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Synthetic biology is emerging as a key approach in chemical synthesis, whose efficiency hinges crucially on the direct engineering of metabolic pathways. In this study, we propose a dual-intervention paradigm to reprogram the industrial fungus Fusarium fujikuroi from a default gibberellic acid (GA3) producer into an exclusive factory for the higher-value gibberellin GA4+7, providing a complementary and orthogonal approach to traditional intra-pathway manipulations. First, by introducing Arabidopsis-derived transporters (Npfs and Sweets), we successfully created a thermodynamic sink that actively depletes intracellular GA4/GA7 pools. With the best candidate protein, Sweet1, the parent strain was converted into an exclusive producer of GA4+7 (with GA3 levels undetectable), a conversion driven by the significantly accelerated dissociation rate (Kdis) for GA7. Concurrently, we uncovered a non-canonical, highly specific regulatory mechanism: overexpression of the Sfp-type 4'-phosphopantetheinyl transferase Ppt1 triggered targeted post-transcriptional silencing of up to 99.9 % of P450-3 mRNA, thereby completely silencing GA3 biosynthesis and again yielding an exclusive producer of GA4+7. Synergistic integration of transporter-driven spatial pulling and Ppt1-mediated gene silencing, coupled with fermentation optimization, propelled the final GA4+7 titer to an unprecedented 3.29 g/L (reaching 0.4 g/L for GA4 and 2.89 g/L for GA7, representing 17.39-, 962.33-, and 125.54-fold increases over the parent strain, respectively). This study achieves indirect metabolic reprogramming not via conventional pathway engineering, but by targeting a membrane transport bottleneck and employing a putative post-transcriptional silencing mechanism.
(R)-3-Aminobutanol is a valuable chiral amino alcohol widely used in the pharmaceutical industry. The amine dehydrogenase (AmDH)-catalyzed synthesis with inorganic ammonia as the amine donor represents one of the most promising routes for its production. However, this approach is limited by the low catalytic efficiency of AmDHs toward the unnatural substrate 4-hydroxy-2-butanone, as well as the high cost associated with coenzyme requirements. To address these issues, a structure-guided engineering strategy integrating loop remodeling with dynamically coupled network analysis was employed to modify an AmDH derived from leucine dehydrogenase (GKGB-AmDH). A pentamutant was obtained with a 7.8-fold increase in catalytic efficiency (kcat/Km) and a 6.8-fold increase in specific activity. Molecular dynamics simulations were performed to elucidate the molecular mechanism underlying the improved catalytic performance. A dual-enzyme co-expression system for GKGB-AmDH-M5 and formate dehydrogenase (FDH) was established for in situ NADH regeneration. As a result, the catalytic efficiency toward 4-hydroxy-2-butanone was enhanced, and the conversion reached 83.8% at a substrate concentration of 300 mM, with an enantiomeric excess (e.e.) of > 99.99%. These results demonstrate the feasibility of this engineering strategy and provide a theoretical basis for the efficient and green biomanufacturing of chiral amino alcohols.
d-Allulose is a valuable low-calorie rare sugar with diverse physiological benefits. Although phosphorylation-dephosphorylation-based multi-enzyme cascades enable efficient d-allulose biosynthesis, the free-enzyme format limits substrate channeling and promotes intermediate diffusion, resulting in byproduct accumulation and reduced cascade efficiency. In this study, peptide-mediated assembly strategies were employed to construct a dual-enzyme complex using d-allulose 6-phosphate epimerase (A6PE) and d-allulose 6-phosphate phosphatase (A6PP) as model enzymes, to mitigate reversible epimerization. Among them, the ReverseTag/ReverseCatcher system was selected due to its positive impact on enzyme activity, as evidenced by the 2.1‑fold and 27.5% increases in activity observed for RCA6PE and RTA6PP, respectively. Successful complex assembly was confirmed by dynamic light scattering and transmission electron microscopy. A five-enzyme complex (RFE) was further constructed by integrating α-glucan phosphorylase, phosphoglucomutase, phosphoglucose isomerase, A6PE, and A6PP to spatially organize an artificial in vitro d-allulose biosynthetic pathway. With 10 g/L maltodextrin as the substrate, the RFE system achieved a d-allulose yield of 63.1%, representing a 37.2% increase over the free-enzyme system. These findings demonstrate that ReverseTag/ReverseCatcher-mediated covalent assembly improves multi-enzyme cascade efficiency and provides a modular platform for engineering artificial in vitro biosynthetic systems.
d-Tagatose is a rare hexose sugar with excellent properties, and its synthesis catalyzed by d-tagatose 4-epimerase (T4E) represents a competitive novel pathway. In this study, EbT4E derived from the Eubacteriales bacterium was screened and systematically characterized. By reshaping the microenvironment of the active pocket, mutant M3(S131D/H410W/T279S) was constructed, which showed a 3.89-fold higher conversion rate compared with the wild-type (WT) enzyme. Kinetic parameter analysis and molecular dynamics (MD) simulations revealed that M3 had enhanced substrate affinity, hydrogen bond network, charge properties, and channel accessibility. Finally, the conversion rates of d-fructose to d-tagatose catalyzed by the purified M3 enzyme and M3 whole-cell catalysts reached 29.46% and 26.2%, respectively. Additionally, the dual-enzyme cascade reaction of M3 with glucose isomerase (GI) TEGI-M-L38M-V137L was constructed, achieving a 13.16% yield of d-tagatose from d-glucose. This study demonstrates that EbT4E-M3 is a promising biocatalyst for d-tagatose production, laying the foundation for its subsequent industrial application.