Beneficial Substitutions Research Articles

Enhancing the thermal stability of ketoreductase ChKRED12 using the FireProt web server

Ketoreductase ChKRED12 catalyzes the asymmetric bioreduction of ethyl 3-oxo-3-(2-thienyl) propanoate to produce the key chiral intermediate of duloxetine. To improve the robustness of the enzyme, we used the FireProt web server to predict potential thermostabilizing amino acid substitutions, and we experimentally confirmed 4 beneficial substitutions (S79P, L128M, V162I and G163A). These substitutions were combined to form a quadruple mutant (M7234), which displayed substantially enhanced thermostability with half-life of inactivation increased to 58 h at 45 °C, approximately 21-fold of that of the wild-type. Moreover, the temperature optimum increased to 45 °C, which is 5 °C higher than that of the wild-type. M7234 was able to catalyze the complete conversion of 100 g/L substrate within 8 h and produce (S)-3-hydroxy-3-(2-thienyl) propanoate at >99% ee, achieving a space-time yield of 289 g/L/d after column chromatography.

Structure-based design of prefusion-stabilized SARS-CoV-2 spikes.

The COVID-19 pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. Here, we characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting ~10-fold higher expression than its parental construct and the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.

Fluorescence-based high-throughput screening system for R-ω-transaminase engineering and its substrate scope extension.

ω-Transaminase (ω-TA) is an attractive alternative to metal catalysts for the stereoselective amination of prochiral ketones. The narrow substrate scope of an R-ω-transaminase from Mycobacterium vanbaalenii (MvTA) limits its application in R-amine synthesis. A fluorescence-based TA activity screening system was developed to extend its substrate scope. The reactions were conducted in microtiter plates (MTPs) and displayed low background interference, high sensitivity (μM magnitude), and a wide dynamic range (ɀ-factor > 0.9). A KnowVolution campaign was performed on this enzyme, and screening ~ 8000 clones with this fluorescence-based screening system resulted in two beneficial substitutions (G68Y and F129A) and three improved variants (M3, M4, and M5). The best variant, MvTA M5 (WT+G68Y+F129A), achieved the highest catalytic efficiency (toward fluorogenic substrate NMA) which was 3.2-fold higher than that of the WT enzyme. MvTA M5 exhibited significantly enhanced activity toward six different prochiral ketones with e.e. > 99% (R). The specific activity of MvTA M5 was more than 100 times higher than that of the WT enzyme toward acetonaphthone (M5: 8.1U/mg, WT: ~ 0.07U/mg), and it showed the highest activity on acetonaphthone, p-ethylacetophenone, and phenylacetone.

Computer-Assisted Recombination (CompassR) Teaches us How to Recombine Beneficial Substitutions from Directed Evolution Campaigns.

A main remaining challenge in protein engineering is how to recombine beneficial substitutions. Systematic recombination studies show that poorly performing variants are usually obtained after recombination of 3 to 4 beneficial substitutions. This limits researchers in exploiting nature's potential in generating better enzymes. The Computer‐assisted Recombination (CompassR) strategy provides a selection guide for beneficial substitutions that can be recombined to gradually improve enzyme performance by analysis of the relative free energy of folding (ΔΔG fold). The performance of CompassR was evaluated by analysis of 84 recombinants located on 13 positions of Bacillus subtilis lipase A. The finally obtained variant F17S/V54K/D64N/D91E had a 2.7‐fold improved specific activity in 18.3 % (v/v) 1‐butyl‐3‐methylimidazolium chloride ([BMIM][Cl]). In essence, the deducted CompassR rule allows recombination of beneficial substitutions in an iterative manner and empowers researchers to generate better enzymes in a time‐efficient manner.

Cover Feature: Computer‐Assisted Recombination (CompassR) Teaches us How to Recombine Beneficial Substitutions from Directed Evolution Campaigns (Chem. Eur. J. 3/2020)

Cover Feature: Computer‐Assisted Recombination (CompassR) Teaches us How to Recombine Beneficial Substitutions from Directed Evolution Campaigns (Chem. Eur. J. 3/2020)

Enhancing Catalytic Efficiency of an Actinoplanes utahensis Echinocandin B Deacylase through Random Mutagenesis and Site-Directed Mutagenesis.

Echinocandin B deacylase (EBDA), from Actinoplanes utahensis ZJB-08196, is capable of cleaving the linoleoyl group from echinocandin B (ECB), forming the echinocandin B nucleus (ECBN), which is a key precursor of semisynthetic antifungal antibiotics. In the present study, molecular evolution of AuEBDA by random mutagenesis combined with site-directed mutagenesis (SDM) and screening was performed. Random mutagenesis on the wild-type (WT) AuEBDA generated two beneficial substitutions of G287Q, R527V. The "best" variant AuEBDA-G287Q/R527V was obtained by combining G287Q with R527V through SDM, which was most active at 35 °C, pH 7.5, with Km and vmax values of 0.68 mM and 395.26 U/mg, respectively. Mutation of G287Q/R527V markedly increased the catalytic efficiency kcat/Km by 290% compared with the WT-AuEBDA.

Direct coupling analysis improves the identification of beneficial amino acid mutations for the functional thermostabilization of a delicate decarboxylase.

The optimization of enzyme properties for specific reaction conditions enables their tailored use in biotechnology. Predictions using established computer-based methods, however, remain challenging, especially regarding physical parameters such as thermostability without concurrent loss of activity. Employing established computational methods such as energy calculations using FoldX can lead to the identification of beneficial single amino acid substitutions for the thermostabilization of enzymes. However, these methods require a three-dimensional (3D)-structure of the enzyme. In contrast, coevolutionary analysis is a computational method, which is solely based on sequence data. To enable a comparison, we employed coevolutionary analysis together with structure-based approaches to identify mutations, which stabilize an enzyme while retaining its activity. As an example, we used the delicate dimeric, thiamine pyrophosphate dependent enzyme ketoisovalerate decarboxylase (Kivd) and experimentally determined its stability represented by a T50 value indicating the temperature where 50% of enzymatic activity remained after incubation for 10 min. Coevolutionary analysis suggested 12 beneficial mutations, which were not identified by previously established methods, out of which four mutations led to a functional Kivd with an increased T50 value of up to 3.9°C.

Quantifying maladaptation during the evolution of sexual dimorphism.

Females and males have distinct trait optima, resulting in selection for sexual dimorphism. However, most traits have strong cross-sex genetic correlations, which constrain evolutionary divergence between the sexes and lead to protracted periods of maladaptation during the evolution of sexual dimorphism. While such constraints are thought to be costly in terms of individual and population fitness, it remains unclear how severe such costs are likely to be. Building upon classical models for the 'cost of selection' in changing environments (sensu Haldane), we derived a theoretical expression for the analogous cost of evolving sexual dimorphism; this cost is a simple function of genetic (co)variances of female and male traits and sex differences in trait optima. We then conducted a comprehensive literature search, compiled quantitative genetic data from a diverse set of traits and populations, and used them to quantify costs of sexual dimorphism in the light of our model. For roughly 90% of traits, costs of sexual dimorphism appear to be modest, and comparable to the costs of fixing one or a few beneficial substitutions. For the remaining traits (approx. 10%), sexual dimorphism appears to carry a substantial cost-potentially orders of magnitude greater than costs of selection during adaptation to environmental changes.

The Effect of the Aquatic Extract of Stevia on the MDA Level and Catalase Activity in the Testicular Tissue of Streptozotocin-Nicotinamide-Induced Diabetic Rats

Background: It is believed that diabetes has undesirable impacts on the function of the reproductive system in men like decreasing plasma testosterone levels and sperm count and making apoptosis in the testicular germ cells. There are some chemical drugs for improving the complications, but it has been proven that chemicals have many side effects in the patients. Hence, herbal medicines due to anti-oxidant properties, less cost, and fewer side effects can be beneficial substitutions for chemical drugs. Nowadays, Stevia rebaudiana Bertoni as a sweet plant has shown to be effective in ameliorating diabetic problems. This experimental study investigated the effects of stevia on suppressing oxidative stress in Streptozotocin-Nicotinamide (STZ-NA)-induced diabetic rats’ testes. Methods: In the present study, adult Wistar rats were separated into three diabetic groups and two sham groups each containing 10 rats: 1. normal group receiving 1 mL of water; 2. diabetic control group receiving 1 mL of water; 3. and 4. diabetic groups treated with the aquatic extracts of stevia (400 mg/kg) and metformin (500 mg/kg), respectively (treated four weeks after confirming diabetes for 14 days); and 5. healthy group treated with the aquatic extract of stevia (400 mg/kg). Finally, after six weeks, the rats were anesthetized, blood samples were collected from the heart for evaluating serum glucose, and testis tissues were separated for measuring Malondialdehyde (MDA) level and Catalase (CAT) activity. Statistical analysis was carried out using one-way analysis of variance (ANOVA), followed by Tukey post hoc multiple-comparison test and data were presented as the mean±standard error of the mean (SEM). Results: This study documented that stevia significantly declined serum glucose (P < 0.001) and MDA level (P = 0.008) and increased catalase activity (P = 0.005) in the treated groups (metformin and stevia) compared to the diabetic groups. Conclusions: The upshot of all, stevia, due to its antioxidant nature, could improve oxidative stress in the testis tissue of diabetic rats.

How to engineer glucose oxidase for mediated electron transfer.

Glucose oxidase (GOx) is of high industrial interest for glucose sensing because of its high β-d-glucose specificity. The efficient and specific electrochemical communication between the redox center and electrodes is crucial to ensure accurate glucose determination. The efficiency of the electron transfer rates (ETR) with GOx, together with quinone diamine based mediators, is low and differs even among mediator derivatives. To design optimized enzyme-mediator couples and to describe a mediator binding model, a joint experimental and computational study was performed based on an oxygen-independent GOx variant V7 and two quinone diimine based electron mediators (QDM-1 and QDM-2), which differ in polarity and size, and ferrocenemethanol (FM). A site saturation library at position 414 was screened with all three mediators and yielded four beneficial substitutions Tyr, Met, Leu, and Val. The variants showed increased mediator activity for the more polar QDM-2 with a simultaneously decreased activity for the less polar and smaller QDM-1 and for FM. The variant GOx V7-I414Y exhibited the biggest change for the quinone diimine derivatives compared with V7 (QDM-1: 55.9 U/mg V7, 33.2 U/mg V7-I414Y; QDM-2: 2.7 U/mg V7, 12.9 U/mg V7-I414Y). TheoreticalETR calculated based on the Marcus theory were in good agreement with the experimental results. Molecular docking studies revealed a preferable binding of the two QD mediators directly in the active site, 3.5 Å away from the N5 atom of the flavin adenine dinucleotide (FAD) and in direct vicinity to position 414. In summary, position 414 in the active site was identified to modulate the electron shuttling from the FAD of the GOx to small water-soluble mediators dependent on the polarity and size of residue 414 and on the polarity and size of the mediator. The presented mediator binding model offers a promising possibility for the design of optimized enzyme-mediator couples.

Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High-Molecular-Weight Hyaluronic Acid.

Hyaluronic acid (HA), with diverse cosmetic and medical applications, is the natural glycosaminoglycan product of HA synthases. Although process and/or metabolic engineering are used for industrial HA production, the potential of protein engineering has barely been realised. Herein, knowledge-gaining directed evolution (KnowVolution) was employed to generate an HA synthase variant from Pasteurella multocida (pmHAS) with improved chain-length specificity and a twofold increase in mass-based turnover number. Seven improved pmHAS variants out of 1392 generated by error-prone PCR were identified; eight prospective positions were saturated and the most beneficial amino acid substitutions were recombined. After one round of KnowVolution, the longest HA polymer (<4.7 MDa), through an engineered pmHAS variant in a cell-free system, was synthesised. Computational studies showed that substitutions from the best variant (T40L, V59M and T104A) are distant from the glycosyltransferase sites and increase the flexibility of the N-terminal region of pmHAS. Taken together, these findings suggest that the N terminus may be involved in HA synthesis and demonstrate the potential of protein engineering towards improved HA synthase activity.

Neutral Theory and the Somatic Evolution of Cancer.

Kimura's neutral theory argued that positive selection was not responsible for an appreciable fraction of molecular substitutions. Correspondingly, quantitative analysis reveals that the vast majority of substitutions in cancer genomes are not detectably under selection. Insights from the somatic evolution of cancer reveal that beneficial substitutions in cancer constitute a small but important fraction of the molecular variants. The molecular evolution of cancer community will benefit by incorporating the neutral theory of molecular evolution into their understanding and analysis of cancer evolution-and accepting the use of tractable, predictive models, even when there is some evidence that they are not perfect.

Molecular engineering of the salicylate-inducible transcription factor Sal7AR for orthogonal and high gene expression in Escherichia coli.

I have previously identified a metagenomic fragment (~4 kb) containing the salicylate (2-hydroxybenzoate)-responsive transcriptional regulator Sal7AR. Taking advantage of the inert nature of salicylate to common genetic switches used in Escherichia coli, here I developed a salicylate-inducible high expression system in E. coli. I first applied a deletion analysis to the metagenomic fragment to identify the core region (~1 kb) necessary for the salicylate-dependent expression. Sal7AR was subjected to an error-prone PCR, and a library was screened for an enhanced expression of a reporter green fluorescent protein (GFP) gene in the presence of 1 mM salicylate, where virtually no growth inhibition was observed. Three beneficial amino acid substitutions were identified (N282K, Q292R, and V295G), each of which improved the expression of GFP relative to the wildtype by several-fold. The three sites were then completely randomized by saturation mutagenesis either individually or combinatorially to identify three variants carrying a single point mutation, N282L, V295F, or V295S; no further improvements were observed by combining these mutations. Salicylate-dependent expression of these mutants was highly repressed in its absence and escalated in response to ~10 μM salicylate, and gradually increased up to 1 mM salicylate; the induction rate was approximately 15 times greater than that achieved with a lactose promoter. Orthogonality to the lactose-based expression system was also confirmed. This salicylate-based expression system should thus be advantageously used for high-level production of recombinant proteins in combination with common lactose-dependent induction systems.

Loop engineering reveals the importance of active-site-decorating loops and gating residue in substrate affinity modulation of arginine deiminase (an anti-tumor enzyme)

Protein engineering of enzyme loop regions is an effective strategy to improve enzymatic properties. Previous studies that aimed to boost the activity of PpADI (an arginine deiminase from Pseudomonas plecoglossicida) under physiological conditions yielded several significantly improved variants that harbor substitutions predominantly located in active-site-decorating loops. A multi-site saturation mutagenesis at four positions in loop 1 (37, 38, 42, and 43) and three positions in loop 4 (402, 403, and 404) was performed to elucidate the importance of these loops in modulating the substrate affinity of PpADI. The identified “best” variant (M6-L1-4) showed a decreased S0.5 (‘KM’) of 0.48 mM compared with the parent M6 (0.81 mM). Subsequently, a rational design to recombine beneficial substitutions within loops 1 and 4 yielded variant L6 with a substantially decreased S0.5 value (0.17 mM). A comprehensive simulation analysis resulted in a conclusion that high loop flexibility (especially the gating residue Arg400) is beneficial for substrate affinity due to less efficient blocking of the active site.

Exploring the full natural diversity of single amino acid exchange reveals that 40\u201360% of BSLA positions improve organic solvents resistance

ObjectivesProtein engineering has been employed to successfully improve organic solvent resistance of enzymes. Exploration of nature’s full potential (how many beneficial positions/beneficial substitutions of the target enzyme) to improve organic solvent resistance of enzymes by a systematic study was performed.ResultsWe report the results of screening the previously generated BSLA (Bacillus subtilis lipase A)-SSM (site saturation mutagenesis) library (covering the full natural diversity of BSLA with one amino acid exchange) in presence of three cosolvents. The potential of single amino acid substitution that nature offers to improve the cosolvent resistance of BSLA was determined by analyzing the number of beneficial positions/substitutions, accessibility and chemical compositions.ConclusionLessons learned from analysis of BSLA-SSM library are: (1) 41–59% of BSLA positions with in total 4–10% of all possible substitutions improve the cosolvent resistance against TFE, DOx, and DMSO; (2) charged substitutions are preferred to improve DOx and TFE resistance whereas polar ones are preferred for DMSO; (3) charged substitutions on the surface predominantly improved resistance while polar ones were preferred in buried “regions”. (4) Interestingly, 58–93% of beneficial substitutions led to chemically different amino acids.

Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis

BackgroundBacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels.ResultsIn total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously.ConclusionsSingle amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an optimization target for successful protein production. Our results further suggest that variants with improved properties might be identified much faster and easier if mutagenesis is prioritized towards elements that contribute to enzymatic activity or structural integrity.

Directed evolution to improve the catalytic efficiency of urate oxidase from Bacillus subtilis.

Urate oxidase is a key enzyme in purine metabolism and catalyzes the oxidation of uric acid to allantoin. It is used to treat hyperuricemia and gout, and also in a diagnostic kit. In this study, error-prone polymerase chain reaction and staggered extension process was used to generate a mutant urate oxidase with improved enzyme activity from Bacillus subtilis. After several rounds of mutagenesis and screening, two mutants 6E9 and 8E279 were obtained which exhibited 2.99 and 3.43 times higher catalytic efficiency, respectively. They also exhibited lower optimal reaction temperature and higher thermo-stability. D44V, Q268R and K285Q were identified as the three most beneficial amino acid substitutions introduced by site-directed mutagenesis. D44V/Q268R, which was obtained through random combination of the three mutants, displayed the highest catalytic activity. The Km, kcat/Km and enzyme activity of D44V/Q268R increased by 68%, 83% and 129% respectively, compared with that of wild-type urate oxidase. Structural modeling indicated that mutations far from the active site can have significant effects on activity. For many of them, the underlying mechanisms are still difficult to explain from the static structural model. We also compared the effects of the same set of single point mutations on the wild type and on the final mutant. The results indicate strong effects of epistasis, which may imply that the mutations affect catalysis through influences on protein dynamics besides equilibrium structures.

Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?

Despite the significant advances in the field of protein engineering, general design principles to improve organic cosolvent resistance of enzymes still remain undiscovered. Previous studies drew conclusions to engineer enzymes for their use in water-miscible organic solvents based on few amino acid substitutions. In this study, we conduct a comparison of a Bacillus subtilis lipase A (BSLA) library—covering the full natural diversity of single amino acid substitutions at all 181 positions of BSLA—with three state of the art random mutagenesis methods: error-prone PCR (epPCR) with low and high mutagenesis frequency (epPCR-low and high) as well as a transversion-enriched Sequence Saturation Mutagenesis (SeSaM-Tv P/P) method. Libraries were searched for amino acid substitutions that increase the enzyme’s resistance to the water-miscible organic cosolvents 1,4-dioxane (DOX), 2,2,2-trifluoroethanol (TFE), and dimethyl sulfoxide (DMSO). Our analysis revealed that 5%–11% of all possible single substitutions (BSLA site-saturation mutagenesis (SSM) library) contribute to improved cosolvent resistance. However, only a fraction of these substitutions (7%–12%) could be detected in the three random mutagenesis libraries. To our knowledge, this is the first study that quantifies the capability of these diversity generation methods generally employed in directed evolution campaigns and compares them to the entire natural diversity with a single substitution. Additionally, the investigation of the BSLA SSM library revealed only few common beneficial substitutions for all three cosolvents as well as the importance of introducing surface charges for organic cosolvent resistance—most likely due to a stronger attraction of water molecules.

Structural and functional innovations in the real-time evolution of new (βα)8 barrel enzymes

New genes can arise by duplication and divergence, but there is a fundamental gap in our understanding of the relationship between these genes, the evolving proteins they encode, and the fitness of the organism. Here we used crystallography, NMR dynamics, kinetics, and mass spectrometry to explain the molecular innovations that arose during a previous real-time evolution experiment. In that experiment, the (βα)8 barrel enzyme HisA was under selection for two functions (HisA and TrpF), resulting in duplication and divergence of the hisA gene to encode TrpF specialists, HisA specialists, and bifunctional generalists. We found that selection affects enzyme structure and dynamics, and thus substrate preference, simultaneously and sequentially. Bifunctionality is associated with two distinct sets of loop conformations, each essential for one function. We observed two mechanisms for functional specialization: structural stabilization of each loop conformation and substrate-specific adaptation of the active site. Intracellular enzyme performance, calculated as the product of catalytic efficiency and relative expression level, was not linearly related to fitness. Instead, we observed thresholds for each activity above which further improvements in catalytic efficiency had little if any effect on growth rate. Overall, we have shown how beneficial substitutions selected during real-time evolution can lead to manifold changes in enzyme function and bacterial fitness. This work emphasizes the speed at which adaptive evolution can yield enzymes with sufficiently high activities such that they no longer limit the growth of their host organism, and confirms the (βα)8 barrel as an inherently evolvable protein scaffold.

Uniparental Inheritance Promotes Adaptive Evolution in Cytoplasmic Genomes.

Eukaryotes carry numerous asexual cytoplasmic genomes (mitochondria and plastids). Lacking recombination, asexual genomes should theoretically suffer from impaired adaptive evolution. Yet, empirical evidence indicates that cytoplasmic genomes experience higher levels of adaptive evolution than predicted by theory. In this study, we use a computational model to show that the unique biology of cytoplasmic genomes—specifically their organization into host cells and their uniparental (maternal) inheritance—enable them to undergo effective adaptive evolution. Uniparental inheritance of cytoplasmic genomes decreases competition between different beneficial substitutions (clonal interference), promoting the accumulation of beneficial substitutions. Uniparental inheritance also facilitates selection against deleterious cytoplasmic substitutions, slowing Muller’s ratchet. In addition, uniparental inheritance generally reduces genetic hitchhiking of deleterious substitutions during selective sweeps. Overall, uniparental inheritance promotes adaptive evolution by increasing the level of beneficial substitutions relative to deleterious substitutions. When we assume that cytoplasmic genome inheritance is biparental, decreasing the number of genomes transmitted during gametogenesis (bottleneck) aids adaptive evolution. Nevertheless, adaptive evolution is always more efficient when inheritance is uniparental. Our findings explain empirical observations that cytoplasmic genomes—despite their asexual mode of reproduction—can readily undergo adaptive evolution.