Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis.

Understanding enzyme catalysis as connected to protein motions is a major challenge. Here, based on temperature kinetic studies combined with isotope effect measurements, we obtain energetic description of C-H activation in NAD-dependent UDP-glucuronic acid C4 epimerase. Approach from the ensemble-averaged ground state (GS) to the transition state-like reactive conformation (TSRC) involves, alongside uptake of heat ( Δ H ‡ = 54 kJ mol-1), significant loss in entropy ( - T Δ S ‡ = 20 kJ mol-1; 298 K) and negative activation heat capacity ( Δ C p ‡ = -0.64 kJ mol-1 K-1). Thermodynamic changes suggest the requirement for restricting configurational freedom at the GS to populate the TSRC. Enzyme variants affecting the electrostatic GS preorganization reveal active-site interactions important for precise TSRC sampling and H-transfer. Collectively, our study captures thermodynamic effects associated with TSRC sampling and establishes rigid positioning for C-H activation in an enzyme active site that requires conformational flexibility in fulfillment of its natural epimerase function.

Reflections on the Origin of Coded Protein Biosynthesis.

The principle of continuity posits that some central features of primordial biocatalytic mechanisms should still be present in the genetically dependent pathway of protein synthesis, a crucial step in the emergence of life. Key bimolecular reactions of this process are catalyzed by DNA-dependent RNA polymerases, aminoacyl-tRNA synthetases, and ribosomes. Remarkably, none of these biocatalysts contribute chemically active groups to their respective reactions. Instead, structural and functional studies have demonstrated that nucleotidic α-phosphate and ß-d-ribosyl 2' OH and 3' OH groups can help their own catalysis, a process which, consequently, has been called "substrate-assisted". Furthermore, upon binding, the substrates significantly lower the entropy of activation, exclude water from these catalysts' active sites, and are readily positioned for a reaction. This binding mode has been described as an "entropy trap". The combination of this effect with substrate-assisted catalysis results in reactions that are stereochemically and mechanistically simpler than the ones found in most modern enzymes. This observation is consistent with the way in which primordial catalysts could have operated; it may also explain why, thanks to their complementary reactivities, ß-d-ribose and phosphate were naturally selected to be the central components of early coding polymers.

The Importance of Spin-Polarized Charge Reorganization in the Catalytic Activity of D-Glucose Oxidase.

The front cover artwork is provided by Prof. Ron Naaman's group at the Weizmann Institute of Science. The image shows that direct electron transfer through GOx is governed by electron spins, which result from the chiral-induced spin selectivity (CISS) effect. Read the full text of the Research Article at 10.1002/cphc.202400033.

Cell-free biosynthesis and engineering of ribosomally synthesized lanthipeptides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with diverse chemical structures and potent biological activities. A vast majority of RiPP gene clusters remain unexplored in microbial genomes, which is partially due to the lack of rapid and efficient heterologous expression systems for RiPP characterization and biosynthesis. Here, we report a unified biocatalysis (UniBioCat) system based on cell-free gene expression for rapid biosynthesis and engineering of RiPPs. We demonstrate UniBioCat by reconstituting a full biosynthetic pathway for de novo biosynthesis of salivaricin B, a lanthipeptide RiPP. Next, we delete several protease/peptidase genes from the source strain to enhance the performance of UniBioCat, which then can synthesize and screen salivaricin B variants with enhanced antimicrobial activity. Finally, we show that UniBioCat is generalizable by synthesizing and evaluating the bioactivity of ten uncharacterized lanthipeptides. We expect UniBioCat to accelerate the discovery, characterization, and synthesis of RiPPs.

Platform-directed allostery and quaternary structure dynamics of SAMHD1 catalysis.

SAMHD1 regulates cellular nucleotide homeostasis, controlling dNTP levels by catalysing their hydrolysis into 2'-deoxynucleosides and triphosphate. In differentiated CD4+ macrophage and resting T-cells SAMHD1 activity results in the inhibition of HIV-1 infection through a dNTP blockade. In cancer, SAMHD1 desensitizes cells to nucleoside-analogue chemotherapies. Here we employ time-resolved cryogenic-EM imaging and single-particle analysis to visualise assembly, allostery and catalysis by this multi-subunit enzyme. Our observations reveal how dynamic conformational changes in the SAMHD1 quaternary structure drive the catalytic cycle. We capture five states at high-resolution in a live catalytic reaction, revealing how allosteric activators support assembly of a stable SAMHD1 tetrameric core and how catalysis is driven by the opening and closing of active sites through pairwise coupling of active sites and order-disorder transitions in regulatory domains. This direct visualisation of enzyme catalysis dynamics within an allostery-stabilised platform sets a precedent for mechanistic studies into the regulation of multi-subunit enzymes.

Boron catalysis in a designer enzyme.

Enzymes play an increasingly important role in improving the benignity and efficiency of chemical production, yet the diversity of their applications lags heavily behind chemical catalysts as a result of the relatively narrow range of reaction mechanisms of enzymes. The creation of enzymes containing non-biological functionalities facilitates reaction mechanisms outside nature's canon and paves the way towards fully programmable biocatalysis1-3. Here we present a completely genetically encoded boronic-acid-containing designer enzyme with organocatalytic reactivity not achievable with natural or engineered biocatalysts4,5. This boron enzyme catalyses the kinetic resolution of hydroxyketones by oxime formation, in which crucial interactions with the protein scaffold assist in the catalysis. A directed evolution campaign led to a variant with natural-enzyme-like enantioselectivities for several different substrates. The unique activation mode of the boron enzyme was confirmed using X-ray crystallography, high-resolution mass spectrometry (HRMS) and 11B NMR spectroscopy. Our study demonstrates that genetic-code expansion can be used to create evolvable enantioselective enzymes that rely on xenobiotic catalytic moieties such as boronic acids and access reaction mechanisms not reachable through catalytic promiscuity of natural or engineered enzymes.

An In Vitro Hybrid Biocatalytic System Enabled by a Combination of Surface-Displayed, Purified, and Cell-Free Expressed Enzymes.

Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to (S)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide-protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (S)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 µM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.

Improved Enzymatic Production of the Fucosylated Human Milk Oligosaccharide LNFP II with GH29B α-1,3/4-l-Fucosidases.

Five GH29B α-1,3/4-l-fucosidases (EC 3.2.1.111) were investigated for their ability to catalyze the formation of the human milk oligosaccharide lacto-N-fucopentaose II (LNFP II) from lacto-N-tetraose (LNT) and 3-fucosyllactose (3FL) via transglycosylation. We studied the effect of pH on transfucosylation and hydrolysis and explored the impact of specific mutations using molecular dynamics simulations. LNFP II yields of 91 and 65% were obtained for the wild-type SpGH29C and CpAfc2 enzymes, respectively, being the highest LNFP II transglycosylation yields reported to date. BbAfcB and BiAfcB are highly hydrolytic enzymes. The results indicate that the effects of pH and buffer systems are enzyme-dependent yet relevant to consider when designing transglycosylation reactions. Replacing Thr284 in BiAfcB with Val resulted in increased transglycosylation yields, while the opposite replacement of Val258 in SpGH29C and Val289 CpAfc2 with Thr decreased the transfucosylation, confirming a role of Thr and Val in controlling the flexibility of the acid/base loop in the enzymes, which in turn affects transglycosylation. The substitution of an Ala residue with His almost abolished secondary hydrolysis in CpAfc2 and BbAfcB. The results are directly applicable in the enhancement of transglycosylation and may have significant implications for manufacturing of LNFP II as a new infant formula ingredient.

Stereoselective amino acid synthesis by photobiocatalytic oxidative coupling.

Photobiocatalysis-where light is used to expand the reactivity of an enzyme-has recently emerged as a powerful strategy to develop chemistries that are new to nature. These systems have shown potential in asymmetric radical reactions that have long eluded small-molecule catalysts1. So far, unnatural photobiocatalytic reactions are limited to overall reductive and redox-neutral processes2-9. Here we report photobiocatalytic asymmetric sp3-sp3 oxidative cross-coupling between organoboron reagents and amino acids. This reaction requires the cooperative use of engineered pyridoxal biocatalysts, photoredox catalysts and an oxidizing agent. We repurpose a family of pyridoxal-5'-phosphate-dependent enzymes, threonine aldolases10-12, for the α-C-H functionalization of glycine and α-branched amino acid substrates by a radical mechanism, giving rise to a range of α-tri- and tetrasubstituted non-canonical amino acids 13-15 possessing up to two contiguous stereocentres. Directed evolution of pyridoxal radical enzymes allowed primary and secondary radical precursors, including benzyl, allyl and alkylboron reagents, to be coupled in an enantio- and diastereocontrolled fashion. Cooperative photoredox-pyridoxal biocatalysis provides a platform for sp3-sp3 oxidative coupling16, permitting the stereoselective, intermolecular free-radical transformations that are unknown to chemistry or biology.

Immobilized Dipeptidase in Manganese Ion-Loaded Polyethylenimine-Induced Calcium Phosphate Nanocrystals for Carnosine Synthesis.

Carnosine is a natural bioactive dipeptide with important physiological functions widely used in food and medicine. Dipeptidase (PepD) from Serratia marcescens can catalyze the reverse hydrolytic reaction of ß-alanine with l-histidine to synthesize carnosine in the presence of Mn2+. However, it remains challenging to practice carnosine biosynthesis due to the low activity and high cost of the enzyme. Therefore, the development of biocatalysts with high activity and stability is of significance for carnosine synthesis. Here, we proposed to chelate Mn2+ to polyethylenimine (PEI) that induced rapid formation of calcium phosphate nanocrystals (CaP), and Mn-PEI@CaP was used for PepD immobilization via electrostatic interaction. Mn-PEI@CaP as the carrier enhanced the stability of the immobilized enzyme. Moreover, Mn2+ loaded in the carrier acted as an in situ activator of the immobilized PepD for facilitating the biocatalytic process of carnosine synthesis. The as-prepared immobilized enzyme (PepD-Mn-PEI@CaP) kept similar activity with free PepD plus Mn2+ (activity recovery, 102.5%), while exhibiting elevated thermal stability and pH tolerance. Moreover, it exhibited about two times faster carnosine synthesis than the free PepD system. PepD-Mn-PEI@CaP retained 86.8% of the original activity after eight cycles of batch catalysis without the addition of free Mn2+ ions during multiple cycles. This work provides a new strategy for the co-immobilization of PepD and Mn2+, which greatly improves the operability of the biocatalysis and demonstrates the potential of the immobilized PepD system for efficient carnosine synthesis.

Novel Catechol O-methyltransferases from Lentinula edodes Catalyze the Generation of Taste-Active Flavonoids.

Due to the increasing demand for natural food ingredients, including taste-active compounds, enzyme-catalyzed conversions of natural substrates, such as flavonoids, are promising tools to align with the principles of Green Chemistry. In this study, a novel O-methyltransferase activity was identified in the mycelium of Lentinula edodes, which was successfully applied to generate the taste-active flavonoids hesperetin, hesperetin dihydrochalcone, homoeriodictyol, and homoeriodictyol dihydrochalcone. Furthermore, the mycelium-mediated OMT activity allowed for the conversion of various catecholic substrates, yielding their respective (iso-)vanilloids, while monohydroxylated compounds were not converted. By means of a bottom-up proteomics approach, three putative O-methyltransferases were identified, and subsequently, synthetic, codon-optimized genes were heterologously expressed in Escherichia coli. The purified enzymes confirmed the biocatalytic O-methylation activity against targeted flavonoids containing catechol motifs.

Synergistic Photoenzymatic Catalysis Enables Synthesis of a-Tertiary Amino Acids Using Threonine Aldolases.

a-Tertiary amino acids are essential components of drugs and agrochemicals, yet traditional syntheses are step-intensive and provide access to a limited range of structures with varying levels of enantioselectivity. Here, we report the α-alkylation of unprotected alanine and glycine by pyridinium salts using pyridoxal (PLP)-dependent threonine aldolases with a Rose Bengal photoredox catalyst. The strategy efficiently prepares various a-tertiary amino acids in a single chemical step as a single enantiomer. UV-vis spectroscopy studies reveal a ternary interaction between the pyridinium salt, protein, and photocatalyst, which we hypothesize is responsible for localizing radical formation to the active site. This method highlights the opportunity for combining photoredox catalysts with enzymes to reveal new catalytic functions for known enzymes.

Photocatalytic regeneration of nicotinamide cofactor biomimetics drives biocatalytic reduction by Old Yellow enzymes.

A key approach in developing green chemistry involves converting solar energy into chemical energy of biomolecules through photocatalysis. Photocatalysis can facilitate the regeneration of nicotinamide cofactors during redox processes. Nicotinamide cofactor biomimetics (NCBs) are economical substitutes for natural cofactors. Here, photocatalytic regeneration of NADH and reduced NCBs (NCBsred) using graphitic carbon nitride (g-C3N4) was developed. The process involves g-C3N4 as the photocatalyst, Cp*Rh(bpy)H2O2+ as the electron mediator, and Triethanolamine as the electron donor, facilitating the reduction of NAD+ and various oxidative NCBs (NCBsox) under light irradiation. Notably, the highest reduction yield of 48.32 % was achieved with BANA+, outperforming the natural cofactor NAD+. Electrochemical analysis reveals that the reduction efficiency and capacity of cofactors relies on their redox potentials. Additionally, a coupled photo-enzymatic catalysis system was explored for the reduction of 4-Ketoisophorone by Old Yellow Enzyme XenA. Among all the NCBsox and NAD+, the highest conversion ratio of over 99 % was obtained with BANA+. After recycled for 8 times, g-C3N4 maintained over 93.6 % catalytic efficiency. The photocatalytic cofactor regeneration showcases its outstanding performance with NAD+ as well as NCBsox. This work significantly advances the development of photocatalytic cofactor regeneration for artificial cofactors and its potential application.

Elucidation of the Catalytic Sequence of Dihydroorotate Dehydrogenase B from Lactoccocus lactis: Evidence for Accumulation of a Flavin Bisemiquinone State in Catalysis.

The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by ∼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.

Myosin-Catalyzed ATP Hydrolysis in the Presence of Disease-Causing Mutations: Mavacamten as a Way to Repair Mechanism.

Hypertrophic cardiomyopathy is one of the most common forms of genetic cardiomyopathy. Mavacamten is a first-in-class myosin modulator that was identified via activity screening on the wild type, and it is FDA-approved for the treatment of obstructive hypertrophic cardiomyopathy (HCM). The drug selectively binds to the cardiac ß-myosin, inhibiting myosin function to decrease cardiac contractility. Though the drug is thought to affect multiple steps of the myosin cross-bridge cycle, its detailed mechanism of action is still under investigation. Individual steps in the overall cross-bridge cycle must be queried to elucidate the full mechanism of action. In this study, we utilize the rare-event method of transition path sampling to generate reactive trajectories to gain insights into the action of the drug on the dynamics and rate of the ATP hydrolysis step for human cardiac ß-myosin. We study three known HCM causative myosin mutations: R453C, P710R, and R712L to observe the effect of the drug on the alterations caused by these mutations in the chemical step. Since the crystal structure of the drug-bound myosin was not available at the time of this work, we created a model of the drug-bound system utilizing a molecular docking approach. We find a significant effect of the drug in one case, where the actual mechanism of the reaction is altered from the wild type by mutation. The drug restores both the rate of hydrolysis to the wildtype level and the mechanism of the reaction. This is a way to check the effect of the drug on untested mutations.

Discovery of Potent Glycosidases Enables Quantification of Smoke-Derived Phenolic Glycosides through Enzymatic Hydrolysis.

When grapes are exposed to wildfire smoke, certain smoke-related volatile phenols (VPs) can be absorbed into the fruit, where they can be then converted into volatile-phenol (VP) glycosides through glycosylation. These volatile-phenol glycosides can be particularly problematic from a winemaking standpoint as they can be hydrolyzed, releasing volatile phenols, which can contribute to smoke-related off-flavors. Current methods for quantitating these volatile-phenol glycosides present several challenges, including the requirement of expensive capital equipment, limited accuracy due to the molecular complexity of the glycosides, and the utilization of harsh reagents. To address these challenges, we proposed an enzymatic hydrolysis method enabled by a tailored enzyme cocktail of novel glycosidases discovered through genome mining, and the generated VPs from VP glycosides can be quantitated by gas chromatography-mass spectrometry (GC-MS). The enzyme cocktails displayed high activities and a broad substrate scope when using commercially available VP glycosides as the substrates for testing. When evaluated in an industrially relevant matrix of Cabernet Sauvignon wine and grapes, this enzymatic cocktail consistently achieved a comparable efficacy of acid hydrolysis. The proposed method offers a simple, safe, and affordable option for smoke taint analysis.

A New Approach in Lipase-Octyl-Agarose Biocatalysis of 2-Arylpropionic Acid Derivatives.

The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base-pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (eep = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity.

Enantiodivergent kinetic resolution of 4-substituted 1,2,3,4-tetrahydroquinolines employing amine oxidase.

Optically pure 1,2,3,4-tetrahydroquinolines (THQs) represent a class of important motifs in many natural products and pharmaceutical agents. While recent advances on redox biocatalysis have demonstrated the great potential of amine oxidases, all the transformations focused on 2-substituted THQs. The corresponding biocatalytic method for the preparation of chiral 4-substituted THQs is still challenging due to the poor activity and stereoselectivity of the available enzyme. Herein, we developed a biocatalytic kinetic resolution approach for enantiodivergent synthesis of 4-phenyl- or alkyl-substituted THQs. Through structure-guided protein engineering of cyclohexylamine oxidase derived from Brevibacterium oxidans IH-35 A (CHAO), the variant of CHAO (Y215H/Y214S) displayed improved specific activity toward model substrate 4-phenyl substituted THQ (0.14 U/mg, 13-fold higher than wild-type CHAO) with superior (R)-stereoselectivity (E > 200). Molecular dynamics simulations show that CHAO Y215H/Y214S allows a suitable substrate positioning in the expanded binding pocket to be facilely accessed, enabling enhanced activity and stereoselectivity. Furthermore, a series of 4-alkyl-substituted THQs can be transformed by CHAO Y215H/Y214S, affording R-isomers with good yields (up to 50 %) and excellent enantioselectivity (up to ee > 99 %). Interestingly, the monoamine oxidase from Pseudomonas fluorescens Pf0-1 (PfMAO1) with opposite enantioselectivity was also mined. Together, this system enriches the kinetic resolution methods for the synthesis of chiral THQs.

Discovery and Characterization of Pyridoxal 5'-Phosphate-Dependent Cycloleucine Synthases.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are the most versatile biocatalysts for synthesizing nonproteinogenic amino acids. α,α-Disubstituted quaternary amino acids, such as 1-aminocyclopentane-1-carboxylic acid (cycloleucine), are useful building blocks for pharmaceuticals. In this study, starting with the biosynthesis of fusarilin A, we discovered a family of PLP-dependent enzymes that can facilitate tandem carbon-carbon forming steps to catalyze an overall [3 + 2]-annulation. In the first step, the cycloleucine synthases use SAM as the latent electrophile and an in situ-generated enamine as the nucleophile for γ-substitution. Whereas previously characterized γ-replacement enzymes protonate the resulting α-carbon and release the acyclic amino acid, cycloleucine synthases can catalyze an additional, intramolecular aldol or Mannich reaction with the nucleophilic α-carbon to form the substituted cyclopentane. Overall, the net [3 + 2]-annulation reaction can lead to 2-hydroxy or 2-aminocycloleucine products. These studies further expand the biocatalytic scope of PLP-dependent enzymes.

Engineering a Carotenoid Cleavage Oxygenase for Coenzyme-Free Synthesis of Vanillin from Ferulic Acid.

One-pot biosynthesis of vanillin from ferulic acid without providing energy and cofactors adds significant value to lignin waste streams. However, naturally evolved carotenoid cleavage oxygenase (CCO) with extreme catalytic conditions greatly limited the above pathway for vanillin bioproduction. Herein, CCO from Thermothelomyces thermophilus (TtCCO) was rationally engineered for achieving high catalytic activity under neutral pH conditions and was further utilized for constructing a one-pot synthesis system of vanillin with Bacillus pumilus ferulic acid decarboxylase. TtCCO with the K192N-V310G-A311T-R404N-D407F-N556A mutation (TtCCOM3) was gradually obtained using substrate access channel engineering, catalytic pocket engineering, and pocket charge engineering. Molecular dynamics simulations revealed that reducing the site-blocking effect in the substrate access channel, enhancing affinity for substrates in the catalytic pocket, and eliminating the pocket's alkaline charge contributed to the high catalytic activity of TtCCOM3 under neutral pH conditions. Finally, the one-pot synthesis of vanillin in our study could achieve a maximum rate of up to 6.89 ± 0.3 mM h-1. Therefore, our study paves the way for a one-pot biosynthetic process of transforming renewable lignin-related aromatics into valuable chemicals.