Yuguo Zheng

Reconstruction and metabolic regulation of O-succinyl-L-homoserine biosynthesis pathway in Escherichia coli W3110.

O-Succinyl-L-homoserine (OSH) plays a pivotal role in L-methionine biosynthesis. Microbial cell factories for high-yield OSH production have been progressively optimized, achieving substantial improvements in fermentation titers. In this study, a multi-step progressive optimization strategy was adopted to construct a high-yield OSH-producing strain. First, the feedback inhibition of the key enzyme HST was relieved, and the key genes involved in the byproduct metabolic pathways were knocked out. This modification enabled engineered strain to produce 9.77 ± 0.27g/L OSH in shake-flask fermentation. Second, ribosome binding site (RBS) engineering, promoter engineering, and dynamic metabolic regulation were integrated to strengthen and balance the intracellular supply of the two core precursors, L-homoserine and succinyl-CoA. These strategies greatly increased the OSH titer to 18.54 ± 0.03g/L. Finally, global optimization of cofactor and energy optimization was carried out to further enhance strain performance, and the engineered strain OSHM40 achieved the OSH titer of 20.15 ± 0.21g/L via shake-flask cultivation, and 104.09 ± 2.06g/L in a 5-L bioreactor under fed-batch fermentation, with a sugar-acid conversion rate of 64.99% and a volumetric productivity of 1.43g/L/h. Notably, the OSH titer and sugar-acid conversion rate of this strain represent the highest levels reported to date among all plasmid-free OSH-producing strains. The plasmid-free system constructed in this study effectively avoids the plasmid-induced metabolic burden and genetic instability. This work demonstrates the prominent advantages and great application potential of plasmid-free modular engineering for the efficient biosynthesis of OSH and other high-value amino acids.

Enhancing d-Pantothenic Acid Production in Corynebacterium glutamicum via Integrating Dynamic Regulation With Structure-Guided Protein Engineering of Ketopantoate Reductase.

This study establishes a robust chassis for sustainable DPA production and provides a generalizable framework for engineering other valuable biochemicals.

Bee ovum-inspired hydrogel programs microenvironment remodeling for diabetic wound healing

Chronic diabetic wounds heal poorly due to a persistently imbalanced microenvironment involving inflammation and bacterial infection. Notably, oxidative stress, driven by mitochondrial damage, perpetuates inflammation and hinders repair. Existing therapeutic materials struggle to simultaneously address infection, inflammation, and oxidative stress through combined drug delivery and targeted activation of mitophagy. To tackle these intertwined challenges, we designed a bee ovum-inspired hydrogel (BOV) that mimics the parasitic wasp egg strategy, firm host adhesion and staged bioactive secretion, to programmatically remodel the wound microenvironment. The BOV consists of a borate-ester-crosslinked hyaluronic acid network providing robust wet adhesion and self-healing properties. It encapsulates gelatin-coated ZIF-8@Myricetin (Myr) nanoparticles and polyhexamethylene biguanide (PHMB), which are released sequentially in response to the wound's acidic, high-reactive oxygen species (ROS), and high-matrix metalloproteinase-9 (MMP-9) microenvironment: PHMB first exerts antibacterial action to control infection, followed by Myricetin release to scavenge ROS and suppress inflammation. Beyond antioxidant effects, BOV further activates the SIRT1/FOXO3a/BNIP3 pathway to promote mitophagy, clearing damaged mitochondria and thereby mitigating oxidative stress at its source. In vivo, BOV reduced bacterial burden, alleviated inflammatory response, and enhanced collagen deposition, and re-epithelialization. This study translates a natural parasitic strategy into a programmable drug-delivery platform, offering a promising approach for refractory diabetic wound therapy through microenvironment-responsive sequential treatment and upstream mitochondrial homeostasis restoration.

Enhanced Adenosine Biosynthesis in Paecilomyces hepiali Through Strain Evolution and Transcriptomic Insights.

Adenosine is a major bioactive nucleoside and quality marker in cordyceps-derived fungal products, but its efficient biosynthesis in Paecilomyces hepiali remains limited by insufficient strain performance and incomplete understanding of metabolic regulation. Here, we developed an integrated strategy combining protoplast-based strain evolution, medium optimization, and transcriptomic analysis to enhance adenosine biosynthesis in P. hepiali. Efficient protoplast preparation was achieved using 48 h seed cultures digested with 1% driselase and 1% yatalase at 28°C for 7 h, and 40 s ultraviolet irradiation was selected for mutant library construction. A stable mutant, P. hepiali A3, produced 65.32 mg/L adenosine, representing a 31.62% increase over the parental strain. Subsequent response surface optimization identified maltose, peptone, and aspartic acid as key nutritional factors, increasing the adenosine titer to 170.41 mg/L in shake flasks and 191.36 mg/L in a 5-L bioreactor. Comparative transcriptomic analysis revealed extensive metabolic remodeling involving central carbon metabolism, ribose precursor supply, purine nucleotide metabolism, and sterol biosynthesis. Upregulation of ribose-5-phosphate isomerase RPIB and downregulation of ADA related to purine degradation were associated with enhanced purine nucleoside accumulation. These results provide transcriptomic insights into adenosine biosynthesis and establish a practical framework for improving fungal nucleoside cell factories.

Transaminases engineering and their cascade systems for natural product synthesis.

Beyond direct pathway engineering: reprogramming Fusarium fujikuroi from a GA3 producer into a GA4+7 factory.

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.

Dynamically Coupled Network-Guided Engineering of Amine Dehydrogenase With Cofactor Recycling for Efficient Biosynthesis of (R)-3-Aminobutanol.

(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.

Construction of a Self-Assembled Multi-Enzyme Cascade for Efficient D-Allulose Biosynthesis.

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.

Active Pocket Engineering of d-Tagatose 4-Epimerase for Improved Catalytic Performance and Efficient Cascade Synthesis of d-Tagatose from d-Glucose.

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.