decrease the free energy of folding predicted by the PoPMuSiC program to stabilize the overall protein structure. A total of 15 single-site mutants were obtained, and four mutants — H209L, Q226V, N302W, and P477V — showed enhanced thermostability. Combinational mutations were subsequently introduced, and the best mutant was triple mutant H209L/Q226V/P477V. Its half-life at 60 °C was 3.8-fold of that of the wild type and displayed a 3.2 °C increase in melting temperature compared with that of the wild type. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50 °C to 55 °C, the optimum pH shifted from 9.5 to 10.0, the stable pH range expanded from 7.0–11.0 to 6.0–12.0, the specific activity increased by 24 %, and the catalytic efficiency (k _cat/K _m) increased from 1.8×10⁴ to 3.5?×?10⁴ l/(g min). Finally, the mechanisms responsible for the increased thermostability were analyzed through comparative analysis of structure models. The structure-based rational design and systems engineering strategies in this study may also improve the thermostability of other industrial enzymes.     

Modeling of transition state by molecular dynamics. Prediction of catalytic efficiency of the mutants of mandelate racemase

Modeling of transition state by molecular dynamic method often requires modification of the force field parameters to describe energy profile accurately. In this work, we avoided the modification by modeling a series of mutants at binding-related site. In predicting the catalytic efficiency (k(cat)/K(m)) of the mutants of mandelate racemase (MR), the prediction performance of three energy subsets was investigated. It was indicated that the interaction-energy subset exhibited better prediction performance than whole-system subset and binding-site subset in both quantity and trend. When prediction error (PE) criterion was equal to 5%, 10 out of 12 samples were predicted correctly within interaction-energy subset, which demonstrated a great application potential of this method in prediction of enzyme catalytic efficiency and enzyme rational design.     

Changes in the catalytic properties and substrate specificity of Bacillus sp. US149 maltogenic amylase by mutagenesis of residue 46

Maltogenic amylase from Bacillus sp. US149 (MAUS149) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of residue Asp46 in the specificity and catalytic properties of MAUS149 by using site-directed mutagenesis. Three mutated enzymes (D46V, D46G and D46N) were constructed and studied. The three mutants were found to be similar to the wild-type MAUS149 regarding thermoactivity, thermostability and pH profile. Nevertheless, the kinetic parameters for all the substrates of the mutant enzymes D46V and D46G were altered enormously as compared with those of the wild type. Indeed, the K _m values of MAUS149/D46G for all substrates were strongly increased. Nevertheless, the affinity and catalytic efficiency of MAUS149/D46V toward β-CD were increased fivefold as compared with those of MAUS149. Molecular modelling suggests that residue D46 forms a salt bridge with residue K282. This bond would maintain the arrangement of side chains of residues Y45 and W47 in a particular orientation that promotes access to the catalytic site and maintains the substrate therein. Hence, any replacement with uncharged amino acids influenced the flexibility of the gate wall at the substrate binding cleft resulting in changes in substrate selectivity.     

Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis

The serine alkaline protease, SAPB, from Bacillus pumilus CBS is characterized by its high thermoactivity, pH stability and high catalytic efficiency (k_cat/K_m) as well as its excellent stability and compatibility with an alkaline environment under harsh washing conditions. Based on sequence alignments and homology-modeling studies, the present study identified five amino acids Leu31, Thr33, Asn99, Phe159 and Gly182 being putatively important for the enzymatic behaviour of SAPB. To corroborate the role of these residues, 12 mutants were constructed by site-directed mutagenesis and then purified and characterized. The findings demonstrate that the single mutants F159T, F159S and G182S and combined double substitutions were implicated in the decrease of the optimum pH and temperature to 8.0–9.0 and 50 °C, respectively, and that mutant F159T/S clearly affected substrate affinity and catalytic efficiency. With regards to the single L31I, T33S and N99Y and combined double and triple mutations, the N99Y mutation strongly improved the half-life times at 50 °C and 60 °C to 660 and 295 min from of 220 and 80 min for the wild-type enzyme, respectively. More interestingly, this mutation also shifted the optimum temperature from 65 °C to 75 °C and caused a prominent 31-fold increase in k_cat/K_m with N-succinyl-l-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF). The L31I and T33S mutants were observed to improve mainly the optimum pH from 11.0 to 11.5 and from 11.0 to 12.0, respectively. Kinetic studies of double and triple mutants showed that the cumulative effect of polar uncharged substitutions had a synergistic effect on the P1 position preference using synthetic peptide substrates, which confirms the implication of these amino acids in substrate recognition and catalytic efficiency.     

Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates

Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.     

Directed Evolution and Structural Analysis of NADPH-Dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) Reductase from Ralstonia eutropha Reveals Two Mutations Responsible for Enhanced Kinetics

NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited k_cat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.     

Engineering of glycerol dehydrogenase for improved activity towards 1, 3-butanediol

The objective of this study was to use protein engineering techniques to enhance the catalytic activity of glycerol dehydrogenase (GlyDH) on racemic 1, 3-butanediol (1, 3-BDO) for the bioproduction of the important pharmaceutical intermediate 4-hydroxy-2-butanone. Three GlyDH genes (gldA) from Escherichia coli K-12, Salmonella enterica, and Klebsiella pneumoniae MGH78578 were shuffled to generate a random mutagenesis library. The nitroblue tetrazolium/phenazine methosulfate high throughput screening protocol was used to select four chimeric enzymes with up to a 2.6-fold improved activity towards 1, 3-BDO. A rational design method was also employed to further improve the enzyme activity after DNA shuffling. Based on the homology model of GlyDH (Escherichia coli), Asp121 was predicted to influence 1, 3-BDO binding and replaced with Ala by site-directed mutagenesis. Combination of the mutations from both DNA shuffling and rational design produced the best mutant with a V _max value of 126.6 U/mg, a 26-fold activity increase compared with that of the wild type GlyDH from E. coli.     

Engineering of choline oxidase from Arthrobacter nicotianae for potential use as biological bleach in detergents

In order to engineer the choline oxidase from Arthrobacter nicotianae (An_CodA) for the potential application as biological bleach in detergents, the specific activity of the enzyme toward the synthetic substrate tris-(2-hydroxyethyl)-methylammonium methylsulfate (MTEA) was improved by methods of directed evolution and rational design. The best mutants (up to 520% wt-activity with MTEA) revealed mutations in the FAD- (A21V, G62D, I69V) and substrate-binding site (S348L, V349L, F351Y). In a separate screening of a library comprising of randomly mutagenised An_CodA, with the natural substrate choline, four mutations were identified, which were further combined in one clone. The constructed clone showed improved activity towards both substrates, MTEA and choline. Mapping these mutation sites onto the structural model of An_CodA revealed that Phe351 is positioned right in the active site of An_CodA and very likely interacts with the bound substrate. Ala21 is part of an α-helix which interacts with the diphosphate moiety of the flavin cofactor and might influence the activity and specificity of the enzyme.     

Engineering an SspB-mediated degron for novel controllable protein degradation

Elegant controllable protein degradation tools have great applications in metabolic engineering and synthetic biology designs. SspB-mediated ClpXP proteolysis system is well characterized, and SspB acts as an adaptor tethering ssrA-tagged substrates to the ClpXP protease. This degron was applied in metabolism optimization, but the efficiency was barely satisfactory. Limited high-quality tools are available for controllable protein degradation. By coupling structure-guided modeling and directed evolution, we establish state-of-the-art high-throughput screening strategies for engineering both degradation efficiency and SspB-ssrA binding specificity of this degron. The reliability of our approach is confirmed by functional validation of both SspB and ssrA mutants using fluorescence assays and metabolic engineering of itaconic acid or ferulic acid biosynthesis. Isothermal titration calorimetry analysis and molecular modeling revealed that an appropriate instead of excessively strong interaction between SspB and ssrA benefited degradation efficiency. Mutated SspB-ssrA pairs with 7–22-fold higher binding K_D than the wild-type pair led to higher degradation efficiency, revealing the advantage of directed evolution over rational design in degradation efficiency optimization. Furthermore, an artificial SspB-ssrA pair exhibiting low crosstalk of interactions with the wild-type SspB-ssrA pair was also developed. Efforts in this study have demonstrated the plasticity of SspB-ssrA binding pocket for designing high-quality controllable protein degradation tools. The obtained mutated degrons enriched the tool box of metabolic engineering designs.