AI Networking Cookbook: Practical recipes for AI-assisted network automation and development
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Gibberellins (GAs) are ubiquitous phytohormones that regulate plant growth and are widely used in agriculture. Among these, GA3 and GA4+7 are the only commercially available products, yet GA4+7 commands a much higher price than GA3, primarily due to its low titer in the industrial fungus Fusarium fujikuroi. To engineer a high-yielding GA4+7 producer, we first deleted p450-3 to block the conversion of GA4 and GA7 to GA1 and GA3. This led to the accumulation of GA4+7 at 0.934 g/L, a 37-fold increase over the wild-type strain, albeit with over half reduction in total GA accumulation. Using this Δp450-3 mutant as a platform, we combined metabolic engineering (overexpressing rate-limiting enzymes) with process optimization (pH, medium composition and fermentation duration). These combinatorial interventions synergistically boosted GA4+7 production. Under optimized conditions, the engineered strain achieved a final titer of 6.08 g/L (a 6.51-fold increase over the Δp450-3 parent), comprising 2.13 g/L GA4 and 3.96 g/L GA7, representing 5.30- and 7.44-fold increases, respectively. Preliminary optimization in a 10 L fermenter yielded 3.51 g/L of GA4+7. Finally, a solid-state fermentation system was developed on wheat bran, yielding 17.34 g GA4+7 per kg and enabling green, in-house, in-situ gibberellin production. In addition to substantially increasing the GA4+7 titer and total GA accumulation, this study demonstrated that the GA4/GA7 ratio can be modulated through both molecular and fermentation strategies.
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.
This study establishes a robust chassis for sustainable DPA production and provides a generalizable framework for engineering other valuable biochemicals.
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.
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.
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.
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