Construction of the chiral 24R-OH squalamine intermediate by an engineered ketoreductase from Rhodococcus sp.

Pinene synthase is a crucial biocatalyst for microbial production of (+)-α-pinene, a natural insect repellent and green pesticide enhancer. However, its low catalytic efficiency hinders industrial application. We employed a combination of homologous sequence alignment and structure-guided engineering to identify key mutations (H345F, S372C, and C527S) that significantly enhanced enzymatic activity. The double mutant H345F/S372C increased (+)-α-pinene titer to 273.66 mg/L, 3.38-fold higher than wild-type, with a 3.10-fold improvement in catalytic efficiency. Structural and computational analyses revealed that mutations optimized the substrate channel and reinforced hydrophobic pocket rigidity, improving substrate binding and enzyme stability. This engineered enzyme offers an efficient biosynthetic route for sustainable agrochemical production.

Polyester formation by polycondensation in water is limited by an esterification-hydrolysis equilibrium that strongly favors hydrolysis under mild conditions. Although aqueous-phase esterification has been demonstrated with chemical catalysts and isolated enzymes, these systems typically target small-molecule esters or rely on preactivated donors. The polyhydroxyalkanoate (PHA) pathway is the only known native metabolic route for polyester biosynthesis, but it is essentially limited to the polymerization of 3-hydroxybutyrate and related short-chain hydroxyalkanoate monomers. Consequently, realizing efficient aqueous-phase polycondensation to high-molar-mass polyesters remains a major challenge. Here, an intracellular CoA activation/acyltransferase cascade (ACOS5 At -WS2 Mh ) for poly-(hydroxy fatty acid) (PHFA) biosynthesis is constructed within a whole-cell catalyst. Mechanistic analysis indicates that a mildly hydrophilic region lining the ACOS5 At substrate tunnel is critical for ω-hydroxy fatty acid (ωHFA) recognition and activation in water. The substantial steric bulk of ω-hydroxyacyl-CoA (ωHFA-CoA) hinders its entry into the WS2 Mh hydroxyl-donor channel, enforcing a hydroxyl-terminal chain-growth mode, whereas the higher diffusivity and lower steric hindrance of short oligomers underlie the low dispersity of the resulting PHFA. Chassis engineering, catalytic optimization, and a substrate-channeling fusion-protein strategy collectively increase the cascade flux and the product titer. Under optimized conditions, a PHFA titer of 1.87 g L-1 (M n,app = 10.8 kDa; Đ = 1.05) was obtained in a 3 L bioreactor, yielding polymers with heterotelechelic hydroxyl/carboxyl chain ends. This work establishes a green, fully aqueous, whole-cell route from unactivated ωHFAs to non-PHA polyesters and provides general design principles for engineering living catalysts capable of overcoming hydrolysis-limited equilibria in condensation polymerization.

Identification of key enzymes and tolerance mechanisms for protocatechuic acid synthesis in Escherichia coli via systematic metabolic engineering.

Enzymatic cascades, defined here as multi-enzymatic sequences operating on a shared reaction pathway and inspired by the spatial and temporal organization of metabolism, have emerged as powerful and versatile tools for sustainable organic synthesis. They minimize intermediate isolation, enhance atom economy and ensure outstanding chemo-, regio- and stereoselectivity, providing efficient alternatives to conventional multistep routes. Here, we highlight the conceptual role of substrate channeling, minimal cells, artificial metabolism and enzyme promiscuity in the translation of enzymatic cascades into synthetic strategies. Special attention is focused on advanced immobilization on functional and renewable supports, which enhance stability and recyclability and introduce new ways for thermodynamic and kinetic control. Hybrid systems integrating enzymes with photocatalysis, electrochemistry and chemical modules expand the catalytic repertoire far beyond biology. Complementary tools in bioinformatics, structural modeling and artificial intelligence may also enable pathway balancing, predictive design and dynamic optimization. Applications span from the valorization of renewable feedstocks to the synthesis of privileged scaffolds and fine chemicals.

Layered engineering of sucrose phosphorylase to control substrate tunnels for efficient industrial bioproduction of 2‑O‑α‑D‑glucopyranosyl‑L‑ascorbic acid.

From biopolymers to microcompartments: A structured review of protein-based scaffolds for enzyme immobilization.

Exploring the multi-protein assembly of the enzymes of the de novo purine nucleotide biosynthetic pathway from Pseudomonas aeruginosa.

Computational dual-loop frameworks bridging single-enzyme design and cascade tunnel network engineering for next-generation biosynthetic systems.

Engineering the substrate tunnel of diacylglycerol acyltransferase ScDGA1 enhances triacylglycerol biosynthesis in Saccharomyces cerevisiae.

The spatial coordination between cellular organelles and metabolic enzyme assemblies represents a fundamental mechanism for maintaining metabolic efficiency under stress. While previous work has shown that membrane-bound organelles regulate metabolic activities and that membrane-less condensates conduct metabolic reactions, the coordination between these two organizations remains unaddressed. By using a combination of proximity labeling, superresolution fluorescence microscopy, and metabolite analyses using isotopic tracing, we investigated the relationships between these metabolic hotspots. Here, we show that nutrient deficiency elongates mitochondria and transforms the ER from a tubular to sheet-like morphology, coinciding with increased mitochondrial respiration and inosine 5'-monophosphate levels. These structural changes promote the colocalization of purinosomes with these organelles, enhancing metabolic channeling. Disruption of ER sheet formation via MTM1 knockout destabilizes purinosomes, impairs substrate channeling, and reduces intracellular purine nucleotide pools without altering enzyme expression. Our findings reveal that organelle morphology and interorganelle contacts dynamically regulate the assembly and function of metabolic condensates, providing a structural basis for coordinated metabolic control in response to nutrient availability.

Plant metabolism is increasingly being demonstrated to be partially controlled by dynamically assembled metabolons-multienzyme complexes that enable substrate channeling, insulate reactive intermediates, and permit rapid, low-energy flux control. Rigorous criteria are defined to distinguish true metabolons from generic assemblies, and evidence is synthesized across cyanogenic glucoside, phenylpropanoid/flavonoid, alkaloid, terpenoid, polyamine, sporopollenin, and auxin pathways. A practical workflow is presented in which AP-MS (Affinity purification mass spectrometry)/Co-IP (Co-immunoprecipitation), proximity labeling, BiFC (Bimolecular fluorescence complementation)/FRET (Förster resonance energy transfer)/Split-luciferase, and isotope-dilution metabolomics are integrated to resolve composition, dynamics, and direct channeling in vivo. In enzyme-based substrate channeling engineering, design rules are distilled for membrane anchoring, modular scaffolds, compartment targeting, and inducible/optogenetic control, and limitations such as metabolic burden, stoichiometry, and leakiness are noted. An AI-assisted loop is outlined in which structure-aware generative models produce binders/interfaces that are coupled to spatial optimization of enzyme order, orientation, and distance. Together, these advances reposition metabolons as a deployable technology for programmable flux in plants, enabling safer handling of labile intermediates and higher titers of valuable natural products.

Distal peptide elongation by a protease-like ligase and two distinct carrier proteins

Computer assisted discovery of novel nicotinamide phosphoribosyltransferase agonists to combat muscle atrophy.

Co-immolization of lipase and bimetallic Cu/Mn aminoclay nanozyme onto montmorillonite for spatially synergistic conversion of p-nitrophenyl esters.

The internal alternative NADH dehydrogenase (Ndi1) is the electron input in the Saccharomyces cerevisiae respirasome.

Design and mechanistic insights into a bifunctional dextransucrase-dextranase fusion enzyme for controlled synthesis of high-molecular-weight dextran.

Engineering a broad-spectrum glucose oxidase via substrate channel and linker design for enhanced lignocellulose bioconversion

Substrate inhibition limits the industrial use of tryptophan hydroxylase (TPH), the key catalyst for 5-hydroxytryptophan (5-HTP) production, by causing tunnel congestion and substrate trapping at high concentrations. We developed a mechanism-guided strategy to overcome this. The crystal structure of the Y235S (MS) variant revealed a 243% expansion of the substrate channel, reducing tunnel congestion and increasing activity 2.38-fold, though substrate affinity decreased. Mechanistic analysis showed loop II acts as a molecular gate controlling cofactor-substrate binding. Its rational stabilization in variant MS4 enhanced loop stability and optimized substrate orientation, increasing catalytic efficiency by over 150% compared to MS and specific activity by 285% compared to wild-type. This approach proved generalizable across TPH orthologs. Combined with a tetrahydrobiopterin regeneration system, MS4 broke through the substrate concentration limitation, achieving >5-fold higher whole-cell 5-HTP production (16.37 mM in 4 h). This work establishes a general framework for relieving tunnel congestion and substrate trapping through integrated structural, computational, and loop engineering.

A CRISPR-Cas6-mediated lycopene synthase assembly regulation strategy was developed to optimize the metabolic pathway of lycopene biosynthesis in Escherichia coli and enhance production efficiency. Leveraging the orthologous properties of EcCas6e and Csy4 within the Cas6 protein family, along with RNA scaffolding, we constructed a protein-RNA complex for enzyme assembly. Sixteen plasmids (LYC-1 to LYC-16) were designed, and the assembly strategy was systematically optimized by varying the gene arrangement, linker length, and RNA scaffold expression. The performance of RNA scaffold-based enzyme assembly was compared with conventional protein linker-based approaches. Lycopene production was quantified via high-performance liquid chromatography (HPLC) to evaluate system performance. The recombinant strain LYC-3-4, which co-localized CrtB and CrtI via EcCas6e-Csy4 protein-RNA complexes, achieved the highest lycopene yield (4.02 mg/L), 58% higher than the control strain LYC-3-5 (2.55 mg/L) with mismatched RNA hybridization regions, and 41% higher than strain LYC-6 (2.86 mg/L), in which the enzymes were expressed separately. This result indicates that protein-RNA-mediated spatial co-localization significantly enhanced the substrate channeling effect, whereas other assembly configurations either failed to improve or even reduced lycopene production. In summary, we exploited the protein assembly capability of CRISPR-Cas6 proteins in combination with RNA scaffolds to achieve efficient enzyme co-localization within the lycopene biosynthetic pathway. This approach offers a convenient, flexible, and scalable tool for enzyme assembly regulation in metabolic engineering, with potential applications in microbial production of lycopene and other valuable metabolites.