Multi-enzyme cascade catalysis has become a pivotal technology in green biomanufacturing and the synthesis of fine chemicals, facilitating efficient and highly selective transformations through the mimicry of natural metabolic pathways. Despite considerable advances in single-enzyme engineering, substantial challenges persist in enhancing the overall catalytic efficiency of multi-enzyme systems. Limitations such as inadequate inter-enzyme synergy and low operational stability significantly impede their scalable implementation. Thus, achieving precise spatial organization of multi-enzyme systems to emulate synergistic functions and improve stability within artificial environments has emerged as a crucial strategy to overcome existing bottlenecks. This review systematically categorizes multi-enzyme cascade reactions into five modular types (linear, parallel, orthogonal, cyclic, and triangular) and classifies assembly materials into non-biological (synthetic polymer and inorganic material) and biological categories (polysaccharide, nucleic acid, protein, and organism). Drawing on current literature, we systematically examine diverse enzyme assembly strategies, highlight state-of-the-art engineering methodologies, and introduce "ordered assembly" as a novel paradigm for constructing advanced multi-enzyme systems. In consideration of the distinct features of various cascade modules, we further elaborate on design principles for customized assembly strategies and illustrate their implementation through representative case studies. Finally, the current challenges and future directions of multi-enzyme assembly technology are discussed. This study aims to establish a theoretical foundation and propose innovative strategies for enhancing the application of multi-enzyme assembly in biocatalysis, thereby contributing to the sustainable manufacturing of fine chemicals.

Oligomerization is a notable post-translational self-assembly process in proteins. While enzyme oligomerization often enhances stability, its mechanisms remain unclear. Nitrilases are particularly prone to oligomerization, making precise regulation of this process critical for advancing the biochemo catalytic route to gabapentin and for broader applications in pharmaceutical synthesis and green chemistry. Here, we applied computational protein engineering to optimize a nitrilase from Acidovorax facilis (AcN). Using AlphaFold 3 for structural analysis and ESM-DLkcat for activity estimation, we first explored C-terminal engineering as a means to modulate AcN oligomerization, identifying proline content and net charge as important contributors to changes in filament length and stability. While C-terminal modifications primarily improved thermal robustness, they were accompanied by reduced or modestly altered catalytic activity. To overcome this limitation, we subsequently applied FoldX-guided mutagenesis to the beta-sheet and active-site regions of the thermally stable AcN Delta C30 variant, yielding the optimized mutant M1_5 (F307Y/F168V/T201M/V305L/I320A). M1_5 exhibited a 36.9% increase in activity and a 6.8-fold longer half-life (40.77 h). Molecular dynamics simulations suggest that improved substrate channel accessibility and enhanced structural rigidity contribute to the observed performance gains. Application of similar active-site redesign principles to an alternative variant (M2) resulted in M2_4, which showed substantially increased activity, highlighting the broader utility of this stepwise engineering framework. Notably, M1_5 enabled complete conversion of 1 M substrate 1-cyanocyclohexane acetonitrile under industrial conditions (an increase from 67.99% to 100%), underscoring its potential for sustainable biomanufacturing.

l -Pipecolic acid ( l -PA) and its hydroxylated derivatives (hydroxypipecolic acids, HPAs) are non-proteinogenic amino acids that serve as valuable chiral building blocks for pharmaceuticals, antibiotics, and natural products. Conventional chemical synthesis of these compounds often suffers from operational complexity, poor environmental compatibility, and insufficient stereochemical control, driving a shift toward biosynthetic approaches. This review covers recent advances in enzyme engineering and synthetic biology aimed at enabling sustainable and efficient production of l -PA and HPAs. For l -PA biosynthesis, various metabolic engineering strategies to enhance its production in microbes are introduced, and enzyme cascades, single enzyme strategy, and immobilized enzyme strategy involved in l -PA production are discussed. Regarding HPAs biosynthesis, which involves the regioselective hydroxylation of l -PA, their structural features, catalytic mechanisms, and recent progress in the biosynthesis of diverse HPAs, the protein engineering of proline hydroxylase is emphasized. Finally, we present future perspectives to accelerate the biosynthetic production of l -PA and HPAs.

Corncob, rich in cellulose and hemicellulose, is a promising feedstock for fermentable sugar production. This study aimed to optimize the release of reducing sugars and the biosynthesis of R-2-(4-hydroxyphenoxy) propionic acid (R-HPPA) from corncob hydrolysate (CCH) using a biofilm-based fermentation strategy with Beauveria bassiana. Dilute acid pretreatment (2.4% H2SO4, 121 degrees C, 29 min) yielded 38.2 g/L reducing sugars (CCH-1). Detoxification with Ca(OH)(2) and activated carbon effectively removed 99.0% furfural and 99.2% 5-hydroxymethylfurfural, with less than 10% sugar loss (CCH-3). To enhance R-HPPA production, different CCH hydrolysates were evaluated: CCH-2 (CCH-1 treated with Ca(OH)(2)), CCH-3 (CCH-1 treated with Ca(OH)(2) and activated carbon), and CCH-4 (CCH-3 concentrated to varying reducing sugar concentrations). The highest R-HPPA titer (15.9 g/L) was achieved when the hydrolysate was neutralized, detoxified, and concentrated to 60 g/L reducing sugars. This process highlights the potential of CCH as a low-cost and renewable substrate for industrial R-HPPA production.

Escherichia coli (E. coli) is a key workhorse of biotechnology. Commonly used CRISPR-Cas9 systems for E. coli genome editing are complex and impose metabolic stress on the host, creating demand for more streamlined strategies. Recent studies identified the IS605 transposon-associated TnpB as a programmable RNA-guided (ωRNA) DNA endonuclease, prompting us to explore whether endogenous TnpB in E. coli (EcoTnpB) could be harnessed for genome editing. Biochemical and cellular analyses demonstrated that EcoTnpB efficiently cleaves both chromosomal and plasmid DNA at custom-specified sites in a TAM-dependent manner. Interestingly, E. coli possesses an endogenous recombination machinery capable of repairing EcoTnpB-induced DNA double-strand breaks (DSBs), challenging the long-held view that bacteria lack efficient homologous recombination systems. Based on these findings, we established a single-plasmid editing system (SPEED) in which genome editing is achieved by simply providing ωRNA and a homologous recombination template. By utilizing endogenous EcoTnpB together with the host HR pathway, this system enabled inducible and seamless genome editing at multiple genomic loci in BL21 (DE3), with editing efficiencies ranging from approximately 29% to 56%. Our results demonstrate for the first time that endogenous TnpB can be harnessed for genome editing and may hold potential for broader applications, such as species-specific antimicrobial development.

Antagonistic epistasis often limits enzyme engineering by causing activity loss when beneficial mutations are combined. Here, we present a mechanism-guided multidimensional feature analysis (MDFA) strategy integrating rational design, structural partitioning, machine learning, and computational simulations to optimize D-amino acid oxidase (DAAO) for D-phosphinothricin (D-PPT). Channel geometry and electrostatics defined preferred screening ranges, while a CNN ensemble with a Random Forest surrogate enabled multisite classification. Computational and experimental analyses suggested that excessive local positive charge and imbalanced flexibility contribute to A. epistasis, whereas spatial partitioning reduces local conflict. OAEMT (N53R-V57R-S233 K-Q339R) increased catalytic efficiency 80.94-fold and converted 85.5% of D-PPT within 5 h in a 2 L reactor. MDFA thus improves combinatorial design within a defined mutational space.

Functional coenzymes, a class of organic small-molecule cofactors, play essential roles in enzyme-catalyzed reactions. They participate in key biological processes, including redox reactions, group transfers, and isomerization, through transient association with enzyme proteins. As vital components for sustaining enzymatic activity and cellular energy metabolism, functional coenzymes are experiencing rapidly growing market demand, driven by global population aging and increasing health awareness worldwide. Biomanufacturing technologies can better meet this market need by enhancing production efficiency, lowering costs, and fostering product innovation, thereby injecting new vitality into the industry. Using β-nicotinamide mononucleotide (NMN) as a representative functional coenzyme, this article systematically reviews the current state of coenzyme synthesis technologies. Special emphasis is placed on multi-strategy approaches applied in the fermentative and enzymatic synthesis of NMN, covering enzyme classification, cofactor regeneration, competitive pathway modulation, and transport system optimization. The screening, engineering, and application of key enzymes, together with interdisciplinary research directions such as systems metabolic engineering and coupled fermentation process optimization, are proposed to drive continuous innovation in coenzyme biomanufacturing. These efforts will provide a solid foundation for the high-quality development of the coenzyme industry and enhance its international competitiveness.

The Bamberger rearrangement is a key route to functionalized aminophenols that are widely used in pharmaceutical synthesis. Hydroxylaminobenzene mutase (HabM) catalyzes a Bamberger-type isomerization in the biosynthesis of the Pranlukast intermediate 3-amino-2-hydroxyacetophenone (3AHAP), but its low catalytic efficiency and unknown structure have limited rational improvement. Here, we combine AI-assisted phylogenetic mining, structural elucidation, protein engineering, and metabolic coordination to enhance 3AHAP biosynthesis. Deep learning-guided screening identified the NRBh-HabMEo pair as the most effective combination, substantially outperforming previously reported systems. Using an integrated dry-wet strategy involving homology template search, spectroscopy, mutagenesis, Size-Exclusion-Chromatography analysis, and AlphaFold3-assisted modeling, we reveal that HabMEo is a Fe-dependent tetramer. Rational mutagenesis supported by molecular dynamics simulations yielded a synergistic triple mutant with improved pocket dynamics, optimized Fe-substrate positioning, and markedly enhanced catalytic efficiency. To alleviate reductive limitations, NAD kinase was introduced to strengthen NADPH cycling; however, increased upstream flux led to intermediate accumulation and by-product formation. This was overcome by implementing a RIAD/RIDD-based scaffold to spatially organize NR, HabM, GDH, and NADK, thereby promoting intermediate channeling and suppressing over-reduction. Overall, this study elucidates the structure of HabM and established a successful paradigm for optimizing complex multi-enzyme cascades for sustainable production of high-value biopharmaceutical intermediates.

Transaminases (TAs) represent a class of enzymes that enable highly stereoselective amination reactions under mild conditions, providing an exceptionally powerful and versatile green chemistry tool for the synthesis of chiral amines and nitrogen-containing compounds. However, most current reviews on transaminases focus on the efficient synthesis of chiral amines, whereas the efficient synthesis of complex natural products has rarely been reported. This review systematically discusses the characteristics of different types of transaminases, covering their underlying catalytic mechanisms, the dynamics and roles of the cofactor involved, and key enzymatic properties that are critical for their function and practical applicability. Recent advances in protein engineering strategies and in the design of multi-enzyme cascade systems are also discussed. A particular focus is directed toward representative applications of transaminase-mediated multi-enzyme cascade systems in natural product synthesis. Based on a systematic review of recent advances in transaminase research, this review focuses on their representative applications in multi‑enzyme cascade systems for natural product synthesis, providing a comprehensive and referenceable framework for green synthetic strategies.

Cytochrome P450 enzymes hold immense potential as biocatalysts for oxidative reactions but often suffer from inefficient electron transfer due to their reliance on transient interactions with redox partners (RPs). To overcome this limitation, we constructed self-sufficient fusion enzymes by fusing the avermectin-oxidizing P450-Ema1-M212A to selected RPs. Linker sequences were primarily optimized experimentally, while computational modeling, including AlphaFold3-predicted multi-protein assemblies and molecular dynamics simulations, was used as an auxiliary tool to assess feasible linker lengths and interpret the structural basis by which optimized linker design enhances electron-transfer efficiency. Based on literature precedent and preliminary structural considerations, a series of fusion constructs with varying linker sequences and lengths was constructed. Molecular dynamics simulations suggested a structural rationale for why a linker length of 9 amino acids exhibited optimal enzymatic activity, as it stabilized a functional conformation, minimized structural fluctuations, and maintained a favorable distance between the heme and Fe₂S₂ clusters for efficient electron transfer. Experimental characterization confirmed that the F4 variant exhibited substantially increased activity over the non-fusion version. Kinetic analysis demonstrated a significantly enhanced kcat/Km ratio, indicating accelerated catalysis without compromising substrate binding. For the synthesis of 4″-oxo-avermectin, a key intermediate of emamectin, the F4 variant achieved 90% conversion within 8 h, far exceeding the performance of the non-fused system. This study establishes an experimentally driven, semi-rational framework for constructing self-sufficient P450 fusion enzymes, highlighting the synergy between computational prediction and protein engineering for developing powerful biocatalysts with high industrial application potential.

Designing biocatalytic cascades with precise control over coenzyme regeneration, efficiency, and selectivity remains a central challenge in biocatalysis. Here, we present a sustainable strategy for constructing covalently bonded multienzyme complexes (CBMEs) that enable efficient redox biosynthesis under mild and environmentally friendly conditions. A quantitative reaction-diffusion model was developed to elucidate spatial organization within CBMEs, introducing a G factor that quantitatively predicts the adaptation efficiency (ε) of dual-enzyme systems by integrating geometric and kinetic parameters. Guided by this model, a dual-enzyme complex for l-amino acid biosynthesis achieved over 99% conversion within 2 h and >99.9% enantiomeric excess, while significantly reducing coenzyme use and reaction waste. This work demonstrates that controlling enzyme spatial arrangement through covalent coupling not only enhances catalytic efficiency and selectivity but also provides a green and sustainable framework for designing high-performance bioredox systems.

Efficient electron supply and cofactor utilization remain key bottlenecks in cytochrome P450-catalyzed oxidations. Here, we report an electro-assisted system that overcomes these limitations, using the P450 Ema1-catalyzed oxidation of avermectin B1a as a model reaction. Under a constant current of 1 mA, > 96% conversion of 2.5 mM (2.2 g/L) substrate was achieved within 4 h, whereas the non-electrified system reached only 68% conversion after 8 h. The electro-assisted system enhanced catalytic efficiency at low substrate concentrations, while the advantage diminished at higher substrate loadings (≥ 5 mM), likely attributable to H2O2 accumulation, indicating a transition between different kinetic regimes. Prolonged electrical input led to enzyme deactivation, revealing a trade-off between catalytic enhancement and enzyme stability. Notably, transient electrical exposure for 1 h produced a catalytic activity gain that was abolished by catalase, persisted after current removal, and was accompanied by minor spectroscopic changes suggesting localized structural rearrangements rather than global conformational reorganization. Finally, we establish that the NADH/NAD+ ratio, rather than absolute NADH concentration, is the master regulator of catalytic activity, with NAD+ acting as a high-affinity allosteric effector. Our work demonstrates a controllable strategy for enhancing P450-catalyzed reactions by integrating electrical input with cofactor regulation, providing insights for the design of electro-biocatalytic processes.

Natural fragrance compounds determine the sensory quality of food, cosmetics, and consumer goods. Driven by the limitations of traditional botanical extraction and petrochemical synthesis, sustainable biomanufacturing empowered by systems metabolic engineering and enzyme biocatalysis has emerged as a compelling alternative. This review systematically summarizes recent advances in the biosynthesis of high-value natural fragrances. First, we delineate the biosynthesis-driven classification of these volatile molecules and elucidate the structure-odor relationship, establishing a rational basis for target selection. Subsequently, we outline the core biosynthetic networks responsible for generating the four major lineages: terpenoids, phenylpropanoids and benzenoids, fatty acid derivatives, and amino acid derivatives. To provide systematic guidance for the biosynthesis of natural fragrance compounds, we comprehensively detail advanced engineering strategies across three progressive hierarchical tiers: the enzyme, metabolic, and cellular levels. Finally, we evaluate current challenges and future directions, with a specific emphasis on biosynthetic bottlenecks, bioprocess scale-up, and the rational design of complex multi-component aromas, thereby providing new insights into the sustainable bioproduction of these high-value natural fragrance compounds.

This study aimed to characterize the G protein-coupled receptor FfGpr1-Gα-AC transduction pathway and its role in regulating gibberellin (GA) metabolism in Fusarium fujikuroi. By constructing Ffgpr1 deletion and constitutively activated FfG2Q204 L mutants, we found that glucose-induced cAMP (cyclic Adenosine MonoPhosphate) synthesis was abolished in Ffgpr1Δ. Bimolecular fluorescence complementation confirmed membrane-localized FfGpr1-FfG2 and FfG2-AC interactions, with intensified fluorescence at septa. Fermentation assays revealed opposing GA3 yields: Ffgpr1Δ produced 21% less GA3 than the wild type, whereas FfG2Q204 L increased yield by 17%. qPCR（quantitative real-time PCR） analysis demonstrated that Ffgpr1Δ upregulated FfCPS/KS, FfP450-2, and FfP450-3 transcription by 6-8-fold while downregulating FfDES by 82%, whereas FfG2Q204 L induced a 6-fold increase in FfCPS/KS mRNA level. Strikingly, FfDES overexpression in Ffgpr1Δ restored GA3 production to wild-type levels but led to GA7 accumulation and suppressed FfP450-3 upregulation, suggesting feedback-regulated metabolic constraints. Glucose-induced cAMP production required FfGpr1. FfGpr1-FfG2 and FfG2-AC interacted on the cell membrane, with enhanced co-localization at the septal region. The FfGpr1-Gα-AC pathway significantly affected GA yield, with complex and noteworthy regulation of GA cluster gene expression.

Continuous-flow reactors have major advantages for biocatalysis, such as better mass transfer, easier scale-up, and continuous production. However, continuous biocatalysis remains in its early stages, with most work focused on modifying enzyme surfaces or carrier materials. In this study, we developed a new continuous biocatalysis system using an engineered anchor peptide called Cg63, which binds strongly to poly(methyl methacrylate) (PMMA). Moreover, binding interactions between the anchor peptide and the material surface were analyzed via molecular dynamics (MD) simulations, based on which a structure-guided directed evolution strategy for anchor peptides was proposed and successfully applied for rational peptide design. The engineered variant Cg63E8N exhibited a 1.57-fold increase in binding efficiency over the original peptide. This immobilization method was applicable to six major enzyme classes (EC1-EC6). Furthermore, we designed a continuousflow microreactor featuring a 1 mm inner-diameter PMMA channel, effectively preventing interference from the reaction environment to the immobilized enzymes. Reynolds number calculations (0.17-0.61) under various solvent conditions confirmed the dominance of molecular diffusion in this laminar flow regime, which is highly favorable for enzymatic catalysis. Importantly, immobilized Caenorhabditis elegans (EC1, CeDAAO) retained 79.81 % of its activity after 10 cycles of use, and CtNHase - Cg63E8N (EC3) reached a high space - time yield of 818.2 mmol/L/day. Overall, this system provides a promising and scalable platform for sustainable industrial biocatalysis.

R-2-(4-hydroxyphenoxy)propionic acid (R-HPPA) is a globally sought-after intermediate for aromatic phenoxy propionic acid herbicides. In this study, a mutant strain of Beauveria bassiana, designated as ZJB23323 (CCTCC NO: M 2023584), was obtained through UV, atmospheric, and room temperature plasma, and N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, exhibiting 7.24-fold improved production of R-HPPA. The concentrations of glucose, yeast extract, and R-2-phenoxypropionic acid (R-PPA) in the initial medium were optimized for R-HPPA production by using response surface methodology with Box-Behnken design. The maximum R-HPPA yield of 25.8 g/L was obtained at optimal concentrations of 64.7 g/L for glucose, 21.9 g/L for yeast extract, and 31.7 g/L for R-PPA, which increased by 48.7% compared to the original medium. Finally, the scaled-up biosynthesis of R-HPPA was successfully carried out in a 6-L plastic tray bioreactor, and the kinetic model and model parameters of the R-HPPA batch fermentation process were described. The metabolic characterization of strain ZJB23323 for the fermentative production of R-HPPA under three modes of batch culture, constant rate feeding, and exponential feeding was further investigated. The results showed that 100 g/L of R-PPA could be completely converted to R-HPPA within 13 days under the exponential feeding culture strategy.

Chiral amide herbicides represent a significant class of agrochemicals, widely used for effective weed control. Prominent examples include S-metolachlor and dimethenamid-P, both of which share the intermediate (S)-1-methoxy-2-propylamine, a key structural component in their synthesis. Developing green and sustainable methods for producing this intermediate is crucial for enhancing the environmental and economic feasibility of herbicide manufacturing. Biosynthesis, with its advantages in sustainability and efficiency, has emerged as a pivotal approach in pesticide production. This review explores the classification and current development status of chiral amide herbicides, including their varieties and applications in the agricultural market. It outlines the synthesis pathways for S-metolachlor and dimethenamid-P, covering both chemical and biosynthetic routes. The review also highlights the functional properties of the key enzymes involved in the biosynthesis of (S)-1-methoxy-2-propylamine, focusing on the potential for enzyme engineering and creation to optimize these pathways. The challenges and future development directions for amide herbicides are discussed, with an emphasis on overcoming synthetic and ecological barriers.

In enzyme engineering, a lot of studies have focused on engineering the active site to broaden substrate specificity or enhance transaminase activity; however, relatively little is known about the mechanisms by which substrates are recognized and enter the binding pocket. Transaminases play a crucial role in the synthesis of chiral amines due to their exceptional stereoselectivity and catalytic efficiency. In this study, we explored how the pedal-like loop at the active site influences (R)-transaminase (ATA) activity and substrate recognition by modulating the substrate channel. The pedal-like loop at the active site was swapped with loops from other well-characterized transaminases, and the best-performing variant exhibited a 5.2-fold increase in activity toward (R)-phenylethylamine ((R)-PEA) and an 11.8-fold increase in activity toward isopropylamine (IPA). Additionally, some variants showed significant changes in substrate preference. Homology modeling and molecular docking analysis provided compelling evidence that the pedal-like loop is a critical determinant of both substrate recognition and catalytic activity in (R)-ATA.

Using Nakaseomyces glabrata as a model organism, we demonstrate that targeting calcineurin can synergize with caspofungin to induce a quorum sensing (QS) effect mediated by farnesol. This QS effect requires calcineurin deficiency, sub-minimum inhibitory concentration (MIC) levels of caspofungin, and a high-density cell population. Cell growth and biofilm formation were significantly inhibited within a specific range of cell density and sub-MIC caspofungin treatment in the calcineurin mutant. The inhibition of biofilm formation follows the 'paradoxical growth,' showing a concentration-dependent response to caspofungin. We show that high cell density triggers two antagonistic effects: overcoming antibiotic inhibition, which promotes cell propagation, and QS-mediated growth inhibition, which negatively regulates cell proliferation. The QS molecule farnesol was detectable only in the calcineurin mutant, where the transcription of the farnesol synthase Dpp3 was significantly up-regulated, and deletion of DPP3 abolished the QS effect in both spot assay and biofilm formation of the calcineurin mutant. Besides this, we identified a Dpp3-dependent, ergosterol-farnesol metabolism-linked Crz1-independent regulatory mechanism that contributes to the calcineurin-mediated multi-stress resistance. We demonstrate that calcineurin, Dpp3, and caspofungin are all involved in regulating ergosterol metabolism and the transcription of ERG11 and FKS genes, leading to significant changes in membrane and cell wall stress tolerance. The cell wall composition undergoes substantial alterations upon deletion of calcineurin or treatment with caspofungin, while caspofungin also increases the levels of β-glucan and short peptides in the medium, tentatively pointing to the release of QS inducers from the cell wall.

In biosynthesis, while focusing on the productivity of individual compounds, the development of high-efficiency bio-components and universal enabling tools for advancing biosynthesis remains a critical and persistent challenge. Plant-derived Integral Membrane Proteins (IMPs) from two distinct families were heterologously expressed in E. coli, inducing filamentous cell growth, increased membrane permeability, polyploidy, and growth arrest. GFP-tagged IMPs were successfully delivered to the cell membrane. Filamentous cells contained significantly elevated DNA content, and displayed a rough surface morphology, an enlarged periplasmic space, and heightened sensitivity against membrane and cell wall stressors. These findings correspond to significantly altered transcription of genes linked to cell membrane and wall integrity, including those regulating cell division, elongation, DNA replication, and IMP delivery. Notably, the observed cellular toxicity could be modulated by chimeric fusion of the N-terminus and a certain number of hydrophobic transmembrane helices, potentially through α-aggregation-mediated membrane disruption. Finally, we demonstrated that IMP expression enhanced biosynthesis in all six tested scenarios, including biocatalysis, fermentation, and mixed-cell catalysis for the production of diverse chemicals. A plant-IMPs toolkit has been developed for versatile biosynthetic applications in E. coli.