Rational enzyme design by reducing the number of hotspots and library size.

Biocatalysts that are eco-friendly, sustainable, and highly specific have great potential for applications in the production of fine chemicals, food, detergents, biofuels, pharmaceuticals, and more. However, due to factors such as low activity, narrow substrate scope, poor thermostability, or incorrect selectivity, most natural enzymes cannot be directly used for large-scale production of the desired products. To overcome these obstacles, protein engineering methods have been developed over decades and have become powerful and versatile tools for adapting enzymes with improved catalytic properties or new functions. The vastness of the protein sequence space makes screening a bottleneck in obtaining advantageous mutated enzymes in traditional directed evolution. In the realm of mathematics, there are two major constraints in the protein sequence space: (1) the number of residue substitutions (M); and (2) the number of codons encoding amino acids as building blocks (N). This feature review highlights protein engineering strategies to reduce screening efforts from two dimensions by reducing the numbers M and N, and also discusses representative seminal studies of rationally engineered natural enzymes to deliver new catalytic functions.

Chiral silver nanoparticles with surface-anchored L(D)-Cys exhibit dissimilar biological characteristics in vitro but not in vivo.

This work investigated the influence of surface chirality on cellular internalization, cytotoxicity, and tissue distribution of silver nanoparticles (AgNPs). D-cysteine and L-cysteine are chiral forms of the amino acid cysteine. These enantiomers exhibit distinct spatial arrangements, with D-cysteine having a different configuration from L-cysteine. This structural dissimilarity can lead to variations in how these forms interact with biological systems, potentially impacting their cytotoxic responses. Four distinct types of AgNPs were synthesized, each possessing a unique surface coating: pristine AgNPs (pAgNPs), L-cysteine coated AgNPs (AgNPs@L-Cys), D-cysteine coated AgNPs (AgNPs@D-Cys), and racemic AgNPs coated with both L-Cys and D-Cys (AgNPs@L/D-Cys). We found chiral-dependent cytotoxicity of AgNPs on J774A.1 cells. Specifically, AgNPs@L-Cys exhibited the highest toxicity, and AgNPs@D-Cys exhibited the lowest toxicity. Meanwhile, the cellular uptake of the AgNPs correlated nicely with their cytotoxicity, with AgNPs@L-Cys being internalized to the greatest extent while AgNPs@D-Cys displays the least internalization. Scavenger receptors and clathrin predominantly mediate the cellular internalization of these AgNPs. Strikingly, the dissimilar cellular internalization and cytotoxicity of AgNPs with different chirality were eliminated upon protein corona coverage. Notably, following intravenous injection in mice, these four types of AgNPs showed similar patterns among various organs due to the inevitable protein adsorption in the bloodstream. These findings underscored the pivotal role of surface chirality in governing the biological interactions and toxicity of AgNPs.

Engineering the activity and thermostability of a carboxylic acid reductase in the conversion of vanillic acid to vanillin.

Vanillin is a valuable natural product that can be used as a fragrance and additive. Recent research in the biosynthesis of vanillin has brought attention to a key enzyme, carboxylic acid reductase (CAR), which catalyzes the reduction of vanillic acid to vanillin. Nevertheless, the biosynthesis of vanillin is hampered by the low activity and stability of CAR. As such, a rational design campaign was conducted on a well-documented carboxylic acid reductase from Segniliparus rugosus (SrCAR), using vanillic acid as the model substrate. After combined active site saturation and iterative site-specific mutagenesis, the best quadruple mutant N292H/K524S/A627L/E1121W (M3) was successfully obtained. In comparison to the wildtype SrCAR, M3 demonstrated a 4.2-fold increase in catalytic efficiency (kcat/Km), and its half-life (t1/2) was enhanced by 3.8 times up to 385.08 minutes at 40 °C. In silico docking and molecular dynamics simulation provided insights into the improved activity and stability. In the subsequent preparative-scale reaction with 100 mM (16.8 g L-1) vanillic acid, the whole cell catalysis utilizing M3 produced 10.15 g·L-1 of vanillin and 1.11 g·L-1 of vanillyl alcohol, respectively. This work demonstrates a dual improvement in the activity and thermal stability of SrCAR, thereby potentially facilitating the application of carboxylic acid reductase in the biosynthesis of vanillin.

[Substitutability of metal-binding sites in an alcohol dehydrogenase].

Covalently anchoring of a ligand/metal via polar amino acid side chain(s) is often observed in metalloenzyme, while the substitutability of metal-binding sites remains elusive. In this study, we utilized a zinc-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) as a model enzyme, analyzed the sequence conservation of the three residues Cys37, His59, and Asp150 that bind the zinc ion, and constructed the mutant library. After experimental validation, three out of 224 clones, which showed comparative conversion and ee values as the wild-type enzyme in the asymmetric reduction of the model substrate tetrahydrofuran-3-one, were screened out. The results reveal that the metal-binding sites in TbSADH are substitutable without tradeoff in activity and stereoselectivity, which lay a foundation for designing ADH-catalyzed new reactions via metal ion replacement.

Unlocking the Stereoselectivity and Substrate Acceptance of Enzymes: Proline-Induced Loop Engineering Test.

Protein stability and evolvability influence each other. Although protein dynamics play essential roles in various catalytically important properties, their high flexibility and diversity makes it difficult to incorporate such properties into rational engineering. Therefore, how to unlock the potential evolvability in a user-friendly rational design process remains a challenge. In this endeavor, we describe a method for engineering an enantioselective alcohol dehydrogenase. It enables synthetically important substrate acceptance for 4-chlorophenyl pyridine-2-yl ketone, and perfect stereocontrol of both (S)- and (R)-configured products. Thermodynamic analysis unveiled the subtle interaction between enzyme stability and evolvability, while computational studies provided insights into the origin of selectivity and substrate recognition. Preparative-scale synthesis of the (S)-product (73 % yield; >99 % ee) was performed on a gram-scale. This proof-of-principle study demonstrates that interfaced proline residues can be rationally engineered to unlock evolvability and thus provide access to new biocatalysts with highly improved catalytic performance.

Biosynthesis of Chiral Amino Alcohols via an Engineered Amine Dehydrogenase in E. coli.

Chiral amino alcohols are prevalent synthons in pharmaceuticals and synthetic bioactive compounds. The efficient synthesis of chiral amino alcohols using ammonia as the sole amino donor under mild conditions is highly desired and challenging in organic chemistry and biotechnology. Our previous work explored a panel of engineered amine dehydrogenases (AmDHs) derived from amino acid dehydrogenase (AADH), enabling the one-step synthesis of chiral amino alcohols via the asymmetric reductive amination of α-hydroxy ketones. Although the AmDH-directed asymmetric reduction is in a high stereoselective manner, the activity is yet fully excavated. Herein, an engineered AmDH derived from a leucine dehydrogenase from Sporosarcina psychrophila (SpAmDH) was recruited as the starting enzyme, and the combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was applied to improve the activity. After three rounds of mutagenesis in an iterative fashion, the best variant wh84 was obtained and proved to be effective in the asymmetric reductive amination of 1-hydroxy-2-butanone with 4-fold improvements in k cat /K m and total turnover number (TTN) values compared to those of the starting enzyme, while maintaining high enantioselectivity (ee >99%) and thermostability (T 50 15 >53°C). In preparative-scale reaction, the conversion of 100 and 200 mM 1-hydroxy-2-butanone catalyzed by wh84 was up to 91-99%. Insights into the source of an enhanced activity were gained by the computational analysis. Our work expands the catalytic repertoire and toolbox of AmDHs.

Engineering of ß-Glucosidase Bgl15 with Simultaneously Enhanced Glucose Tolerance and Thermostability To Improve Its Performance in High-Solid Cellulose Hydrolysis.

In this study, a Petri-dish-based double-layer high-throughput screening method was established to improve glucose tolerance of ß-glucosidase Bgl15. Two beneficial mutations were identified, and the joint mutant 2R1 improved the half-maximal inhibitory concentration of glucose from 0.04 to 2.1 M. The crystal structure of 2R1 was subsequently determined at 2.7 Å. Structure analysis revealed that enhancement of glucose tolerance may be due to improved transglycosylation activity made possible by a hydrophobic binding site for glucose as an acceptor and more stringent control of a putative water channel. To further ameliorate the application potential of the enzyme, it was engineered to increase the half-life at 50 °C from 0.8 h (Bgl15) to 180 h (mutant 5R1). Furthermore, supplementation of 5R1 to the cellulase cocktail significantly improved glucose production from pretreated sugar cane bagasse by 38%. Consequently, this study provided an efficient approach to enhance glucose tolerance and generated a promising catalyst for cellulose saccharification.

Improving Galactooligosaccharide Synthesis Efficiency of ß-Galactosidase Bgal1-3 by Reshaping the Active Site with an Intelligent Hydrophobic Amino Acid Scanning.

There are ongoing interests in improving the galactooligosaccharide (GOS) synthesis efficiency of ß-galactosidase by protein engineering. In this study, an intelligent double-hydrophobic amino acid scanning strategy was proposed and employed to target nine residues forming the glycon-binding site (-1 subsite) of ß-galactosidase Bgal1-3. Two mutants C510V and H512I with significantly improved GOS synthesis efficiency were obtained. When 40% (w/v) lactose was used as a substrate, Bgal1-3 reached a maximum GOS yield of 45.3% at 16 h, while the mutants reached higher yields in a much shorter time (59.1% at 10 h for C510V, 51.5% at 2 h for H512I). When skim milk was treated with these enzymes, more GOS was produced (19.9 g/L for C510V, 12.7 g/L for H512I) than that for Bgal1-3 (10.3 g/L) at a lactose conversion of 90%. These results validated hydrophobicity scanning as an efficient method to engineer ß-galactosidases into promising catalysts for the preparation of GOS and GOS-enriched milk.

Tat-Independent Secretion of Polyethylene Terephthalate Hydrolase PETase in Bacillus subtilis 168 Mediated by Its Native Signal Peptide.

Widespread utilization of polyethylene terephthalate (PET) has caused critical environmental pollution. The enzymatic degradation of PET is a promising solution to this problem. In this study, PETase, which exhibits much higher PET-hydrolytic activity than other enzymes, was successfully secreted into extracellular milieu from Bacillus subtilis 168 under the direction of its native signal peptide (named SPPETase). SPPETase is predicted to be a twin-arginine signal peptide. Intriguingly, inactivation of twin-arginine translocation (Tat) complexes improved the secretion amount by 3.8-fold, indicating that PETase was exported via Tat-independent pathway. To the best of our knowledge, this is the first report on the improvement of Tat-independent secretion by inactivating Tat components of B. subtilis 168 in LB medium. Furthermore, PET film degradation assay showed that the secreted PETase was fully active. This study paves the first step to construct an efficient engineered strain for PET degradation.

Enhancing the Thermostability of Highly Active and Glucose-Tolerant ß-Glucosidase Ks5A7 by Directed Evolution for Good Performance of Three Properties.

A high-performance ß-glucosidase for efficient cellulose hydrolysis needs to excel in thermostability, catalytic efficiency, and resistance to glucose inhibition. However, it is challenging to achieve superb properties in all three aspects in a single enzyme. In this study, a hyperactive and glucose-tolerant ß-glucosidase Ks5A7 was employed as the starting point. Four rounds of random mutagenesis were then performed, giving rise to a thermostable mutant 4R1 with five amino acid substitutions. The half-life of 4R1 at 50 °C is 8640-fold that of Ks5A7 (144 h vs 1 min). Meanwhile, 4R1 had a higher specific activity (374.26 vs 243.18 units·mg-1) than the wild type with a similar glucose tolerance. When supplemented to Celluclast 1.5L, the mutant significantly enhanced the hydrolysis of pretreated sugar cane bagasse, improving the released glucose concentration by 44%. With excellent performance in thermostability, activity, and glucose tolerance, 4R1 will serve as an exceptional catalyst for industrial applications.

Improving the Secretion Yield of the ß-Galactosidase Bgal1-3 in Pichia pastoris for Use as a Potential Catalyst in the Production of Prebiotic-Enriched Milk.

In this study, three kinds of milk were treated with the ß-galactosidase Bgal1-3 (4 U/mL), resulting in 7.2-9.5 g/L galactooligosaccharides (GOS) at a lactose conversion of 90-95%. Then, Bgal1-3 was secreted from Pichia pastoris X33 under the direction of an α-factor signal peptide. After cultivation for 144 h in a flask culture with shaking, the extracellular activity of Bgal1-3 was 4.4 U/mL. Five more signal peptides (HFBI, apre, INU1A, MF4I, and W1) were employed to direct the secretion, giving rise to a more efficient signal peptide, W1 (11.2 U/mL). To further improve the secretion yield, recombinant strains harboring two copies of the bgal1-3 gene were constructed, improving the extracellular activity to 22.6 U/mL (about 440 mg/L). This study successfully constructed an engineered strain for the production of the ß-galactosidase Bgal1-3, which is a promising catalyst in the preparation of prebiotic-enriched milk.