Protein representations: Encoding biological information for machine learning in biocatalysis.

Enzymes offer a more environmentally friendly and low-impact solution to conventional chemistry, but they often require additional engineering for their application in industrial settings, an endeavour that is challenging and laborious. To address this issue, the power of machine learning can be harnessed to produce predictive models that enable the in silico study and engineering of improved enzymatic properties. Such machine learning models, however, require the conversion of the complex biological information to a numerical input, also called protein representations. These inputs demand special attention to ensure the training of accurate and precise models, and, in this review, we therefore examine the critical step of encoding protein information to numeric representations for use in machine learning. We selected the most important approaches for encoding the three distinct biological protein representations - primary sequence, 3D structure, and dynamics - to explore their requirements for employment and inductive biases. Combined representations of proteins and substrates are also introduced as emergent tools in biocatalysis. We propose the division of fixed representations, a collection of rule-based encoding strategies, and learned representations extracted from the latent spaces of large neural networks. To select the most suitable protein representation, we propose two main factors to consider. The first one is the model setup, which is influenced by the size of the training dataset and the choice of architecture. The second factor is the model objectives such as consideration about the assayed property, the difference between wild-type models and mutant predictors, and requirements for explainability. This review is aimed at serving as a source of information and guidance for properly representing enzymes in future machine learning models for biocatalysis.

Increasing Vitamin K2 Synthesis in Bacillus subtilis by Controlling the Expression of MenD and Stabilizing MenA.

As an indispensable member of the family of lipid vitamins, vitamin K2 (MK-7) plays an important role in blood coagulation, cardiovascular health, and kidney health. Microbial fermentation is favored due to its high utilization rate of raw materials, simple operation, and moderate conditions. However, the biosynthesis pathway of vitamin K2 in microorganisms is highly complex, which hinders its industrial production in microbial cell factories. One of the major challenges is the stable expression and deregulation of key enzymes in the vitamin K2 biosynthesis pathway, which remains unclear and has undergone little investigation. In this study, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid synthase (MenD) and 1,4-dihydroxy-2-naphthoate polyprenyltransferase (MenA) were identified as pivotal enzymes in the biosynthesis of vitamin K2. To investigate the catalytic efficiency of MenD in the biosynthesis pathway of vitamin K2, structure-based mutation design and site-directed mutagenesis were performed. Three mutation sites were identified in MenD: A115Y, R96 M, and R323M, which improve the expression level and protein stability. Meanwhile, the MenA mutant T290M, which exhibits improved protein stability, was obtained by modifying its hydrophobic stacking structure. Finally, an engineered strain noted ZQ13 that combinatorially overexpressed MenD (A115Y) and MenA (T290M) mutants was constructed and achieved 338.37 mg/L vitamin K2 production in a 3-L fermenter.

Enhanced degradation of insoluble chitin: Engineering high-efficiency chitinase fusion enzymes for sustainable applications.

N-acetyl-D-glucosamine and its dimer are degradation products of chitin waste with great potential in therapeutic and agricultural applications. However, the hydrolysis of insoluble chitin by chitinases remains a major bottleneck. This study investigated the biochemical properties and catalytic mechanisms of PoChi chitinase obtained from Penicillium oxalicum with a focus on enhancing its efficiency during the degradation of insoluble chitin. Recombinant plasmids were engineered to incorporate chitin-binding (ChBD) and/or fibronectin III (FnIII) domains. Notably, PoChi-FnIII-ChBD exhibited the highest substrate affinity (Km = 2.7 mg/mL) and a specific activity of 15.4 U/mg, which surpasses those of previously reported chitinases. These findings highlight the potential of engineered chitinases in advancing industrial biotechnology applications and offer a promising approach to more sustainable chitin waste management.

Nitrile hydratase as a promising biocatalyst: recent advances and future prospects.

Amides are an important type of synthetic intermediate used in the chemical, agrochemical, pharmaceutical, and nutraceutical industries. The traditional chemical process of converting nitriles into the corresponding amides is feasible but is restricted because of the harsh conditions required. In recent decades, nitrile hydratase (NHase, EC 4.2.1.84) has attracted considerable attention because of its application in nitrile transformation as a prominent biocatalyst. In this review, we provide a comprehensive survey of recent advances in NHase research in terms of natural distribution, enzyme screening, and molecular modification on the basis of its characteristics and catalytic mechanism. Additionally, industrial applications and recent significant biotechnology advances in NHase bioengineering and immobilization techniques are systematically summarized. Moreover, the current challenges and future perspectives for its further development in industrial applications for green chemistry were also discussed. This study contributes to the current state-of-the-art, providing important technical information for new NHase applications in manufacturing industries.

Characterization and rational engineering of an alkaline-tolerant azoreductase derived from Roseibium sp. H3510 for enhanced decolorization of azo dyes.

rAzoR2326, an azoreductase derived from Roseibium sp. H3510, functions as an FMN-dependent homodimer utilizing NADH as cofactor. It demonstrated maximum activity at 45 °C and retained moderate activity above 50 °C, exhibiting stability from pH 7-10. Evolution and structure guided rational design of wild-type rAzoR2326 (WT) efficiently yielded 6 single-point mutants with improved thermostability and activity from a 22-variant library. Further combinatorial mutation led to mutant M20 with substantially enhanced thermostability (15-fold longer half-life at 50 °C) and activity (3.24-fold higher kcat/Km). M20 exhibited superior catalytic properties for decolorizing Allura Red compared to WT. Specifically, its decolorization capacity at pH 10.0 was 4.26-fold higher than WT. Additionally, M20 demonstrated remarkable thermostability, retaining 76.83 % decolorization activity for Allura Red after 120 min at 50 °C, whereas WT nearly lost all catalytic activity under the same conditions. Molecular dynamics simulations revealed the structural changes in M20, such as improved hydrogen bonding and a new C-H···π interaction, led to a more compact and rigid enzyme structure. This resulted in a more stable FMN-binding pocket and substrate tunnel, thereby improving the catalytic stability and activity of M20. Given its enhanced dye decolorization ability and alkaline tolerance, M20 shows promise as a biocatalyst for treating azo dye effluents.

[Teaching reform and practice of the postgraduate course "Biocatalysis and Enzyme Engineering" under the "dual-drive and dual-guide" model].

Value shaping and innovation capability improvement are two important goals of postgraduate course teaching. Biocatalysis and Enzyme Engineering is a core course for postgraduates majoring in bioengineering. Our teaching team established a "dual-drive and dual-guide" teaching model, that is, "double drive" (value-driven + innovation-driven) as the traction, this model integrated professional quality with industry needs, engineering ethics, and scientist spirit, combined theoretical teaching with research frontiers, industrial cases, and engineering practice, and incorporated thematic lectures, group peer evaluation, and review papers into course assessment. The teaching under this model achieved the teaching objectives of "double guide" (guiding ideology and ability), and improved the postgraduates' value identification, innovation awareness, engineering thinking, and ability to solve practical engineering problems.

Biosensor-Guided Engineering of a Baeyer-Villiger Monooxygenase for Aliphatic Ester Production.

Esters are valuable aroma compounds and can be produced enzymatically by Baeyer-Villiger monooxygenases (BVMOs) from (aliphatic) ketone precursors. However, a genetically encoded biosensor system for the assessment of BVMO activity and the detection of reaction products is missing. In this work, we assembled a synthetic enzyme cascade - featuring an esterase, an alcohol dehydrogenase, and LuxAB - in the heterologous host Escherichia coli. Target esters are produced by a BVMO, subsequently cleaved, and the corresponding alcohol oxidized through the artificial pathway. Ultimately, aldehyde products are detected in vivo by LuxAB, a luciferase from Photorhabdus luminescens that emits bioluminescence upon the oxidation of aldehydes to the corresponding carboxylates. This biosensor system greatly accelerated the screening and selection of active BVMO variants from a focused library, omitting commonly used low-throughput chromatographic analysis. Engineered enzymes accepted linear aliphatic ketones such as 2-undecanone and 2-dodecanone and exhibited improved ester formation.

Advancing sustainable biotechnology through protein engineering.

The push for industrial sustainability benefits from the use of enzymes as a replacement for traditional chemistry. Biological catalysts, especially those that have been engineered for increased activity, stability, or novel function, and are often greener than alternative chemical approaches. This Review highlights the role of engineered enzymes (and identifies directions for further engineering efforts) in the application areas of greenhouse gas sequestration, fuel production, bioremediation, and degradation of plastic wastes.

Improving regioselectivity of phenylalanine aminomutase from Taxus chinensis by semi-rational mutagenesis for the biocatalytic amination of cinnamates.

The occupancy of the binding pocket by the substrate ultimately determines the outcome of enzyme catalysis. Previous engineering and substrate scope of phenylalanine aminomutase from Taxus chinensis (TcPAM) has generated valuable knowledge about the regioselectivity with biocatalytic potentials for the preparation of α- and ß-phenylalanine and their derivatives. However, the significantly different regioselectivity during the amination of cinnamates by TcPAM is not fully understood. In this study, we take a reconstruction approach to change the whole binding pocket of TcPAM for probing the factors affecting the regioselectivity, resulting in variant C107S/Q319M/I431V reaching a 25.5-fold enhancement of the ß/α product ratio toward trans-cinnamate acid. Furthermore, when substituted cinnamates were used as substrates, the regioselectivity was strongly correlated with various changes in the binding pocket, and value-added 2-Cl-α-Phe (100% α-selectivity) and 4-CH3-ß-Phe (98% ß-selectivity) were individually verified by the mutants L104A and Q319M at a preparative scale, exemplifying the application feasibility of our engineering strategy. The present study uncovered the cooperative connection between aromatic binding and carboxylate binding to affect the regioselectivity, which provides new insights into the determinants of the regioselectivity possessed by TcPAM and paves the way for its biocatalytic applications on phenylalanine derivatives.

Supercharged Phosphotriesterase for improved Paraoxon activity.

Phosphotriesterases (PTEs) represent a class of enzymes capable of efficient neutralization of organophosphates (OPs), a dangerous class of neurotoxic chemicals. PTEs suffer from low catalytic activity, particularly at higher temperatures, due to low thermostability and low solubility. Supercharging, a protein engineering approach via selective mutation of surface residues to charged residues, has been successfully employed to generate proteins with increased solubility and thermostability by promoting charge-charge repulsion between proteins. We set out to overcome the challenges in improving PTE activity against OPs by employing a computational protein supercharging algorithm in Rosetta. Here, we discover two supercharged PTE variants, one negatively supercharged (with -14 net charge) and one positively supercharged (with +12 net charge) and characterize them for their thermodynamic stability and catalytic activity. We find that positively supercharged PTE possesses slight but significant losses in thermostability, which correlates to losses in catalytic efficiency at all temperatures, whereas negatively supercharged PTE possesses increased catalytic activity across 25°C-55°C while offering similar thermostability characteristic to the parent PTE. The impact of supercharging on catalytic efficiency will inform the design of shelf-stable PTE and criteria for enzyme engineering.

Efficient conversion of xylan to l-arabinose by multi-enzymatic cascade reaction including d-xylulose 4-epimerase as a new stereoselectivity-exchange enzyme.

l-Arabinose has been produced by hydrolyzing arabinan, a component of hemicellulose. However, l-arabinose has limitations in industrial applications owing to its relatively high cost. Here, d-xylulose 4-epimerase as a new-type enzyme was developed from d-tagaturonate 3-epimerase from Thermotoga petrophila using structure-guided enzyme engineering. d-Xylulose 4-epimerase, which epimerized d-xylulose to l-ribulose, d-xylulokinase and sugar phosphatase, which overcame the equilibrium of d-xylose isomerase, were included to establish a new efficient conversion pathway from d-xylose to l-arabinose. l-Arabinose at 34 g/L was produced from 100 g/L xylan in 45 h by multi-enzymatic cascade reaction using xylanase and enzymes involved in the established conversion pathway. As l-ribulokinase was used instead of d-xylulokinase in the established conversion pathway, an efficient reverse-directed conversion pathway from l-arabinose to d-xylose and the production of d-xylose from arabinan using arabinanase and enzymes involved in the proposed pathway are proposed.

Microbial production of an aromatic homopolyester.

We report the development of a metabolically engineered bacterium for the fermentative production of polyesters containing aromatic side chains, serving as sustainable alternatives to petroleum-based plastics. A metabolic pathway was constructed in an Escherichia coli strain to produce poly[d-phenyllactate(PhLA)], followed by three strategies to enhance polymer production. First, polyhydroxyalkanoate (PHA) granule-associated proteins (phasins) were introduced to increase the polymer accumulation. Next, metabolic engineering was performed to redirect the metabolic flux toward PhLA. Furthermore, PHA synthase was engineered based on in silico simulation results to enhance the polymerization of PhLA. The final strain was capable of producing 12.3 g/l of poly(PhLA), marking it the first bio-based process for producing an aromatic homopolyester. Additional heterologous gene introductions led to the high level production of poly(3-hydroxybutyrate-co-11.7 mol% PhLA) copolymer (61.4 g/l). The strategies described here will be useful for the bio-based production of aromatic polyesters from renewable resources.

[Enzyme engineering in microbial biosynthesis of terpenoids: progress and perspectives].

Terpenoids, known for their structural and functional diversity, are highly valued, especially in food, cosmetics, and cleaning products. Microbial biosynthesis has emerged as a sustainable and environmentally friendly approach for the production of terpenoids. However, the natural enzymes involved in the synthesis of terpenoids have problems such as low activity, poor specificity, and insufficient stability, which limit the biosynthesis efficiency. Enzyme engineering plays a pivotal role in the microbial synthesis of terpenoids. By modifying the structures and functions of key enzymes, researchers have significantly improved the catalytic activity, specificity, and stability of enzymes related to terpenoid synthesis, providing strong support for the sustainable production of terpenoids. This article reviews the strategies for the modification of key enzymes in microbial synthesis of terpenoids, including improving enzyme activity and stability, changing specificity, and promoting mass transfer through multi-enzyme collaboration. Additionally, this article looks forward to the challenges and development directions of enzyme engineering in the microbial synthesis of terpenoids.

A growth-based screening strategy for engineering the catalytic activity of an oxygen-sensitive formate dehydrogenase.

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli. This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H (EcFDH-H). Using an EcFDH-H-deficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active EcFDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using EcFDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 EcFDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes.IMPORTANCEOxygen-sensitive metalloenzymes are highly potent catalysts that allow the reduction of chemically inert substrates such as CO2 and N2 at ambient pressure and temperature and have, therefore, been considered for the sustainable production of biofuels and commodity chemicals such as ammonia, formic acid, and glycine. A proven method to optimize natural enzymes for such applications is enzyme engineering using high-throughput variant library screening. However, most screening methods are incompatible with the oxygen sensitivity of these metalloenzymes and thereby limit their relevance for the development of biosynthetic production processes. A microtiter plate-based assay was developed for the screening of metal-dependent formate dehydrogenase that links the activity of the tested enzyme variant to the growth of the anaerobically grown host cell. The presented work extends the application range of growth-based screening to metalloenzymes and is thereby expected to advance their adoption to biosynthesis applications.

Biodegradation: the best solution to the world problem of discarded polymers.

The widespread use of polymers has made our lives increasingly convenient by offering a more convenient and dependable material. However, the challenge of efficiently decomposing these materials has resulted in a surge of polymer waste, posing environment and health risk. Currently, landfill and incineration treatment approaches have notable shortcomings, prompting a shift towards more eco-friendly and sustainable biodegradation approaches. Biodegradation primarily relies on microorganisms, with research focusing on both solitary bacterial strain and multi-strain communities for polymer biodegradation. Furthermore, directed evolution and rational design of enzyme have significantly contributed to the polymer biodegradation process. However, previous reviews often undervaluing the role of multi-strain communities. In this review, we assess the current state of these three significant fields of research, provide practical solutions to issues with polymer biodegradation, and outline potential future directions for the subject. Ultimately, biodegradation, whether facilitated by single bacteria, multi-strain communities, or engineered enzymes, now represents the most effective method for managing waste polymers.

Rational design of a flavoenzyme for aerobic nicotine catabolism.

Enzymatic therapy with nicotine-degrading enzyme is a new strategy in treating nicotine addiction, which can reduce nicotine concentrations and weaken withdrawal in the rat model. However, when O2 is used as the electron acceptor, no satisfactory performance has been achieved with one of the most commonly studied and efficient nicotine-catabolizing enzymes, NicA2. To obtain more efficient nicotine-degrading enzyme, we rationally designed and engineered a flavoenzyme Pnao, which shares high structural similarity with NicA2 (RMSD = 1.143 Å) and efficiently catalyze pseudooxynicotine into 3-succinoyl-semialdehyde pyridine using O2. Through amino acid alterations with NicA2, five Pnao mutants were generated, which can degrade nicotine in Tris-HCl buffer and retain catabolic activity on its natural substrate. Nicotine-1'-N-oxide was identified as one of the reaction products. Four of the derivative mutants showed activity in rat serum and Trp220 and Asn224 were found critical for enzyme specificity. Our findings offer a novel avenue for research into aerobic nicotine catabolism and provide a promising method of generating additional nicotine-catalytic enzymes. IMPORTANCE: Nicotine, the main active substance in tobacco, results in cigarette addiction and various diseases. There have been some attempts at using nicotine oxidoreductase, NicA2, as a therapeutic for nicotine cessation. However, it uses cytochrome c as it is electron acceptor, which is impractical for therapeutic use compared with using O2 as an oxidant. Thus, amino acid alteration was performed on Pnao using NicA2 as model. Five of the mutants generated degraded nicotine at a rate similar to NicA2, and one of the catabolic compounds was identified as nicotine-1'-N-oxide. Our research highlights a new direction in developing enzymes that efficiently catabolize nicotine without co-enzymes and suggests that structure-similar human original MAOA (or B) may assist with nicotine cessation after being engineered.

Improving the catalytic activity and thermostability of Aspergillus niger xylanase through computational design.

Xylanase plays the most important role in catalyzing xylan to xylose moieties. GH11 xylanases have been widely used in many fields, but most GH11 xylanases are mesophilic enzymes. To improve the catalytic activity and thermostability of Aspergillus niger xylanase (Xyn-WT), we predicted potential key mutation sites of Xyn-WT through multiple computer-aided enzyme engineering strategies. We introduce a simple and economical Ni affinity chromatography purification method to obtain high-purity xylanase and its mutants. Ten mutants (Xyn-A, Xyn-B, Xyn-C, E45T, Q93R, E45T/Q93R, A161P, Xyn-D, Xyn-E, Xyn-F) were identified. Among the ten mutants, four (Xyn-A, Xyn-C, A161P, Xyn-F) presented improved thermal stability and activity, with Xyn-F(A161P/E45T/Q93R) being the most thermally stable and active. Compared with Xyn-WT, after heat treatment at 55 °C and 60 °C for 10 min, the remaining enzyme activity of Xyn-F was 12 and 6 times greater than that of Xyn-WT, respectively, and Xyn-F was approximately 1.5 times greater than Xyn-WT when not heat treated. The pH adaptation of Xyn-F was also significantly enhanced. In summary, an improved catalytic activity and thermostability of the design variant Xyn-F has been reported.

[Advances in using artificial intelligence for predicting protein-ligand binding affinity].

The binding of proteins and ligands is a crucial aspect of life processes. The calculation of the protein-ligand binding affinity (PLBA) offers valuable insights into protein function, drug screening targets protein receptors, and enzyme modifications. In recent years, artificial intelligence (AI) has experienced rapid advancements, becoming widely used in PLBA prediction. This is attributed to its robust feature extraction ability, superior algorithm accuracy, and speedy calculations. Our paper aims to provide a comprehensive overview of AI predication process, associated resources, application scenarios, challenges, and potential solutions, serving as a valuable reference for the relevant research endeavors.

Application of synthetic biology strategies to promote biosynthesis of fatty acids and their derivatives.

Fatty acids and their derivatives are indispensable biomolecules in all organisms, and can be used as intermediates in the synthesis of pharmaceuticals, biofuels and pesticides, and thus their demand has increased dramatically in recent years. In addition to serving as structural components of cell membranes and metabolic energy, fatty acids and their derivatives can also be used as signal transduction and regulatory bioactive molecules to regulate cell functions. Biosynthesis of fatty acids and their derivatives through microbial catalysis provides green and alternative options to meet the goal. However, the low biosynthetic titer of fatty acids and their derivatives limits their industrial production and application. In this review, we first summarize the metabolic pathways and related enzymes of fatty acids and their derivatives biosynthesis. Then, the strategies and research progress of biosynthesis of fatty acids and derivatives through metabolic and enzyme engineering were reviewed. The biosynthesis of saturated fatty acids (medium chain fatty acids and long chain fatty acids), bioactive fatty acids (PUFAs, oxylipins, ether lipids), and their derivatives with microbial and enzymatic catalysis were respectively summarized. Finally, synthetic biology strategies to improve fatty acids and their derivatives production through enzyme rational design, carbon metabolism flux, cofactors balance, and metabolic pathways design were discussed. The review provides references and prospects for fatty acids and their derivatives biosynthesis and industrial production.

Efficient and easible biocatalysts: Strategies for enzyme improvement. A review.

Owing to the environmental friendliness and vast advantages that enzymes offer in the biotechnology and industry fields, biocatalysts are a prolific investigation field. However, the low catalytic activity, stability, and specific selectivity of the enzyme limit the range of the reaction enzymes involved in. A comprehensive understanding of the protein structure and dynamics in terms of molecular details enables us to tackle these limitations effectively and enhance the catalytic activity by enzyme engineering or modifying the supports and solvents. Along with different strategies including computational, enzyme engineering based on DNA recombination, enzyme immobilization, additives, chemical modification, and physicochemical modification approaches can be promising for the wide spread of industrial enzyme usage. This is attributed to the successful application of biocatalysts in industrial and synthetic processes requires a system that exhibits stability, activity, and reusability in a continuous flow process, thereby reducing the production cost. The main goal of this review is to display relevant approaches for improving enzyme characteristics to overcome their industrial application.