3-Fucosyllactose (3-FL) is a key functional human milk oligosaccharide with significant applications in infant nutrition and functional foods. However, its microbial high-level production is constrained by inefficient product export and imbalanced precursor metabolic flux. This study developed a combined strategy of efflux pump engineering and metabolic flux reallocation to enhance 3-FL production in Escherichia coli. Systematic screening of multidrug efflux pumps combined with ribosome binding site (RBS) tuning identified MdfA as a transporter associated with enhanced 3-FL secretion. Considering the limited substrate compatibility of native MdfA with 3-FL, a structure-guided protein engineering was employed to generate the optimized C139A/R190L variant with enhanced transport performance. Molecular docking analysis suggested that this variant facilitates the transmembrane transport of 3-FL by altering the local microenvironment and the conformational properties of the substrate-binding cavity, thereby helping to alleviate the secretion bottleneck during 3-FL biosynthesis. Building on this, modular promoter engineering was applied to the gmd-wcaG and manC-manB gene clusters to rewire GDP-L-fucose precursor flux, achieving coordinated enhancement of transport efficiency and metabolic pathway performance. Ultimately, in 3 L fed-batch fermentation, the engineered strain reached a total 3-FL titer of 65.19 g/L, with up to 97% extracellular accumulation, representing the highest level reported to date. This study establishes a semi-rational engineering system for 3-FL efflux pump modification, providing a theoretical foundation for the efficient microbial synthesis of human milk oligosaccharides and other complex oligosaccharides.

In this study,a novel fructosyltransferase(StFFase)was screened from Streptococcus troglodytae through gene mining.The enzyme was then engineered via computer-aided rational design to improve both its catalytic efficiency and thermostability.Based on EVCoupling predictions and experimental validation,three mutants(D104E,I232S,and S684P)with significantly enhanced enzymatic activity were selected from potential candidates.Their enzymatic activities were 2.20,2.65,and 1.53 times that of the wild-type,respectively.Notably,the I232S mutant maintained high relative enzyme activity over a temperature range of 40～70℃,and its half-life at 50℃was extended to 6.60 h,demonstrating superior ther-mostability.Kinetic analysis revealed that the I232S mutant exhibited improved affinity for the substrate sucrose,with the Km value decreasing to 171 mmol/L,and a significantly enhanced catalytic efficiency.Furthermore,a conversion rate of 42.17%for 1-kestose was achieved using this mutant under conditions of 50%sucrose concentration(w/w)and an enzyme dosage of 15 U/g sucrose.Structural simulations revealed that the mutations improved catalytic performance and thermosta-bility by eliminating electrostatic repulsion(D104E),forming new hydrogen bonds(I232S),and stabilizing β-turn confor-mations(S684P).This study provides a new strategy for the rational design of fructosyltransferases and highlights the potential of the engineered enzyme for industrial fructooligosaccharide production.

Background The dual challenges of lactose intolerance and environmental pollution from whey disposal necessitate the efficient valorization of lactose. Cellobiose 2-epimerases (CEases) have emerged as promising biocatalysts for converting lactose into high-value functional sugars, specifically lactulose and epilactose. Scope and approach This review provides a comprehensive overview of the microbial sources, biochemical characteristics, structural features, and catalytic mechanisms of CEases. Furthermore, it critically analyzes recent advancements in enhancing enzymatic performance through structure-guided dynamic engineering, improving operational stability via innovative carrier-free and nano-matrix immobilization technologies, and optimizing scalable production using GRAS microbial hosts and dynamic control systems. Key findings and conclusions CEases have demonstrated great potential in the dairy industry and the synthesis of functional sugars. Advancements in AI-assisted mining have significantly accelerated the discovery of novel candidates. Mechanistically, computational breakthroughs have advanced our understanding from foundational epimerization to a dynamic “swaying mechanism” governing the dual activities of bifunctional CEases. Building on these mechanistic insights, the rational engineering of structural dynamics has significantly improved CEase catalytic efficiency and product selectivity. Moreover, the transition to GRAS expression systems enables safe, food-grade production. Future research should further integrate computational biology with robust, heavy-metal-free immobilization platforms to establish scalable, zero-waste biocatalytic processes for the functional sugar industry.

Compared with the counterparts with mononuclear metal sites, catalysts featuring dual-metal sites with suitable metal-metal distance have shown superiority in enhancing catalytic performance. However, most of heterogeneous dual-metal catalysts are characterized by poor long-range order structures, particularly when obtained via pyrolysis. Furthermore, sophisticated and complicated design of ligand is usually involved for the synthesis of catalysts with dual-metal sites. Herein, a series of mesoporous ZnCo zeolitic imidazolate frameworks were prepared through a facile in-situ hydrogel templated method. Partially replacement of Zn antenna by Co ions leads to boosted CO2 photocatalytic conversion. CO yield over optimized ZnCo reached 2064.9 μmol·g-1·h-1 during the second hour of light illumination, which is 67.9-fold higher than the value of corresponding bare bulk Zn-base ZIF under the same reaction conditions. Parallel control experiments confirmed the critical roles of Zn/Co active sites and the synergistic effect between them, which were further elucidated by in situ characterizations. This work demonstrates a straightforward strategy for construction mesoporous dual-metal-sites MOFs and highlights their potential as an ideal platform for CO2 conversion.

Tyrosinase (TYR), a copper-containing oxidase pivotal in melanin synthesis, is widely distributed across animals, plants, and microorganisms. Despite its significant potential in biotechnology and industry, its practical application is hampered by limitations such as low catalytic efficiency and poor stability. To address these constraints, a highly active type III TYR from Nitrosospira (Sp2) was identified through systematic genomic mining in this study. Based on the structural features of type III TYR, two C-terminal truncated mutants were constructed. Among them, the truncated mutant TYR-Sp2-276 achieved an enzyme activity of 317 U/mg, which is a 15% increase compared to the wild-type. Subsequent protein engineering adopted a distal design strategy, which rationally targets residues remote from the catalytic center coupled with computational simulations to construct a combinatorial mutant library. The combinatorial mutant TYR-Sp2-276-G73A/M106D/Q152A/M231P exhibited a 2.37-fold enhancement in enzymatic activity, reaching 654 U/mg. Its melting temperature (Tm) increased by 4.59 °C, while the kcat value showed a 2.55-fold improvement. Structural predictions from AlphaFold 3 and molecular docking indicate that changes in structural rigidity and microscopic interactions such as hydrogen bonding may be responsible for the enhancement of its catalytic activity and thermal stability. This work demonstrates that rational distal design is an effective strategy for optimizing enzyme properties, offering valuable insights for engineering industrially relevant microbial enzymes.

Converting non-food cellulosic sugars into programmable amylose offers a route to higher-value carbohydrate materials. α-Glucan phosphorylase (αGP) controls glucose-1-phosphate (G-1-P)-dependent chain elongation in cellobiose-to-amylose cascades. Here, Nicotiana attenuata αGP (NicαGP) was engineered by integrating AlphaFold3-based structural modeling, ProteinMPNN, EVcouplings, structural priors, and DynaMut2 filtering. Of 916 candidate substitutions, 37 were selected for experimental validation, and combinatorial screening identified L226Y/V437L/F751H as the optimal triple mutant, with 2.10-fold higher relative activity than the same-batch wild-type (WT) control and clear positive epistasis. The mutant further shifted the optimal temperature from 40 to 45 °C, increased thermal transition midpoint from 56.2 to 58.8 °C, and enhanced catalytic efficiency toward both maltotetraose and G-1-P. In the Clostridium thermocellum cellobiose phosphorylase (CtCBP)-NicαGP cascade, L226Y/V437L/F751H reached 38.86 ± 2.06 % conversion at 72 h versus the previously reported WT value of 35.84 ± 1.04 % at 84 h, increasing apparent productivity by 26.5 %. Docking, CAVER, and molecular dynamics (MD) analyses suggested that these gains may be associated with optimization of structural regions surrounding the conserved catalytic core. This work provides an engineered αGP for amylose synthesis from cellobiose and supports peripheral regulatory engineering as a practical strategy for improving phosphorylase-based biocatalytic cascades.

Zero-valent iron (Fe0) is widely applied for reductive dehalogenation but is limited by inefficient electron utilization and rapid corrosion in aqueous environments. Herein, 3D printing was employed to engineer the structural and interfacial properties of Fe0 for enhanced reductive transformation of florfenicol (FLO) under anoxic conditions. Compared with pristine Fe0 powders, the 3D-printed Fe0 (3DP-Fe0) exhibited a hierarchical porous architecture, lattice expansion, and enhanced hydrophobicity, which collectively regulated Fe0 corrosion behavior and interfacial electron transfer. These structural and interfacial modifications improved electron utilization efficiency toward FLO dehalogenation while suppressing non-productive hydrogen evolution. Mechanistic investigations revealed that atomic hydrogen was the dominant reactive species responsible for sequential FLO dechlorination. Benefiting from regulated corrosion and preserved Fe0 reactivity, 3DP-Fe0 maintained high FLO removal efficiency during repeated cycles and prolonged anoxic aging, accompanied by substantially reduced Fe0 consumption and Fe leaching. Transformation products generated through sequential dechlorination exhibited markedly decreased antibacterial activity, indicating effective toxicity reduction during FLO degradation. Furthermore, 3DP-Fe0 retained robust performance in complex water matrices and enabled efficient removal of other recalcitrant pharmaceuticals, demonstrating its broad applicability. Overall, this study highlights 3D printing as an effective strategy to enhance Fe0 reactivity and stability for the efficient reductive treatment of emerging contaminants.

Lacto-N-neotetraose (LNnT) is a core functional component of human milk oligosaccharides (HMOs) and exhibits a wide range of physiological activities. Efficient and cost-effective microbial biosynthesis is critical for the large-scale deployment of LNnT. This study constructed a high-yield Escherichia coli strain for LNnT production using a multi-level metabolic engineering strategy. The de novo biosynthesis pathway of LNnT was constructed via systematic metabolic engineering to strengthen the transport and supply of precursors, and competitive metabolic pathways were knocked out to reduce substrate flux diversion and loss. Semi-rational engineering of LgtA and LgtB was performed based on EVcouplings co-evolutionary analysis, combined with 5’UTR engineering to optimize translation efficiency. The final engineered strain ZBASU-49 achieved an LNnT titer of 39.85 g/L in 3-L fed-batch fermentation, providing a reproducible innovative strategy and technical support for the efficient industrial microbial biosynthesis of LNnT.

The development of economical, highly efficient, and stable bifunctional electrocatalysts for both the oxygen evolution reaction (OER) and the oxygen reduction reaction (ORR) remains a critical focus in advancing rechargeable metal-air battery systems. Significant progress has been made in the design of high-performance bifunctional electrocatalysts, the development of novel oxygen electrode architectures, and the in-depth understanding of electrocatalytic mechanisms through combined experimental and computational studies. This work provides a comprehensive review of recent advancements in design strategies for oxygen catalysts, including homogeneous electrodes, asymmetric electrodes, and biomimetic electrodes, are thoroughly discussed and summarized. Then, the advanced catalyst modification strategies for ORR/OER are summarized, focusing on critical factors such as enhancement effect of metal/nonmental and synergistic enhancement effect in multiple catalyst. Subsequently, a representative performance evaluation is presented, based on the reported oxygen electrodes used in rechargeable metal-air battery applications. By focusing on these key areas, the review outlines the current challenges and future prospects for the development of bifunctional oxygen electrocatalysts, aiming to guide the design of high-performance bifunctional electrocatalysts and to elucidate the underlying mechanisms involved.

Human lactoferrin is widely used in medical and nutritional fields. However, current production methods, which rely heavily on extraction from bovine or ovine milk, suffer from long production cycles, high costs, and structural and functional differences from the human protein. To address these challenges, this study constructed a Komagataella phaffii (K. phaffii) cell factory using methanol as the sole carbon source, aiming to develop an efficient and sustainable biosynthetic route for human lactoferrin. During the expression design phase, a novel configuration formed by the "CAAAAC" Kozak sequence and the PHO11α signal peptide achieved a secreted yield of 26.1 mg/L (strain K1). Building on this, multi-copy integration using rDNA non-transcribed regions increased the yield to 60.5 mg/L, but also revealed a production bottleneck associated with simply increasing copy number. To address this, fluorescence protein monitoring technology was introduced to precisely dissect the bottleneck, guiding subsequent multi-layer metabolic engineering that further increased the yield to 90.3 mg/L. Critically, AFT1 and SEC12P were identified for the first time in K. phaffii as novel positive regulatory targets. Additionally, morphological engineering confirmed that deletion of the PHA1 gene synergistically enhanced protein accumulation. Ultimately, the engineered strain integrating these multi-layer strategies achieved a human lactoferrin titer of 690 mg/L in 3-L fed-batch fermentation, a leading level among reported heterologous synthesis systems. This study not only provides a new efficient route for human lactoferrin production but also offers new insights for the advanced engineering of K. phaffii as a protein synthesis factory.

A predictive linkage between operating variables and amylose architecture is needed because current phosphorylase-based syntheses from non-food cellulose, though valuable, lack a process-structure-property map for design and manufacturing. The dual-enzyme cascade reaction for amylose synthesis was investigated by systematic variation of temperature, pH, enzyme stoichiometry and addition timing, and the cellobiose/maltotetraose primer ratio, coupled with time-resolved monitoring and multiscale characterization. Under the optimized conditions (pH 5.0, 50 °C), varying the cellobiose/maltotetraose ratio from 1:0.125 to 1:32 enabled the CtCBP-NicαGP cascade to produce amylose spanning 1821 ± 20.06-69,252 ± 30.02 g/mol, with a maximum yield of 35.84%. The concurrent delineation of quantitative distribution curves for cellobiose, glucose-1-phosphate, glucose, and inorganic phosphate revealed the synthesis reaction to be influenced by glucose threshold and delineated into three stages. The availability of primer was found to be the primary variable in programming chain length, polymorph selection (V-B → V-C → A-V), morphology (granules → aggregates → films), and thermal degradation and gelatinisation properties. Overall, this study provides a predictive framework for synthesising amylose with controllable structure and properties, while also outlining the pathway and theoretical basis for the large-scale conversion of non-food cellulose into high-value carbohydrates.

Fucosylated human milk oligosaccharides (FHMOs), a pivotal subclass of human milk oligosaccharides, are essential for neonatal health, performing diverse core physiological functions: prebiotic activity, pathogen adhesion inhibition, immune modulation, intestinal barrier integrity maintenance, and neurocognitive development promotion via the gut-brain axis. Representative FHMOs including 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL) and lacto-N-fucopentaose I (LNFP I) have obtained safety certification from the U.S. Food and Drug Administration and been commercially utilized in infant formula as functional additives. This review focuses on the structural classification and diversity of FHMOs. It comprehensively summarizes the molecular mechanisms underlying their multifunctional roles, systematically elaborates on core biomanufacturing strategies including enzymatic synthesis and microbial metabolic engineering, and reviews advances in their safety assessment, global regulatory status and commercial applications. Aiming at the industrial bottlenecks including low synthesis efficiency of structurally complex FHMOs and urgent demand for key enzyme optimization, this review analyzes the feasibility and development trends of various biological preparation methods, summarizes the cutting-edge interdisciplinary research progress, and prospects the future research directions and potential technological breakthroughs of FHMOs. This review is expected to provide a comprehensive theoretical reference and practical guidance for the in-depth research, development and industrialization of FHMOs.

Non-food cellulose bioconversion to amylose offers a promising avenue for sustainable biomanufacturing while maximizing non-food biomass utilization. Nevertheless, current alpha-glucan phosphorylases (alpha GPs) exhibit insufficient thermostability and suboptimal catalytic performance, thereby limiting their feasibility for industrial implementation. In this study, a novel thermostable alpha GP from Dictyoglomus sp. NZ13-RE01 (Dsp alpha GP) was identified and characterized through computational genome mining. The specific enzyme activity of Dsp alpha GP was 36.3 f 2.1 U/mg under the optimal conditions of temperature 70 degrees C and pH 5.0. The enzyme thermal melting temperature (Tm) was 82.26 degrees C, and the residual activity retained 55.5 % after stored at 70 degrees C for 12 h. These results demonstrated the enzyme an exceptional catalytic efficiency and remarkable thermal stability. Kinetic analysis revealed superior substrate specificity toward maltotriose and maltotetraose with kcat/Km values of 1.96 and 1.83 mM-1s-1, respectively. Integration of Dsp alpha GP with Clostridium thermocellum cellobiose phosphorylase established a dual-enzyme cascade system capable of synthesizing amylose with tunable molecular weight (7392-16,632 g/mol) by adjusting the cellobiose/G4 ratio, a conversion efficiency of 35.67 % was achieved, significantly surpassing existing systems. Site-directed mutagenesis combined with molecular docking revealed that conserved residues Gly32, Arg364, Lys370, Tyr432, and Lys464 play critical roles in substrate binding and catalytic processes. This study provides enhanced process efficiency and operational stability for sustainable carbohydrate bioconversion applications and promoting valorization of non-edible cellulose resources.

α-1,3-Fucosyltransferase (α-1,3-FucT), the rate-limiting enzyme in the biosynthesis of the human milk oligosaccharide 3-fucosyllactose (3-FL), suffers from limited industrial applicability due to its low catalytic activity and poor thermostability. Herein, we systematically optimized the catalytic performance and thermostability of M32-H10 using computer-aided design coupled with a greedy combinatorial strategy. Four computational tools identified 107 potential mutation sites, and subsequent library screening yielded six beneficial single mutants (S98R, K132I, E134M, D147P, N199F and K301P), exhibiting 28%-84% higher specific activity and improved thermostability (ΔTm increased by 0.22 ∼ 4.87 °C). A stepwise combinatorial approach further generated a quadruple mutant (K132I-E134M-N199F-K301P), which demonstrated synergistic effects: a 116% increase in enzymatic activity over the wild-type, an extended half-life (8.05 h at 40 °C), and robust catalytic efficiency across a broad pH range (5.0 ∼ 8.5). Structural analysis revealed that the mutations optimized substrate binding and conformational stability by remodeling the hydrophobic cluster (I132-M134-F199-P301), strengthening the hydrogen-bond network, and fine-tuning the local rigidity-flexibility balance. This study provides an efficient strategy for enzyme rational design and highlights the critical role of combinatorial mutations in industrial enzyme engineering.

6'-Sialyllactose (6 '-SL) is the most abundant sialylated oligosaccharide in breast milk, recognized for its beneficial effects on human health, particularly in infant brain development. This study focused on enhancing the de novo synthesis of 6 '-SL in engineered E. coli. A glutamine cycling system was established, and genes encoding collateral pathways in the 6 '-SL metabolic pathway were inhibited. To overcome the bottleneck posed by the rate-limiting enzyme alpha 2,6-sialyltransferase (alpha 2,6-siaT), a multi-level combinatorial strategy was employed, including screening for ribosome-binding site (RBS) substitutions, fusion protein creation, and multi-copy gene expression. This approach facilitated the efficient utilization of alpha 2,6-siaT in the 6 '-SL biosynthetic pathway. Additionally, coordinated expression of cytidine triphosphate (CTP) cofactor engineering optimized the intracellular supply of the precursor cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) and synchronized the synthesis rate of 6 '-SL, resulting in the high-yielding strain KA34. Strain KA34 achieved a titer of 3.85 g/L in shake flask cultures and 25.31 g/L in a 3-L bioreactor, with a productivity of 0.36 g/L/h and a lactose conversion yield of 0.55 mol 6'-SL/mol. This study demonstrates a successful strategy for optimizing the microbial production of 6'SL.

Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) is crucial for lactulose production; however, its low isomerization activity and poor thermostability limit its industrial application. Herein, four single-point mutants (S70D, F171W, S173L, and L354F) with significantly improved isomerization activity were obtained using a computer-aided semi-rational design, and combined mutants were introduced. The isomerization specific activities of the mutants CsCE-F171W/S173L/L354F and CsCE-S70D/F171W/S173L/L354F were 2.10 and 2.05 times higher than that of wild-type CsCE, respectively. Enzymatic properties revealed that the mutant CsCE-F171W/S173L/L354F demonstrated a 2.50-fold increase in t1/2 at 80 °C and a 2.70-fold improvement in kcat, while the mutant CsCE-S70D/F171W/S173L/L354F exhibited a 2.91-fold increase in t1/2 at 80 °C and a 3.19-fold improvement in kcat. Three-dimensional structural analysis suggested that conformational changes in the flexible ring induced by mutation could enhance isomerization activity, while increased structural rigidity, hydrophobic contacts, van der Waals forces, and favorable electrostatic potentials may promote thermostability. These findings provide novel insights into the molecular modification of CsCE and significantly enhance its potential for industrial applications.

3-Fucosyllactose (3-FL), a functional oligosaccharide in human milk, holds great promise due to its health benefits. However, its microbial production is limited by the low activity of α-1,3-fucosyltransferase and the limited GDP-l-fucose supply. This study combined structure-based modeling and evolutionary analysis to identify five double-site mutants with enhanced 3-FL production. When the best-performing mutant N199V/K301P was expressed in double-copy, the 3-FL titer increased by 35 % (4.92 g/L) compared to the wild-type (3.65 g/L). To improve carbon flux and GDP-l-fucose supply, the engineered Escherichia coli strain L (BL21(DE3)ΔlacZΔwcaJΔnudDΔlon) was further modified by deleting mtlD, wcaE, and wcaI-the latter two functionally validated for the first time in 3-FL biosynthesis. Through the synergistic optimization of these strategies, the best strains l-MEI carrying double-copy S98R/D340E or N199V/K301P achieved 3-FL titers of 53.88 g/L and 54.64 g/L in 3 L fed-batch fermentations, which are at relatively high levels among currently reported titers.

Fucoidanase have received widespread attention as important tool enzymes for degrading fucoidan. This study engineered FcnA, a GH107-family fucoidanase from Mariniflexile fucanivorans, to enhance its catalytic efficiency and thermal stability. Through rational design and screening, we obtained several improved variants including four single mutants (L46R, Y290F, D361T, N374D), two double mutants (L46R/Y290F, D361T/N374D), and one triple mutant (L46R/Y290F/N374D). Of these, L46R/Y290F/N374D showed the greatest increase in enzyme activity, which was 2.67 times that of the WT. Thermal stability was significantly improved in all combinatorial mutants, with L46R/Y290F/N374D showing the greatest enhancement. Notably, mutant D361T/N374D exhibited a shift in optimum pH from 7.5 to 6.0. The enzyme activities were facilitated by Fe2+. Kinetic analysis revealed superior catalytic efficiency in mutants L46R/Y290F (kcat/Km = 122.97 mL s- 1 center dot mg- 1) and L46R/ Y290F/N374D (131.33 mL s- 1 center dot mg- 1) compared to WT (64.3 mL s- 1 center dot mg- 1). Analysis of the surface electrostatic potential and intermolecular interaction forces before and after mutation at each mutation site showed corroboration with the thermal stability results. By performing MD simulations on FcnA, the results showed that the structural rigidity increased and the structure became more stable after the mutation. This work establishes a framework for engineering robust fucoidanase, significantly advancing their industrial utility.

Abundant porosity, sufficient active sites, and appropriate stability are crucial factors for metal-organic frameworks (MOFs) designed for photocatalysis. Herein, a controllable partial pyrolysis strategy was employed to synthesize hierarchically porous UiO-66-X (X denotes the mass ratio of H2BDC-NH2 in ligands) with adjustable coordinated unsaturated zirconium sites and interconnected mesoporous structure. By taking full advantage of linker instability in MOFs, which is usually viewed as an undesirable trait of MOFs, this controllable thermolysis involved heat treatment of the original UiO-66-X, leading to selective partial decomposition and permanent mesopores within the structure. Meanwhile, dangling functional amino groups could serve as anchoring sites for cocatalyst and CO2 molecules. Cu nanoclusters were successfully incorporated into hierarchically porous MOFs by a structure engineering approach, yielding a novel photocatalyst. A mutually active mechanism was put forward and illustrated in this work. Mechanistic investigation reveals that mesoporous structures in a catalyst not only offer three-dimensional (3D) interconnected gas transport channels but also provide sufficient space for accommodating the introduced Cu nanoclusters, which served as active sites and efficiently induced a CO2 photoreduction reaction. At the optimal ratio, the photocatalyst exhibited superior photocatalytic activity, achieving a CO yield of 121.64 μmol g-1 under 5 h of the stimulated solar irradiation without any photosensitizer.

The enzymatic synthesis of amylose is a sustainable solution for the valorisation of non-food cellulose. However, the limited thermostability and catalytic efficiency of existing α-glucan phosphorylases (αGPs) hinders its potential for industrial scalability. In this study, we identified and characterised a novel αGP from Nicotiana attenuata (NicαGP) via genome mining. It exhibited superior catalytic performance, with a specific activity of 82.1 U/mg under optimised conditions (40 °C and pH 5.0), and notable temperature and pH stability. Supplementation of Mg2+ further enhanced its activity by 5 %. Kinetic analysis revealed a catalytic efficiency (kcat/Km) of 27.46 mM-1 s-1 for maltotetraose (G4). Furthermore, a dual-enzyme cascade system integrating NicαGP with Clostridium thermocellum cellobiose phosphorylase achieved up to 28.4 % cellulose conversion, synthesising amylose with tunable molecular weights (6602 ± 47.13-67,154 ± 47.28 g/mol) by modulating ratios of cellobiose/G4. Molecular docking identified the conserved residues (Gly196, Arg706, Lys711, and Lys821) critical for substrate binding and pyridoxal 5'-phosphate-mediated catalysis. Overall, this study provides a new enzyme component for the artificial synthesis of amylose from non-edible biomass, facilitating the widespread application of non-edible cellulosic resources for food conversion.