Substrate Channeling Research Articles

Exploring the impact of sheet thickness scaling on Nanosheet FET gate electrostatics using k.p based simulations

This work explores the impact of sheet thickness scaling on gate electrostatics of NsFETs using k.p simulation. It is shown that thin channel NsFETs exhibit higher threshold voltage irrespective of the substrate orientation and channel material. However, the influence of geometrical confinement varies among different substrate orientations and channel materials due to variations in carrier quantization mass. It is also shown that thin channel NsFETs deliver higher inversion charges at equivalent gate over-drive voltages, thereby offering enhanced gate electrostatics. However, the advantage of gate electrostatics in thin channel NsFETs is limited by quantum capacitance. Optimizing the sub-band structure through strategic selection of substrate orientations and channel materials is essential to regulate quantum capacitance and to fully exploit the benefits of sheet thickness scaling in NsFETs.

An Intelligent Electrochemical Multi-Enzyme Molecular Machine for Chiral Chemical.

In vitro multi-enzyme synthesis pathways harness the core elements of cellular synthesis while simplifying the complexities of cellular processes, facilitating the production of high-value chemicals. However, these in vitro synthesis processes often operate like a "black box," with limited monitoring of each reaction step, leading to a low substrate conversion efficiency. In this study, we present an intelligent multi-enzyme molecular machine(iMEMM) as a model system for achieving the deracemization of D, L-phosphinothricin (D, L-PPT). The entire system leverages electrochemical power and enzyme-catalyzed (cascade) reactions to establish substrate channel and enhance efficiency. By modularizing each reaction step and using electrochemical real-time monitoring of the reaction process, a single-step electrobiotransformation efficiency of up to 98% and a chiral target L-PPT synthesis efficiency exceeding 99% have been achieved. This iMEMM eliminates the need for intermediate separation, enabling a "substrate in, product out" process. Our research paves the way for future green, intelligent, and automated biological manufacturing.

Molecular Understanding of Activity Changes of Alcohol Dehydrogenase in Deep Eutectic Solvents.

Deep eutectic solvents (DESs) have emerged as promising solvents for biocatalysis. While their impact on enzyme solvation and stabilization has been studied for several enzyme classes, their role in substrate binding is yet to be investigated. Herein, molecular dynamics (MD) simulations of horse-liver alcohol dehydrogenase (HLADH) are performed in choline chloride-ethylene glycol (ChCl-EG) and choline chloride-glycerol (ChCl-Gly) at varying water concentrations. In the DES solutions, the active site was significantly constricted, and its flexibility reduced when compared to the aqueous medium. Importantly, the cavity size follows a similar trend as the catalytic activity of HLADH and as such explains previously observed activity changes. To understand the impact on the binding of the substrate (cyclohexanone), an umbrella sampling (US) setup was established to calculate the free energy changes along the substrate binding tunnel of HLADH. The US combined with replica exchange and NADH in its cofactor pocket provided the best sampling of the entire active site, explaining why the cyclohexanone binding on HLADH is reduced with increasing DES content. As different components in these multicomponent mixtures influence the substrate binding, we additionally applied the US setup to study the ability of the DES components to be present inside the substrate tunnel. The presented approach may become useful to understand enzyme behaviors in DESs and to enable the design of more enzyme-compatible and tunable solvents.

Rational Design Engineering of 5-Aminolevulinate Synthase with Activity and Stability Enhancement.

5-Aminolevulinic acid synthase (ALAS) is the key rate-limiting enzyme in the synthesis of the vital biosynthetic intermediate 5-aminolevulinic acid (ALA). However, its catalytic efficiency is compromised due to its low activity and poor stability. Here, we obtained the mutant I325M/V390Y/H391I (T6), which exhibited a 7.0-fold increase in specific activity (2.53 U/mg) compared to the wild type through the application of isothermal compressibility (βT) perturbation engineering in conjunction with two thermal stability prediction algorithms. Moreover, molecular dynamics simulations indicate that positive changes in intermolecular interactions, the substrate channel, and the binding pocket account for the improved catalytic activity of T6. Furthermore, T6 was immobilized on magnetic chitosan nanoparticles, maintaining 73.5% of its original activity after 10 reaction cycles. Overall, combination approaches were employed to construct a superior ALAS variant, providing a novel concept for the synthesis of ALA and a valuable benchmark for optimizing industrial enzymes in related fields.

Structural analysis of a membrane-associated delta-6 fatty acid desaturase from Prorocentrum micans.

Delta-6 fatty acid desaturases, which play key roles in the biosynthesis of polyunsaturated fatty acids (PUFAs), are membrane-associated enzymes that present significant challenges for isolation and purification, complicating their structural characterization. Here we report the identification and structure-function analysis of a novel Δ6 fatty acid desaturase (PmD6) from the marine alga Prorocentrum micans with substrate preference to α-linolenic acid (18:3n-3). Structural modeling revealed a mushroom-like structure of PmD6 formed by four transmembrane α-helices as a stem and three cytoplasmic domains as a cap. Structural alignment identified several key residues positioned around the substrate tunnel and catalytic center in PmD6. Functional analysis of these residues by site-directed mutagenesis showed that Tyr226, Trp235, Phe345, and Ser349, facing the middle region of the substrate tunnel of PmD6, played critical roles in defining the structure for acceptance of substrates. Thr200, Leu391, and Met389, surrounding the end of the substrate tunnel, had roles in interaction with the methyl end of substrates. Asp255, close to a metal iron in the catalytic center, was essential for catalytic reaction by supporting the regional structure. These results have provided mechanistic insights into the structure-function relationship of membrane-bound front-end fatty acid desaturases.

Semi-rational design of an aromatic dioxygenase by substrate tunnel redirection.

Lignin valorization is crucial for achieving economic and sustainable biorefinery processes. However, the enzyme substrate preferences involved in lignin degradation remain poorly understood, and low activity toward specific substrates presents a significant challenge to the efficient utilization of lignin. In this study, we investigated the substrate promiscuity of ThAdo, a key enzyme involved in lignin valorization. Pre-reaction state analysis revealed that a hydrogen bond network is critical in determining substrate selectivity. By performing targeted saturation mutagenesis on residues surrounding the substrate tunnels, we identified the Y205W and Y205Q mutants, which demonstrated 0.73-fold and 0.72-fold enhancements in activity, respectively. Structural analysis indicated that the redirection of the original substrate tunnel may be responsible for the improved activity. Our study provides essential insights into the substrate preference mechanisms of lignin degrading enzymes and suggests that this tunnel-redirection strategy can be extended to other promiscuous enzymes.

Structural and functional insights into UDP-N-acetylglucosamine-enolpyruvate reductase (MurB) from Brucella ovis.

The peptidoglycan biosynthetic pathway involves a series of enzymatic reactions in which UDP-N-acetylglucosamine-enolpyruvate reductase (MurB) plays a crucial role in catalyzing the conversion of UDP-N-acetylglucosamine-enolpyruvate (UNAGEP) to UDP-N-acetylmuramic acid. This reaction relies on NADPH and FAD and, since MurB is not found in eukaryotes, it is an attractive target for the development of antimicrobials. MurB from Brucella ovis, the causative agent of brucellosis in sheep, is characterized here. The FAD cofactor in MurB of B. ovis is reduced to the hydroquinone state without semiquinone stabilization with an estimated Eox/hq of -260 mV. MurB from B. ovis catalyzes the oxidation of NADPH in a slow process that is positively influenced by the presence of the second product, UNAGEP. The crystallographic structure of the MurBox:UNAGEP complex confirms its folding into three domains and the binding of UNAGEP, positioning its enolpyruvyl group for hydride transfer from FAD. MurB shows a complex thermal unfolding pathway that is influenced by UNAGEP and NADP+, confirming its ability to bind both molecules. Molecular dynamics (MD) simulations predict that the nicotinamide of NADP+ is more stable at the active site than the enolpyruvyl of UNAGEP, and suggests that MurB can simultaneously accommodate NADPH and UNAGEP in the substrate channel, increasing overall protein-ligand flexibility. Sequence and evolutionary analyses show that MurB from B. ovis conserves all motifs predicted to be involved in catalysis within the Type IIa family.

Modular cascade with engineered HpaB for efficient synthesis of hydroxytyrosol.

Hydroxytyrosol, a naturally occurring chemical with antioxidant and antiviral properties, is widely used in the nutrition, pharmaceutical, and cosmetic industries. In the present study, a modularized cascade composed of Modules 1 and 2 was designed and implemented to convert l-tyrosine to hydroxytyrosol. Module 1 was a four-enzymatic cascade for converting l-tyrosine to tyrosol. Engineering Module 1 by fine-tuning the expression of the desired enzymes resulted in a robust whole-cell catalyst, BL21 (M1-13), which converted l-tyrosine to tyrosol at high substrate loading. Module 2 involved a 4-hydroxyphenylacetate 3-monooxygenase (HpaBC)-catalyzed reaction to hydroxylate tyrosol to form hydroxytyrosol. The rational design of the HpaB subunit led to a positive variant, HpaB-Mu (T292S/R474A), which was subsequently applied to Module 2 for tyrosol hydroxylation, yielding a robust whole-cell catalyst, BL21 (M2-05). The two designed modules were merged for one-pot conversion of l-tyrosine to hydroxytyrosol by adjusting the ratio and total amount of whole-cell catalyst loading, capable of converting 40mM of l-tyrosine to 35.8mM of hydroxytyrosol with a high space-time yield (1.38g/L/h). The current study proved that engineering HpaB at the substrate tunnel was a feasible way to boost its activity and proposed an effective method for synthesizing hydroxytyrosol from low-cost substrates, which has great economic potential.

Computational design of coevolutionary residues for improved stability and activity of nitrile hydratase.

Nitrile Hydratase (NHase), an industrially significant enzyme, catalyzes the conversion of nitriles into amides. High activity and thermostability are crucial for its broad applications. Compared with classical evaluation and subsequent combination of single-point mutations, redesigning coevolutionary residues offers a more precise approach by targeting key functional sites and facilitating efficient computational design and iteration. Here, we proposed an optimized strategy for redesigning coevolutionary residues to enhance the robustness of NHase, a heterotetrameric protein. We conducted an extensive analysis of 80 coevolutionary residue pairs in NHase from Pseudonocardia thermophila JCM3095 (PtNHase) and identified 21 hotspot designable residue pairs lacking explicit interactions. Virtual saturating combinatorial mutations were applied to these pairs, yielding 27 positive candidates from 8379 theoretical mutations based on changes in folding free energy. After screening and iterative combinations, the optimal mutant A3 (αG86Y/αK57L/αE183F) was obtained, whose specific activity toward acrylonitrile and half-life at 65°C were increased from 1656.8±21.2 U/mg and 20.1min in WT to 2370.1±102.7 U/mg and 62.3min, respectively. Benefiting from higher activity and thermostability, the whole-cell catalyst of A3 significantly facilitated the bioconversion of acrylonitrile to acrylamide. Molecular dynamics simulations further revealed that the newly formed inter-residue interactions stabilized the active site and enhanced the flexibility of the substrate channel, thereby improving both activity and thermostability. This study not only developed a highly robust NHase, but also established a framework for the design of other industrial enzymes.

Structure-Guided Engineering Unveils Deeper Substrate Channel in Processive Endoglucanase EG5C-1 Contributing to Enhanced Catalytic Efficiency and Processivity.

Processive endoglucanases have generated significant interest due to their bifunctionality in the degradation of cellulose and low product inhibition. However, enhancing their catalytic efficiency through engineering remains a formidable challenge. To address this bottleneck, our engineering efforts targeted loop regions located in the substrate channel of processive endoglucanase EG5C-1. Guided by a comparative analysis of characteristic structural features of the substrate channels in cellobiohydrolase, endoglucanase, and processive endoglucanase, a highly active triple mutant CM6 (N105H/T205S/D233L) was generated that had a 5.1- and 4.7-fold increase in catalytic efficiency toward soluble substrate carboxymethyl cellulose-Na and insoluble substrate phosphoric acid-swollen cellulose (PASC), compared with wild-type EG5C-1. Furthermore, this mutant exhibited greater processivity compared to EG5C-1. Molecular dynamics simulations unveiled that the mutations in the loop regions reshaped the substrate channel, leading to a deeper cleft, resembling the closed channel configuration of cellobiohydrolases. The increased compactness of the substrate channel induced changes in the substrate binding mode and substrate deformation, thereby enhancing both binding affinity and catalytic efficiency. Moreover, metadynamics simulations demonstrated that the processive velocity of cellulose chain through the binding channel in mutant CM6 surpassed that observed in EG5C-1.

Machine Learning Reveals Signatures of Promiscuous Microbial Amidases for Micropollutant Biotransformations.

Organic micropollutants, including pharmaceuticals, personal care products, pesticides, and food additives, are widespread in the environment, causing potentially toxic effects. Human waste is a direct source of micropollutants, with the majority of pharmaceuticals being excreted through urine. Urine contains its own microbiota with the potential to catalyze micropollutant biotransformations. Amidase signature (AS) enzymes are known for their promiscuous activity in micropollutant biotransformations, but the potential for AS enzymes from the urinary microbiota to transform micropollutants is not known. Moreover, the characterization of AS enzymes to identify key chemical and enzymatic features associated with biotransformation profiles is critical for developing benign-by-design chemicals and micropollutant removal strategies. Here, to uncover the signatures of AS enzyme-substrate specificity, we tested 17 structurally diverse compounds against a targeted enzyme library consisting of 40 AS enzyme homologues from diverse urine microbial isolates. The most promiscuous enzymes were active on nine different substrates, while 16 enzymes had activity on at least one substrate and exhibited diverse substrate specificities. Using an interpretable gradient boosting machine learning model, we identified chemical and amino acid features associated with AS enzyme biotransformations. Key chemical features from our substrates included the molecular weight of the amide carbonyl substituent and the number of formal charges in the molecule. Four of the identified amino acid features were located in close proximity to the substrate tunnel entrance. Overall, this work highlights the understudied potential of urine-derived microbial AS enzymes for micropollutant biotransformation and offers insights into substrate and protein features associated with micropollutant biotransformations for future environmental applications.

Closing the Loop in the Carbon Cycle: Enzymatic Reactions Housed in Metal-Organic Frameworks for CO2 Conversion to Methanol.

The preparation of value-added chemicals from carbon dioxide (CO2) can act as a way of reducing the greenhouse gas from the atmosphere. Industrially significant C1 chemicals like methanol (CH3OH), formic acid (HCOOH), and formaldehyde (HCHO) can be formed from CO2. One sustainable way of achieving this is by connecting the reactions catalyzed by the enzymes formate dehydrogenase (FDH), formaldehyde dehydrogenase (FALDH), and alcohol dehydrogenase (ADH) into a single cascade reaction where CO2 is hydrogenated to CH3OH. For this to be adaptable for industrial use, the enzymes should be immobilized in materials that are extraordinarily protective of the enzymes, inexpensive, stable, and of ultra-large surface area. Metal-organic frameworks (MOFs) meet these criteria and are expected to usher in the much-awaited dispensation of industrial biocatalysis. Unfortunately, little is known about the molecular behaviour of MOF-immobilized FDH, FALDH, and ADH. It is also yet not known which MOFs are most promising for industrial enzyme-immobilization since the field of reticular chemistry is growing exponentially with millions of hypothetical and synthesized MOF structures reported at present. This review initially discusses the properties of the key enzymes required for CO2 hydrogenation to methanol including available cofactor regeneration strategies. Later, the characterization techniques of enzyme-MOF composites and the successes or lack thereof of enzyme-MOF-mediated CO2 conversion to CH3OH and intermediate products are discussed. We also discuss reported multi-enzyme-MOF systems for CO2 conversion cognizant of the fact that at present, these systems are the only chance of housing cascade-type biochemical reactions where strict substrate channelling and operational conditions are required. Finally, we delve into future perspectives.

Tailored Metal–Organic Framework‐Based Nanozymes for Enhanced Enzyme‐Like Catalysis

AbstractThe global crisis of bacterial infections is exacerbated by the escalating threat of microbial antibiotic resistance. Nanozymes promise to provide ingenious solutions. Here, we reported a homogeneous catalytic structure of Pt nanoclusters with finely tuned metal–organic framework (ZIF‐8) channel structures for the treatment of infected wounds. Catalytic site normalization showed that the active site of the Pt aggregates structure with fine‐tuned pore modifications structure had a catalytic capacity of 14.903×105 min−1, which was 18.7 times higher than that of the Pt particles in monodisperse state in ZIF‐8 (0.793×105 min−1). In situ tests revealed that the change from homocleavage to heterocleavage of hydrogen peroxide at the interface of the nanozyme was one of the key reasons for the improvement of nanozyme activity. Density‐functional theory and kinetic simulations of the reaction interface jointly determine the role of the catalytic center and the substrate channel together. Metabolomics analysis showed that the developed nanozyme, working in conjunction with reactive oxygen species, could effectively block energy metabolic pathways within bacteria, leading to spontaneous apoptosis and bacterial rupture. This pioneering study elucidates new ideas for the regulation of artificial enzyme activity and provides new perspectives for the development of efficient antibiotic substitutes.

Tailored Metal-Organic Framework-Based Nanozymes for Enhanced Enzyme-Like Catalysis.

The global crisis of bacterial infections is exacerbated by the escalating threat of microbial antibiotic resistance. Nanozymes promise to provide ingenious solutions. Here, we reported a homogeneous catalytic structure of Pt nanoclusters with finely tuned metal-organic framework (ZIF-8) channel structures for the treatment of infected wounds. Catalytic site normalization showed that the active site of the Pt aggregates structure with fine-tuned pore modifications structure had a catalytic capacity of 14.903×105 min-1, which was 18.7 times higher than that of the Pt particles in monodisperse state in ZIF-8 (0.793×105 min-1). In situ tests revealed that the change from homocleavage to heterocleavage of hydrogen peroxide at the interface of the nanozyme was one of the key reasons for the improvement of nanozyme activity. Density-functional theory and kinetic simulations of the reaction interface jointly determine the role of the catalytic center and the substrate channel together. Metabolomics analysis showed that the developed nanozyme, working in conjunction with reactive oxygen species, could effectively block energy metabolic pathways within bacteria, leading to spontaneous apoptosis and bacterial rupture. This pioneering study elucidates new ideas for the regulation of artificial enzyme activity and provides new perspectives for the development of efficient antibiotic substitutes.

Decreases in metabolic ATP open KATP channels and reduce firing in an auditory brainstem neuron: A dynamic mechanism of firing control during intense activity

Cartwheel (CW) neurons are glycinergic interneurons in the dorsal cochlear nucleus (DCN) that exhibit spontaneous firing, resulting in potent tonic inhibition of fusiform neurons. CW neurons expressing open ATP-sensitive potassium (KATP) channels do not fire spontaneously, and activation of KATP channels halts spontaneous firing in these neurons. However, the conditions that regulate KATP channel opening in CW neurons remain unknown. Here, we tested the hypothesis that fluctuations in metabolic ATP levels modulate KATP channels in CW neurons. Using whole-cell patch-clamp recordings in CW neurons from young rat brain slices (p17-22) with an ATP-free internal solution, we observed that the mitochondrial uncoupler CCCP hyperpolarized the membrane potential, reduced spontaneous firing, and generated an outward current, which was inhibited by the KATP channel antagonist tolbutamide. Additionally, a glucose-free external solution quickly activated KATP channels and ceased spontaneous firing. We hypothesized that intense membrane ion ATPase activity during strong depolarization would deplete intracellular ATP, leading to KATP channel opening. Consistent with this, depolarizing CW neurons with a 250 pA DC did not increase spontaneous firing because the depolarization activated KATP channels; however, the same depolarization after tolbutamide administration increased firing, suggesting that ATP depletion triggered KATP channel opening to limit action potential firing. These results indicate that KATP channels in the DCN provide dynamic control over action potential firing, preventing excessive excitation during high-firing activity.

Construction of Chitinase Complexes Using Self-Assembly Systems for Efficient Hydrolysis of Chitin.

Chitin biomass is the second most abundant natural polysaccharide after cellulose on the earth, yet its recalcitrance to degrade and utilize severely limits its application. However, many microorganisms, such as Serratia marcescen, can secrete a range of free chitinases to degrade chitin, though their activity is typically insufficient to meet industrial demands. In this study, we employed self-assembly systems, named SpyTag/SpyCatcher and SnoopTag/SnoopCatcher, to modularize the molecular design of CHB, ChiB, ChiC, and CBP21 derived from S. marcescens ATCC14756, and we successfully constructed a variety of chitinase complexes. The assembled complexes showed higher chitinolytic activity and stability, compared to free chitinase mixture. Moreover, the distinct arrangements and combinations of chitinases within these complexes led to varied activities, suggesting that the spatial proximity and substrate channeling effects contribute to the synergy of chitinase complexes. The findings lay a solid technical foundation for the application of chitinosome in the industrial production of N-acetylglucosamine and chitooligosaccharides.

The faucet knob effect of DptE crotonylation on the initial flow of daptomycin biosynthesis

We propose here that acylation modification of actinomycete proteins is a restrictive system that limits the excessive synthesis of secondary metabolites, its mechanism has not been clearly elucidated before. We used crotonylation as an example to investigate the acylation effect in the daptomycin biosynthesis by Streptomyces roseosporus. Our experiments revealed abundant crotonylation of numerous secondary metabolic enzymes in Streptomyces roseosporus, a daptomycin producer. DptE, which initiates daptomycin biosynthesis, is crotonylated at K454. We experimentally identified the corresponding DptE crotonyltransferase Kct1 and decrotonylase CobB. Further studies consistently confirmed that decrotonylation increases DptE activity. Decrotonylation functions like loosening a faucet knob, increasing substrate channel throughput and the initial flow of daptomycin biosynthesis. Moreover, DptE catalytic activity was enhanced via K454 and neighboring residues K184 and Q420 mutation, increasing daptomycin yield by 132%; daptomycin biosynthesis related metabolism activities also increased. Substrate channel prediction revealed 38% higher throughput for mutant DptE (K454I/K184Q/Q420N) than crotonylated DptE. Molecular dynamics (MD) simulations revealed significant increases in flexibility and substrate affinity of the mutant. In summary, we elucidated the faucet knob effect of DptE crotonylation on the initial flow of daptomycin biosynthesis and adopted decrotonylation to generate high-yield industrial strains.

Engineering peroxisomal surface display for enhanced biosynthesis in the emerging yeast Kluyveromyces marxianus

The non-conventional yeast Kluyveromyces marxianus is a promising microbial host for industrial biomanufacturing. With the recent development of Cas9-based genome editing systems and other novel synthetic biology tools for K. marxianus, engineering of this yeast has become far more accessible. Enzyme colocalization is a proven approach to increase pathway flux and the synthesis of non-native products. Here, we engineer K. marxianus to enable peroxisomal surface display, an enzyme colocalization technique for displaying enzymes on the peroxisome membrane via an anchoring motif from the peroxin Pex15. The native KmPex15 anchoring motif was identified and fused to GFP, resulting in successful localization to the surface of the peroxisomes. To demonstrate the advantages for pathway localization, the Pseudomonas savastanoi IaaM and IaaH enzymes were co-displayed on the peroxisome surface; this increased production of indole-3-acetic acid 7.9-fold via substrate channeling effects. We then redirected pathway flux by displaying the violacein pathway enzymes VioE and VioD from Chromobacterium violaceum, increasing selectivity of proviolacein to prodeoxyviolacein by 2.5-fold. Finally, we improved direct access to peroxisomal acetyl-CoA and increased titers of the polyketide triacetic acid lactone (TAL) by 2-fold through concurrent display of the proteins Cat2, Acc1, and the type III PKS 2-pyrone synthase from Gerbera hybrida relative to the same three enzymes diffusing in the cytosol. We further improved TAL production by up to 2.1-fold through engineering peroxisome morphology and lifespan. Our findings demonstrate that peroxisomal surface display is an efficient enzyme colocalization strategy in K. marxianus and applicable for improving production of a wide range of non-native products.

Directionally co-immobilizing glucose oxidase and horseradish peroxidase on three-pronged DNA scaffold and the regulation of cascade activity

In traditional multienzyme random co-immobilization, it is difficult to precisely locate and regulate the relative positions between two enzyme molecules, resulting in low cascade efficiency between the two enzymes and limiting the application of multienzyme cascade catalysis technology. This study prepared PVAC@Y-dsDNA@GOD/HRP magnetic co-immobilized multienzyme by constructing a three-pronged DNA scaffold for co-coupling glucose oxidase (GOD) and horseradish peroxidase (HRP), which achieved directional co-immobilization of dual enzymes and precise regulation of inter-enzyme distance. Compared with traditional random co-immobilization of multienzyme, PVAC@Y-dsDNA@GOD/HRP could shorten the distance between GOD and HRP to the nanoscale and form substrate channeling, which greatly improved the cascade activity between the two enzymes. The inter-enzyme spacing between GOD and HRP could be precisely regulated by changing the length of DNA strands. When the inter-enzyme spacing was 10.08 nm, PVAC@Y-dsDNA@GOD/HRP exhibited high cascade activity of 707 U/mg. The inter-enzyme spacing that was too large or too small would reduce the cascade activity, indicating a distance-dependence of multienzyme cascade activity. PVAC@Y-dsDNA@GOD/HRP showed good reusability, indicating that the three-pronged DNA scaffold constructed by DNA double strands hybridization could firmly immobilize enzyme on carrier, with less enzyme leakage.

Reengineering the Substrate Tunnel to Enhance the Catalytic Efficiency of Squalene Epoxidase.

Squalene epoxidase plays a pivotal role in the biosynthesis of ergosterol, its derivatives, and other triterpenoid compounds by catalyzing the transformation of squalene into 2,3-oxidosqualene. However, its low catalytic efficiency remains a primary bottleneck for the microbial synthesis of triterpenoids. In this study, the catalytic activity of the squalene epoxidase from Saccharomyces cerevisiae was significantly improved by reshaping its substrate tunnel, resulting in a marked increase in the yield of the final product, ergosterol. First, the amino acid in the catalytic pocket of squalene epoxidase was replaced with alanine (Ala), effectively reducing the steric hindrance, and thus, enhancing the affinity of the enzyme with its substrate. Then, the V249H/L343A mutant was obtained by redesigning the substrate tunnel of dominant mutant L343A, thus, increasing the titer of ergosterol. The study also elucidated the mechanism behind the increased catalytic activity of the V249H/L343A mutant through substrate tunnel parameter analysis and molecular dynamics simulations. Finally, a titer of 3345 mg/L of ergosterol was achieved by strains containing V249H/L343A in a 5 L bioreactor, with a specific yield of 84 mg/g dry cell weight (DCW), marking a 64% increase compared with the titer achieved by wild type strains. This study established a strong foundation for improving the synthetic efficiency of ergosterol and other triterpenoid compounds.