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.

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.

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.

d-Pantothenic acid (DPA) is an essential vitamin with broad applications. In this study, we engineered Corynebacterium glutamicum for high-titer DPA production by integrating dynamic pathway regulation with structure-guided protein engineering. Functional characterization revealed that endogenous CgPanE and BsPanE2 function as α-hydroxy acid dehydrogenases rather than ketopantoate reductases, whereas heterologous EcPanE and BsPanE, bifunctional activity toward both ketoisovalerate and ketopantoate, possess larger active cavities. A stationary-phase promoter (P4-N14) was employed to delay ketopantoate reductase expression, reducing precursor consumption. Multiple screening strategies were employed to identify candidate residues for alanine scanning and saturation mutagenesis, which yielded two beneficial mutants, T119I and I183S. Notably, I183S exhibited the most prominent improvements, with a 2.25-fold increase in specific activity and a 1.67-fold higher kcat/Km ratio than the wild-type. Molecular dynamics simulations indicated that the mutations enhanced catalytic efficiency by providing a more stable catalytic environment, tighter binding with the catalytic units, expanding the active cavity, and shortening the substrate tunnel length. The final engineered strain achieved a DPA titer of 36.12 g/L in a 5 L bioreactor. This study establishes a robust chassis for sustainable DPA production and provides a generalizable framework for engineering other valuable biochemicals.

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.

Rhamnolipids are biosurfactants with broad industrial applications, yet their production remains limited by low yield and high cost. Herein, the non-pathogenic P. putida KT2440 was engineered for enhanced rhamnolipid production through systematic medium optimization and metabolic engineering. Using response surface methodology, an optimal medium was developed with mixed carbon sources (glucose and glycerol) and nitrogen sources (yeast extract and NaNO3). The optimized medium increased the rhamnolipid titer from 1.5 g/L (in Luria-Bertani medium with 1% glucose) to 6.1 g/L in shake flasks, a 306.7% improvement. Transcriptional analysis revealed that medium optimization enhanced the Entner-Doudoroff pathway flux, reduced periplasmic pathway flux, and improved nutrient uptake, while the rhamnolipid synthesis pathway itself was not significantly upregulated. To address this bottleneck, metabolic engineering was applied to enhance the supply of rhamnolipid precursors. By overexpressing genes involved in rhamnose (rmlBDAC) and fatty acid (accA, accD, fabD) synthesis, the engineered strain, accAD-fabD-R, achieved a rhamnolipid titer of 9.8 g/L in shake flasks and 40.2 g/L in 5 L bioreactor, with a productivity of 0.56 g/L/h, representing the highest reported levels for engineered P. putida. This study demonstrates an effective strategy for highly efficient rhamnolipid production in P. putida, highlighting its potential for industrial-scale biomanufacturing.

Objective To establish a non-invasive predictive radiomics model of microvascular invasion (MVI) in hepatocellular carcinoma (HCC) and to explore the correlation between MVI status and prognosis of HCC patients after radiofrequency ablation (RFA). Methods A total of 142 HCC patients with or without MVI were randomly allocated into the training set (n=99) and the validation set (n=43). After preprocessing of tri-phase contrast-enhanced MRI, 1688 radiomics features were extracted per lesion. LASSO-Logistic regression was employed for featureselection and radiomics model construction to predict MVI in HCC. The model performance was evaluated using discrimination metrics. Additionally , 58 HCC patients who underwent RFA were enrolled. Univariate and multivariate logistic regression analyses were performed to investigate the impact of MVI on post-RFA prognosis of HCC. Results The radiomics model, especially that derived from the portal venous phase, exhibited effective performance in MVI prediction, achieving an area under the curve (AUC) of 0.888 in the training set and 0.769 in the validation set. Furthermore, the radiomics score was identified as an independent risk factor for recurrence after RFA. Conclusions This non-invasive radiomics model enables preoperative identification of MVI and prediction of post-RFA recurrence risk in HCC patients, thereby providing valuable evidence for formulating individualized treatment strategies.

A novel plasmid-free engineered E. coli strain was developed to significantly increase l -cysteine yield, achieving a titer of 35.54 g L −1 in a 5 L bioreactor.

Heparin, a clinically essential anticoagulant, has long been derived from animal sources, posing risks of contamination and supply chain instability. Bioengineered heparin, synthesized via microbial fermentation and enzymatic modification, offers a promising alternative with enhanced safety, homogeneity, and scalability. This review highlights recent advances in heparosan biosynthesis, enzymatic sulfation strategies, and analytical characterization for bioengineered heparin. Critical challenges remain, including precise control of heparosan molecular weight, optimization of sulfation patterns, demonstration of structural and functional equivalence to animal-derived heparin, and industrial-scale process validation. By combining synthetic biology with advanced bioprocessing and quality control, structure-defined bioengineered heparin is poised to become a sustainable, high-performance replacement for traditional heparin active pharmaceutical ingredient (API).

Vitamin B5 is a crucial water-soluble vitamin widely used in pharmaceuticals, food and animal feed. Traditionally, chemical enzymatic methods, which are not environmentally benign, predominate in the industrial production of VB5. Herein, we present a metabolically engineered Escherichia coli platform for sustainable VB5 production. The key strategies included (1) reinforcing the R-pantoate biosynthetic pathway, (2) redirecting carbon flux from the TCA cycle, and (3) constructing a one-carbon module to enhance precursor supply. Rational design of the acetohydroxyacid isomeroreductase (AHAIR), a critical metabolic node, improved its specificity for acetyl-lactate, thereby increasing the R-pantoate flux. The optimized strain DPAC4 achieved 148.31 g L-1 VB5 in a 5 L bioreactor over 96 hours with beta-alanine supplementation, yielding 0.43 g g-1 glucose and a productivity of 1.54 g L-1 h-1. To eliminate dependence on the beta-alanine supplement, a fully biosynthetic route (FBRV) was developed in strain DPAS3, enabling 65.12 g L-1 VB5 production in 60 hours (0.93 g L-1 h-1) without supplementation. This work demonstrates the synergy of the modular pathway and enzyme engineering for precise metabolic control, advancing the industrial feasibility of VB5 biomanufacturing. Our approach provides a blueprint for sustainable chemical synthesis through tailored microbial chassis design.

Gibberellic acid (GA3) is a critical plant hormone with significant agricultural applications, yet its production in Fusarium fujikuroi is constrained by competition for metabolic precursors, particularly acetyl-CoA, which is essential for GA3 biosynthesis. The genome of F. fujikuroi harbors numerous secondary metabolite biosynthetic gene clusters that divert acetyl-CoA away from the GA3 pathway, thereby limiting its yield. To address this challenge, we employed the CRISPR/Cas9 system to delete the bikaverin and fusarubin biosynthesis gene clusters, which are known to compete with GA3 biosynthesis for acetyl-CoA. This genetic intervention resulted in a substantial increase in GA3 production, with the ΔBIKΔFSR strain yielding 31.67 % more GA3 compared to the wild-type strain. Notably, the deletion of these gene clusters not only enhanced GA3 biosynthesis but also improved mycelial growth and carbon assimilation, as evidenced by increased consumption of reducing sugars during fermentation. We further employed qRT-PCR to assess comparative expression levels of genes associated with the glycohydrolysis, glycolysis, and the TCA pathway in engineered strain. Results indicated that removing by-product gene clusters enhances the glycohydrolase system, accelerating carbon assimilation. Given the presence of dozens of secondary metabolite biosynthetic gene clusters in the F. fujikuroi genome, the strategy reported here offers a promising avenue for further enhancing GA3 production by targeting additional non-essential gene clusters.

Synthetic biotechnology has boosted the manufacture of biobased chemicals. However, the development of sustainable synthetic routes for compounds with complex metabolic pathways remains challenging. In this study, we propose a metabolic node engineering approach to reprogram Escherichia coli for L-cysteine biosynthesis. By dissection of the metabolic module into input, process, and output nodes, a systematic optimization of key components in L-cysteine production was achieved. First, the input node was redirected by modifying the glucose utilization pathway, expanding the carbon supply pool. Subsequently, the process nodes glycerone phosphate and O-acetyl-l-serine were improved by rational metabolic engineering to enhance synthetic flux and block branched pathways. Furthermore, the L-cysteine output efficiency was enhanced by the construction of a dynamic efflux channel, which served to minimize the overflow of L-serine. Finally, the engineered strain EC21-1/pE(X5) produced 20.21 g/L L-cysteine in a 5-L bioreactor. This study provides a systematic method for optimizing biobased chemical synthesis, demonstrating the potential of biotechnology in environmental and resource conservation.

9 alpha-Hydroxyandrost-4-ene-3,17-dione (9-OHAD) is a representative precursor in steroid drug synthesis. However, during the 9-OHAD production from phytosterol in Mycolicibacterium neoaurum (M. neoaurum), the current manufacturing processes suffer from low productivity and purity due to the generation of a variety of byproducts. In this study, we genetically modified the phytosterol catabolism in M. neoaurum and achieved 13.4 g/L and 91.1 % molar yield of 9-OHAD with no detectable by-products at a high phytosterol concentration of 20 g/L in shake flask. It was achieved by inactivating Opccr, SalA, TeB, and overexpressing KshA1 and ChsH1-2 to eliminate the production of 20-hydroxymethyl-9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HBC), 4-androstene-3,17-dione (4-AD) and 3-oxo-4-pregnene-9-OH-20-carboxylic methyl ester (9-OH-3-OPCM) as well as overcoming the production limitations of 9-OHAD. Notably, when scaled up in a 5 L bioreactor with 45 g/L phytosterol concentration, the modified M. neoaurum achieved 24.5 g/L and 74.5 % molar conversion of 9-OHAD by using soybean oil and (2-hydroxypropyl)-beta-cyclodextrin (HP-beta-CD) as a co-solvent and antifoam agent. Therefore, our findings demonstrate a method to improve the efficiency and purity of 9-OHAD biosynthesis in M. neoaurum.

Pathway reconstitution for high-efficiency chemical production in engineered strains often leads to unbalanced intracellular redox and energy states. To overcome this limitation, we systematically redesigned central metabolism in the engineered strain to enhance redox homeostasis and energy regeneration. This strategy enabled increased production of D-Pantothenic acid (D-PA), an essential vitamin and coenzyme A precursor with applications in feed, pharmaceuticals and other industries. Specifically, genetic modifications targeting NADPH regeneration was conducted. Flux balance analysis (FBA) and flux variability analysis (FVA) were used to predict carbon flux distributions in the Embden-Meyerhof-Parnas (EMP), Pentose Phosphate (PPP), Entner-Doudoroff (ED), and tricarboxylic acid (TCA) pathways. With multi-module coordinated engineering of EMP, PPP and ED pathways, balanced intracellular redox state was established that maximized D-PA production while maintained robust growth, with the D-PA/OD600 increased from 0.84 to 0.88. By further conducting engineered electron transport chain coupled with overexpressing a heterologous transhydrogenase system from S. cerevisiae, intracellular redox state and energy supply was synchronously optimized, and 6.71 g/L D-PA was produced in flask, higher than original 5.65 g/L. Additionally, the 5,10-MTHF pool was optimized via optimized serineglycine system, ensuring sufficient supply of one-carbon unit for D-PA productions. Integrated implementing of these strategies under a temperature-sensitive switch for decoupling the cell growth and D-PA productions, the achieved strain produced 124.3 g/L D-PA with yield of 0.78 g/g glucose in fed-batch fermentation. These studies demonstrated the importance of maintaining intracellular redox balance and adequate energy supply on highefficient microbial production of useful chemicals, after the preliminary pathway reconstitutions.

d-Pantothenate (DPA), an essential functional compound, has experienced increasing market demand due to its widespread applications across the pharmaceutical, cosmetic, and animal feed industries. While numerous microbial classic strains have been engineered for DPA synthesis via microbial fermentation as an alternative to conventional chemoenzymatic synthesis, a comprehensive analysis of the metabolic engineering strategies employed for these microbial chassis cells for DPA production remains absent. This review systematically delineates the DPA metabolic pathway, encompassing β-alanine and pantoate metabolic modules, the regulatory network, and transport systems, and highlights the main regulatory mechanisms of operons and genes involved in the DPA biosynthesis pathway. The current research status in metabolic engineering strategies for manipulating DPA-producing strains is summarized and analyzed to elucidate the current trends and insights for further engineering. Finally, current challenges and future perspectives for the sustainable production of DPA are discussed, and guidelines for reducing production costs are proposed.

Synthetic biology is a crucial tool for the development of the bio-industry and bio-economy, representing a significant aspect of new quality productive forces. As a core course for graduate students in bioengineering, Synthetic Biology plays a vital role in ensuring the supply of essential talents for the development of the bio-industry in the new era. To better serve regional economic development and provide high-level talents for China's progress in the bio-industry, we analyzed typical issues encountered in the past teaching activities, set up a multi-disciplinary teaching team, optimized the course contents, adjusted the teaching mode, and mobilized students' learning interest. With the application of scientific research project as the starting point, we guided students to think and discuss deeply through the simulation of application writing and project defense, which improved students' critical thinking and innovative thinking. With industrialization as a focus, we explored a new training model combining production, education, and research through the joint practice base of the university and enterprises introduced typical cases of biomanufacturing to encourage students to engage in scientific research. The teaching reform significantly enhances the comprehensive abilities and national sentiments of graduate students. This paper hopes to serve as a reference for colleagues engaged in teaching in this field.

Polydatin is a bioactive stilbene with pharmaceutical potential, but its limited availability from natural sources poses a challenge for large-scale production. Here, we constructed an Escherichia coli strain capable of de novo biosynthesis of polydatin. A key bottleneck identified was the poor expression level of stilbene synthase (STS), which was addressed by screening heat shock proteins and promoter engineering, achieving a 50-fold increase in conversion capacity. Further optimization through chromosomal integration of glucosyltransferase led to an initial polydatin titer of 205.9 ± 4.9 mg/L. Enhancements in precursor supply, inactivation of competing pathways, and manipulation of the shikimate pathway improved polydatin titer to 3.8 ± 0.1 g/L. Finally, scale-up production in a 3-L fermentor coupled with optimization of key enzymes, malonyl-CoA availability and culture conditions, achieved a final titer of 14.9 ± 0.3 g/L. These results provide a robust framework for the efficient biosynthesis of high-value bioactive compounds in microbial cell factories.

O-acetyl-L-homoserine(OAH)is a promising platform compound for the production of L-methionine and other valuable compounds,while its low yield and low conversion rate limit the industrial application.To solve these problems,we constructed a strain for high OAH production with the previously constructed L-homoserine producer Escherichia coli HS33 as the chassis by systematic metabolic engineering.Firstly,PEP accumulation,pyruvate utilization,and OAH synthesis pathway(overexpressing aspB,aspA,and thrAC1034T)were enhanced to obtain an initial strain accumulating 13.37 g/L OAH.Subsequently,the co-factor synthesis genes were integrated to supply reducing power and energy,which increased the yield to 15.79 g/L.The OAH yield of the engineered strain OAH28 was further increased to 17.49 g/L by strengthening the acetic acid reuse pathway,improving the supply of acetyl-CoA,and regulating the expression of MetX from different sources.Finally,in a 5 L fermenter,OAH28 achieved an OAH titer of 47.12 g/L,with a glucose conversion rate of 32％and productivity of 0.59 g/(L·h).The results lay a foundation for increasing the OAH production by metabolic engineering and give insights into the industrial production of OAH.

D-pantothenic acid (D-PA) is a vital water-soluble vitamin with diverse industrial applications, driving the demand for efficient microbial production. Here, we rationally engineered an Escherichia coli strain to enhance D-PA production through metabolic engineering. First, to enhance carbon utilization efficiency, competing byproduct pathways were deleted and the pentose phosphate pathway was downregulated. Next, the glucose and β-alanine transport systems were strategically enhanced, and cofactor availability was improved through engineering NADPH regeneration and ATP recycling pathways. Subsequently, pathway engineering was applied to fine-tune the expression of heterologous enzymes, thereby enhancing the metabolic pull toward D-PA biosynthesis. To enhance the supply of one-carbon donor required by the rate-limiting enzyme ketopantoate hydroxymethyltransferase (KPHMT), a heterologous 5,10-methylenetetrahydrofolate biosynthesis module was introduced. Finally, dynamic regulation of isocitrate synthase and pantothenate kinase was implemented to balance cell growth and D-PA production. As a result of the integrated metabolic engineering strategies, the final strain DPZ28/P31 achieved a D-PA titer of 98.6 g/L and a yield of 0.44 g/g glucose in a two-stage fed-batch fermentation. These findings provide valuable insights for industrial-scale production of D-PA and related compounds.