Given the high incidence of epidural‑related maternal fever (ERMF) among women with epidural analgesia and the key roles of glycoproteins, an efficient nanoplatform is needed for N‑glycoproteomic analysis to better understand ERMF. Herein, we constructed an ultra-hydrophilic magnetic MXene-based nanoplatform (MMX-Cys) for efficient N-glycopeptide enrichment. By integrating the high surface area of MXene, the hydrophilicity of L-cysteine, MMX-Cys demonstrated ultra-hydrophilicity with a water contact angle of 0°. It exhibits high sensitivity (1 fmol·µL⁻¹), excellent selectivity (HRP: BSA = 1:100), and good reproducibility in enriching glycopeptides from standard protein digests. Applied to plasma samples from ERMF patients and healthy controls, the MMX-Cys based nanoplatform enabled the identification of 1195 N-glycopeptides corresponding to 91 glycoproteins. Differential expression analysis revealed 18 significantly altered glycopeptides from 10 glycoproteins, predominantly involved in humoral immunity and complement activation, including immunoglobulin heavy chains (IGHG1, IGHG2, IGHA1, IGHA2) and complement component C2. This study presents an effective tool for N-glycopeptide determination and provides glycoproteomic evidence supporting the inflammatory pathogenesis of ERMF. The MMX-Cys based nanoplatform holds great potential in offering a foundation for future biomarker discovery and mechanistic studies.

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.

O-succinyl-L-homoserine (OSH), a key C4 platform compound, has broad applications across the pharmaceutical, pesticide, food, and feed industries. Metabolic engineering strains for efficient OSH production have been extensively developed. However, most engineered strains reported to date depend on plasmids, which may impose a metabolic burden and necessitate the use of antibiotics.This study aimed to develop a plasmid-free Escherichia coli strain for OSH biosynthesis. Initially, the metabolic flux of the primary OSH pathway was balanced. Subsequently, the pathways of the organic acid branch were systematically inhibited, and the glucose transport system was optimized to improve the efficiency of carbon source utilization. Finally, OSH accumulation was facilitated by increasing the supply of succinyl-CoA and enhancing the capacity for NADPH regeneration. The final strain, OSHFY-32, was fermented in a 5-L bioreactor using a fed-batch process. The OSH titer of this strain reached 132.28 g/L, with a yield of 0.55 g/g and a productivity of 1.28 g/L/h. Furthermore, successful scale-up was achieved in a 50-L fermenter, with titers, yield, and productivity exceeding the highest levels reported in the literature for plasmid-free strains, reaching 134.16 g/L, 0.54 g/g, and 1.26 g/L/h, respectively.The strategy used in this study can be applied to the production of other amino acids in the L-aspartate family.

Rhamnolipid manufacturing is transitioning from empirical cultivation to programmable, multi-scale molecular bioengineering. This review highlights the transition toward safe, non-pathogenic cell factories via genetic stabilization and precursor expansion. We elucidate how integrating structural biology with machine learning-using algorithms like UniESA and MLSmut-enables structure-guided engineering of rhamnosyltransferases (RhlA/B/C), achieving a fivefold increase in noncanonical congeners and tailored chain lengths. At the bioprocess scale, interfacial engineering strategies decouple oxygen transfer from intensive foaming; bubble-free membrane aeration delivers oxygen transfer rates up to 175 mmol L-1 h-1, while multi-stage froth separation loops drive biomass densities to 117.2 g L-1 with over 90% cell recovery. Finally, we connect upstream multi-omics tuning with downstream separation cascades and quantitative techno-economic assessments to guide the scalable production of application-specific rhamnolipid portfolios, ranging from environmental technical-grades to high-purity pharmaceutical surfactants.

Echinocandin B (ECB), a fungal non-ribosomal lipopeptide, serves as the exclusive natural precursor of the front-line antifungal anidulafungin. Despite its clinical importance, ECB production remains suboptimal due to incomplete understanding of its biosynthesis mechanism. The global regulator LaeA has been implicated in secondary metabolite production, yet its specific role in ECB biosynthesis remains unexplored. To address this, we successfully constructed laeA deletion and overexpression strains, and demonstrated that LaeA functionally couples morphological development with ECB productivity. Transcriptomic analysis revealed LaeA directly activated the sterigmatocystin cluster via pathway-specific regulator AflR, but indirectly influenced ECB through iron-heme cofactor synchronization rather than direct gene cluster activation. LaeA overexpression upregulated siderophore iron transporters and heme biosynthetic genes, with supplementation of Fe2 + or 5-ALA further boosting titers to 2487 ± 123 and 2697 ± 16 mg/L, respectively. To overcome P450s catalytic constraints, we screened out a novel cytochrome P450 reductase CPR2 as the optimal redox partner. Co-expression of CPR2 with bacterial hemoglobin VHb achieved synergistic enhancement, improving the ECB titer to 3170 ± 41 mg/L. This study provided a practical strategy for improving ECB production and offering insights into the versatile regulatory modes of global secondary metabolite regulators.

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 ± 0.27 g/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 ± 0.03 g/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 ± 0.21 g/L via shake-flask cultivation, and 104.09 ± 2.06 g/L in a 5-L bioreactor under fed-batch fermentation, with a sugar-acid conversion rate of 64.99% and a volumetric productivity of 1.43 g/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.

D-pantothenic acid (DPA) is a commercially vital bioproduct, yet its fermentation titer is often constrained by the lack of precise, real-time process regulation. In this study, a two-stage hybrid modeling framework was developed to achieve real-time prediction and dynamic optimization of Escherichia coli DPAT6 fermentation. First, a forward artificial neural network (ANN) was designed as a soft sensor to map environmental conditions (time, temperature, pH) to critical process state variables. These predicted states were then fed into a support vector machine with a radial basis function kernel (SVM-RBF), which exhibited superior predictive performance (RMSE = 5.03g/L, R2 = 0.982). Leveraging this hybrid model, a three-stage dynamic control strategy for temperature and pH was generated using particle swarm optimization (PSO) and genetic algorithms (GA). Experimental validation confirmed that this model-guided strategy increased the D-pantothenic acid titer to 125.98g/L, a 17.4% improvement over the experimentally optimized static (constant temperature and pH) control strategy (30.0℃, pH 6.80). This work demonstrates the potential of integrating machine learning with dynamic process control to enhance industrial fermentation efficiency.

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.

p-Coumaric acid (p-CA) is a phenylpropanoid-derived platform molecule widely used in food, cosmetic, and nutraceutical products, and microbial biosynthesis represents a sustainable alternative to plant extraction. Tyrosine ammonia-lyase (TAL) catalyzes the single-step, cofactor-independent conversion of l-tyrosine to p-CA; however, natural TALs often suffer from low efficiency and limited robustness. Here, we established an "evolution-to-design" workflow integrating evolutionary analysis, terminal-region engineering, and active learning-guided semi-rational design to optimize TAL from Rhodotorula glutinis (RglTAL). Selection pressure analysis revealed strong positive selection at both termini. N-terminal truncation (28 residues) increased whole-cell activity by 31.0% and raised Tm by 1.0°C, while N-terminal replacements further enhanced activity, indicating an N-terminal conformational constraint. AI-assisted screening identified three distal substitutions (G30N, Q387A, L609A); the triple variant improved specific activity by 63.7% (0.31 U/mg) and raised Tm by 3.4°C. Combining these mutations with N-terminal truncation further increased activity to 0.36 U/mg. Molecular dynamics simulations suggest these gains arise from synergistic stabilization, active-site pocket tuning, and truncation-like N-terminal remodeling by G30N. This work provides an improved TAL biocatalyst and a generalizable framework bridging evolutionary insights and data-driven design for bioprocess-oriented enzyme optimization.

cAMP receptor protein (CRP) plays an important role in transcriptional regulation in Escherichia coli. Here, we demonstrate that in addition to regulating carbon metabolism, CRP also modulates sulfur metabolism in E. coli. When cultured in M9s medium until glucose depletion, E. coli W3110 produced H2S, whereas a crp knockout strain did not. We discovered that persulfidation at CRP's Cys179 enhances its specific binding and upregulates the expression of sulfur metabolism-related genes (e.g., tnaA, mstA), thereby affecting production of the signaling molecule H2S. This suggests CRP can adjust gene expression in response to carbon-sulfur fluctuations under certain conditions. Bioinformatic analyses reveal CRP homologs are widespread among bacteria, implying that CRP's sulfur-sensing ability may be a general mechanism balancing carbon and sulfur metabolism.

Escherichia coli Nissle 1917 (EcN) is a well-established probiotic chassis with an excellent safety profile; however, its application as an industrial microbial cell factory remains limited, largely due to genetic instability and antibiotic-dependent expression systems. In this study, we developed an antibiotic-free and genetically stable metabolic engineering strategy for vitamin B5 (VB5) biosynthesis in EcN by integrating global regulatory rewiring with a cryptic plasmid-based pathway coupling platform. To enhance precursor supply, the VB5 biosynthetic pathway was reinforced by coordinated expression of AlsS, PanB, and PanC, leading to improved ketopantoate accumulation. Deletion of the global transcriptional regulators CRP and CRA significantly reshaped carbon metabolism, as revealed by transcriptomic analysis, resulting in enhanced transcription of key pathway genes and increased specific VB5 productivity despite reduced cellular growth. To overcome plasmid instability and metabolic burden associated with conventional expression systems, native cryptic plasmids (pMUT1 and pMUT2) were engineered as modular expression platforms. Positional integration of pathway genes into cryptic plasmids enabled tunable gene expression and ensured long-term genetic stability without antibiotic selection. The optimized strain harboring the complete VB5 pathway on engineered cryptic plasmids produced 98 mg/L VB5 in shake-flask cultures with stable productivity over multiple passages. In a 5-L fed-batch bioreactor, VB5 titer reached 1.15 g/L, representing an 11.8-fold improvement compared with flask cultivation. This work establishes a robust cryptic plasmid-based pathway engineering framework for probiotic E. coli, highlighting its potential as a safe, stable, and scalable chassis for antibiotic-free production of value-added metabolites.

This study presents a dynamic conformation-aware protein language modeling (DC-PLM) strategy for engineering Candida antarctica lipase B (CALB), a lipase whose catalytic efficiency is constrained by lid-dependent structural transitions. By integrating dual 3Di structural alphabet sequences derived from both open- and closed-lid conformations, together with a differential feature derived from dual-state SaProt embeddings and modulated via a Feature-wise Linear Modulation (FiLM) mechanism, the model effectively learns the coupled relationship between the sequence, structure, conformation and function. Iterative PLM-experiment feedback across five rounds led to the identification of high-performing mutants, culminating in a quintuple variant (M5) exhibiting an 8.6-fold increase in Vmax and a 16.5-fold enhancement in kcat. Molecular dynamics simulations revealed that beneficial mutations facilitate lid opening, stabilize the open conformation, and promote catalytically competent substrate orientations. When applied to industrial waste oil, engineered mutants significantly accelerated long-chain fatty acid hydrolysis, with M4 and M5 achieving reaction equilibrium within 1 hour compared to 5 hours for the wild type. These results demonstrate that incorporating dynamic structural information into PLMs enables mechanism-guided optimization of enzymes and provides a new framework for protein engineering.

Rhamnolipids (RLs) are green biosurfactants composed of structurally diverse congeners, each with distinct physicochemical properties. However, the tailored synthesis required to meet specific industrial demands remains a major challenge. Here, a highly tunable RLs production platform in the non-pathogenic Pseudomonas putida KT2440 was constructed by integrating orthogonal genetic regulation with precursor engineering. An initial dual-inducer system employing PrhaB and Ptac promoters was constructed, which, however, was limited in producing high-purity mono-RLs due to the inherent leakiness of Ptac promoter. To address this, a strictly orthogonal PrhaB/ParaB promoter system was implemented, enabling independent and precise regulation of rhlAB (for mono-RLs synthesis) and rhlC (for di-RLs extension), and thereby facilitating dynamic and programmable control of di-RLs fraction across an unprecedented range from 9.3% to 100%. This system represents the first reported full-range homologue control in a single chassis. Metabolic engineering revealed that enhancing fatty acid supply (fadD) increased titer without compromising ratio control, whereas augmenting the sugar donor supply (rmlBDAC) perturbed the homologue distribution, suggesting complex precursor allocation mechanisms. In a 5 L bioreactor, a total RLs titer of 30.2 g/L was achieved with di-RLs purity of 98.2%, which stands as the highest di-RLs titer and purity reported in P. putida. Functional characterization confirmed that di-RLs exhibit superior emulsification and foam stability, whereas mono-RLs show stronger antimicrobial activity. This work establishes a scalable and programmable platform to produce tailor-made rhamnolipids for specific industrial applications.

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.

Aspartate-α-decarboxylase (ADC) catalyzes the decarboxylation of L-aspartate to produce β-alanine, while was restricted for applications by its mechanism-based inactivation. Here the flexibility of the β-sheet containing Y58, a critical residue contributing to the mechanism-based inactivation, was rationally engineered for reducing its interaction with another β-sheet which contained catalytic residue S25. For the variants, enzyme activities of variant P61A, P61K, and R64P exhibited over 75

ABSTRACT The microbial production of pantothenic acid (d-PA) is critically limited by feedback inhibition and low activity of the key enzyme ketopantoate hydroxymethyltransferase (KPHMT). To overcome this, rational enzyme mining based on computational prediction was established. Following sequence conservation analysis, molecular dynamics simulations, and binding free energy (ΔG) calculations, five representative native KPHMT enzymes were selected for experimental validation: EcKPHMT from Escherichia coli, CgKPHMT from Corynebacterium glutamicum, MpKPHMT from Mangrovibacter plantisponsor, EpKPHMT from Enterovibrio pacificus, and BsKPHMT from Bacillus subtilis. EpKPHMT and BsKPHMT exhibited 4.25- and 4.60-fold times that of EcKPHMT, respectively, with relieved pantoate feedback inhibition (IC50: 14.07 and 19.86 mM vs. 1.08 mM) and virtually no inhibition by d-PA. The strain expressing BsKPHMT enhanced d-PA and pantoate titers by 74.30% (3.12 g/L) and 140.0% (0.84 g/L) in shake flasks, and further increased d-PA production by 55.4% in a 5 L bioreactor, over the control. This work established a predictive framework for mining superior enzymes based on in silico prediction, offering a valuable strategy for metabolic engineering of high-value chemicals.IMPORTANCEThe industrial-scale biosynthesis of pantothenic acid (d-PA) is often bottlenecked by the strict feedback inhibition of its key biosynthetic enzyme, ketopantoate hydroxymethyltransferase (KPHMT). This study describes a computational strategy for the mining and selection of naturally occurring KPHMTs with reduced feedback inhibition, providing superior genetic parts for metabolic applications. This approach provides a novel and rational framework for mining allostery-free enzymes, successfully delivering two highly efficient biocatalysts, BsKPHMT and EpKPHMT. These biocatalysts exhibit immediate potential for industrial applications, offering a direct solution to enhance the production of both pantoate and d-PA. Collectively, the integrated methodology demonstrates effective translation from fundamental discovery to practical application, presenting a generalizable model for overcoming similar metabolic bottlenecks.

Abstract In recent years, the principles of cine-substitution have been reinvigorated through transition-metal-catalyzed cross-coupling reactions, offering a modern approach to site-selective molecular editing. Compared with traditional ipso-coupling, cine-coupling provides complementary advantages in regioselectivity and structural diversification, enabling bond formation at positions adjacent to the leaving group that are often inaccessible by conventional methods. These transformations frequently employ readily available aryl and alkenyl derivatives and, through unconventional mechanistic pathways, directly construct structurally distinct molecular frameworks. The mechanistic complexity of cine-coupling is particularly noteworthy: metal migration, radical intermediates, and dynamic rearrangements not only account for the different regioselectivity but also create opportunities for reaction discovery and catalyst design. Despite the growing number of reports, a unifying framework to control selectivity between ipso- and cine-coupling pathways remains lacking. This review summarizes the major classes of cine-coupling reactions of alkenyl and aryl derivatives, highlights their mechanistic features, and discusses recent synthetic applications, aiming to stimulate conceptual development and expand the scope of transition-metal-catalyzed molecular-editing strategies.

Gibberellic acid (GA3) is a crucial phytohormone involved in plant growth regulation with widespread applications in agriculture and other fields. In this study, GA3 synthesis in an industrial GA3 producer F. fujikuroi strain FF00 was improved with a strategy by reprogramming the regulatory network and overexpressing key genes. Three positive regulators AreA (the nitrogen regulator), Lae1 (the velvet complex component), and Hat1 (the histone modification regulator) were identified by screening regulators affecting GA3 biosynthesis. Mutant FF18-4 with GA3 titer of 2.61g/L in shake-flask fermentation was constructed by co-overexpressing AreA, Lae1, and Hat1 in strain FF00. Comparative transcriptomic analysis revealed that reprogramming of the regulatory network resulted in the down-regulation of two key genes (Ggs2 and Cps/Ks) in the GA3 biosynthetic pathway which reduced the supply of GA3 precursor geranylgeranyl pyrophosphate. Ggs2 and Cps/Ks genes were further co-overexpressed to enhance metabolic flux for GA3 biosynthesis. The GA3 titer of the resulting mutant FF19-5 reached 2.73g/L, which was 49.2% higher than that of strain FF00. Therefore, the GA3 biosynthesis of strain FF00 was significantly improved by metabolic network reprogramming and metabolic balance. The established strategy provided the basis for GA3 over-producer construction and may be helpful for the synthesis of other chemicals with microbial cell factories.

Gene knockdown is a pivotal genetic manipulation technique, particularly when targeting lethal genes or genes involved in product synthesis pathways, where complete gene knockout is not a viable option. This approach is particularly valuable in multinucleate species, such as Fusarium fujikuroi, where generating homogeneous gene knockouts is notoriously difficult. To address these limitations, we first screened a set of repression domains, and then leveraged the optimal candidates to construct a CRISPR/dCas9-mediated knockdown platform for F. fujikuroi. By targeting erg9, which encodes squalene synthase, the first committed enzyme in the mevalonate pathway for ergosterol biosynthesis, we successfully diverted a portion of the metabolic flux from sterol production to gibberellic acid (GA) biosynthesis. This strategy minimizes carbon loss to competing pathways while retaining phenotypically normal growth. Additionally, CRISPR/dCas9-mediated knockdown of the dehydrogenase gene des enhanced GA4 production by 2.62-fold and eliminated the intermediate GA7, generating a GA3+4-producing strain and fine-tuning its metabolic profile. Using our CRISPRi system, we achieved a 70-89 % reduction in erg9 mRNA levels and a 67- 84 % reduction in des mRNA levels. Our findings establish a tailored CRISPRi platform for effective gene repression in F. fujikuroi.

L-arginine is a highly nitrogenous amino acid with a wide range of applications in feed, pharmaceuticals and food. Here we developed an Escherichia coli chassis strain for L-arginine production by enhancing the L-arginine synthesis pathway, which produced 58.8 g/L in 5-L bioreactor with a yield of 0.30 g/g glucose. With subsequent systematic engineering strategy focusing on enhancing the nitrogen utilization by adjusting the NH4+ uptake and synchronization-related precursor carbamoyl phosphate pathway, the L-arginine production was increased by 37%, however, the cell growth was inhibited by these gene modifications. To conquer this, Nucleoside Triphosphate (NTP) supply was firstly enhanced by regulating the purine pathway, which was beneficial for cellular repair and enhancing strain's stress resistance. Meanwhile, weakening the L-arginine import system reduces L-arginine consumption and conserves ATP. The final strain produced 102.4 g/L L-arginine, with a yield of 0.48 g/g glucose in 5-L bioreactor. The innovative approach of synergistic adjusting ammonia uptake and the purine pathway, balanced cell growth and boosted L-arginine production, offering valuable guidance for industrial microbial synthesis of other nitrogen-rich chemicals.