Digital twin (DT) technology is emerging as a promising approach for predictive analysis of real-world processes on the computer screen and transforming the optimization process. In food fermentation industry, feedstock variability, environmental deviations, as well as microbial dynamics together make it a very rigorous, tedious, and time-consuming process. This is where digital twin technology can change the fate of the industry. In this review paper, the theoretical approach along with its components and technological enablers such as soft sensors, cloud-integration platforms for data analysis and storage, process analytical tools, machine learning, artificial intelligence, Internet of Things, etc. of DT are discussed, which combine together to form the digital twin system. This study emphasizes the opportunities that DTs provide to the food fermentation industries, ranging from real-time monitoring and control of the microbial processes, adaptive control, contamination prediction, evaluating metabolites and by-products concentration to the simulation of the physical fermenter. While the technology provides promising results at the bench-scale, several limitations persist, which have been critically examined, such as biological variability, lack of standardized frameworks and common data platforms for diverse datasets. It also highlights the future prospects of the digital twins in the food fermentation industry by inducing modifications in the system and feeding pilot-scale data, which could pave the way for their incorporation, developing a sustainable and cost-effective technology.

The use of chemical pesticides has been facing obstacles due to tightening regulatory restrictions and rapidly emerging disease resistance. In this context, bio-based products are an effective alternative to be used in biocontrol. Lipopeptides are biosurfactants with powerful antimicrobial activity and are produced by different microorganisms, including those of the genus Bacillus. In this work, a biosurfactant-producing Bacillus subtilis strain exhibited inhibitory activity against the phytopathogens Rhizoctonia solani, Corynespora cassiicola, Colletotrichum truncatum, Sclerotinia sclerotiorum, and Aspergillus flavus, qualifying it as a potential microorganism for biocontrol. First, the kinetics of bacterial growth was investigated in flasks to monitor the basic parameters of the bioprocess. With these data, different nitrogen and carbon sources were tested as a mean to optimize bacterial spore formation and biosurfactant production. Yeast extract and glucose or sugarcane molasses presented the best results. With the new optimized medium, the process was carried out in a 10 L bioreactor and reached 4.99 × 10⁹ CFU/mL, 1.73 × 10⁹ spores/mL, and crude biosurfactant concentration of 2.80 g/L. LC–ESI–MS analysis confirmed the production of lipopeptides by B. subtilis DEBB B-328, with detected m/z values consistent with fengycin homologues (m/z 1489.8–1491.8) and a surfactin isoform (m/z 1022.6). These results indicate that the product based on B. subtilis DEBB B-328 is a promising alternative to chemical pesticides, with healthier and environmentally friendlier features.

Rising global energy demands, together with the need for alternative, sustainable, and scalable energy production and storage systems, have attracted industry attention. One such potential solution is bioelectric systems, such as biofuel cells (BFCs) and bio-based batteries (BBBs). BFCs are systems that utilize redox-active biomolecules and biopolymers derived from renewable biological sources such as plants, algae, and bacteria to generate electricity. Generally categorized by their catalysts: microbial biofuel cells and enzymatic biofuel cells (EFCs). BBBs are a subclass of BFCs, which replace an active feeding system with an internal fuel that is discarded once depleted. The varying physical properties, biocompatibility, and operation on renewable biological fuels expanded their use across fields such as waste management, healthcare, agriculture, and robotics. Despite active research and development, regulatory bodies lack policies and regulations governing the production and commercial use of BFC. Efforts to commercialize the technology are held back by technical limitations, economic challenges, and the lack of solid policies surrounding green energy. The failure of previous attempts to commercialize biobatteries has highlighted the gap between experimental feasibility and real-world implementation. Future progress in the field is expected to rely on improved integration of biobatteries into existing hybrid energy systems, advancement in the stability of bio-based materials, and the development of supportive regulatory and market infrastructures.

Sphingolipids are essential membrane constituents composed of aliphatic amino alcohols. They are widely distributed in eukaryotes and certain prokaryotes, acting as key signaling molecules. Bacteroidota are the only known group of gut bacteria capable of synthesizing sphingolipids, which dominate their membrane lipid composition and have been shown to modulate host immune responses and metabolic homeostasis. However, it remains unclear whether Bacteroidota synthesize sphingolipids via a eukaryote-like sphinganine-dependent pathway and/or a Caulobacter crescentus-like oxidized dihydroceramide pathway. In this study, two genes bt3032 and bt3075 in Bacteroides thetaiotaomicron were predicted to be related to sphingolipid biosynthesis by homologic alignments with the enzymes sphinganine acyltransferase and ceramide reductase characterized in C. crescentus. The genes Spt (bt0870), Kdsr (bt0972), bt3032 and bt3075 were deleted individually in B. thetaiotaomicron VPI 5482, resulting the mutant strains ΔSPT, ΔKDSR, ΔBT3032 and ΔBT3075, respectively. The lipids were isolated from these mutants and analyzed, using thin-layer chromatography and liquid chromatography–mass spectrometry. Deletion of Spt resulted in complete loss of sphinganine, dihydroceramide, and downstream sphingolipids, confirming its role as the serine palmitoyltransferase catalyzing the first step of dihydroceramide biosynthesis. Deletion of Kdsr caused accumulation of 3-ketodihydrosphingosine while sphingolipid synthesis was partially retained, indicating an alternative dihydroceramide biosynthetic route. In contrast, deletion of bt3032 abolished dihydroceramide and derived sphingolipids, demonstrating its essential role as a dihydroceramide synthase. Deletion of bt3075 reduced sphingolipid levels. Collectively, these results reveal multiple ceramide biosynthetic pathways in B. thetaiotaomicron.

N-acetylserotonin O-methyltransferase (ASMT) is a key rate-limiting enzyme in microbial melatonin biosynthesis, and its low catalytic efficiency hinders high-yield production. To enhance its catalytic performance, virtual saturation mutagenesis scans were conducted on all residues located within the binding substrate 6 Å in this study, and then the mutant library was screened based on computational tools. Rosetta Cartesian_ddg was subsequently used to evaluate the effects of mutations on protein folding stability, allowing the exclusion of structurally destabilizing variants. Next, the binding free energy changes of enzyme-substrate complexes were calculated using the MM-GBSA method to identify mutations with the potential to improve substrate affinity. Through this strategy, a high-activity mutant, M105W/Y108W, was identified, exhibiting a 6.1-fold increase in catalytic efficiency (k_cat/K_ₘ) compared to the wild type (WT). Molecular dynamics simulations and structural analysis revealed that the improved activity resulted from enhanced structural stability, optimized microenvironment within the binding pocket, and strengthened key interactions. When introduced into Escherichia coli co-expressing serotonin N-acetyltransferase (SNAT), and following optimization of expression elements and fermentation conditions, this engineered strain achieved a melatonin production of 742.13 mg/L in shake-flask fermentation, representing the highest level reported at this scale. This study not only obtained a highly efficient engineered ASMT variant, but also proposed a broadly applicable strategy for the rational remodeling of enzyme substrate-binding pockets.

Antarctica hosts one of Earth’s most extreme yet biologically rich microbial diversity, where cold-adapted fungi have evolved unique metabolic capabilities with exceptional bioprospecting potential. This systematic review integrates evidence from diverse Antarctic habitats including oligotrophic soils, glacial ice, marine sediments, sponges, krill, and angiosperms to assess the breadth of bioactive metabolites and their therapeutic relevance. A total of over 100 fungal taxa, predominantly belonging to Penicillium, Pseudogymnoascus, Geomyces, Aspergillus, and several endophytic genera have beenreported to produce structurally diverse compounds such as carotenoids, diketopiperazines, phenolics, eremophilane sesquiterpenes, peptides, and chromones. These metabolites exhibited wide-ranging biological activities, including potent antibacterial, antifungal, antiviral, antioxidant, cytotoxic, antitumoral, antiprotozoal, antiparasitic, herbicidal, antiallergic, and anti-inflammatory effects. Several species demonstrated activity at remarkably low inhibitory concentrations, highlighting their pharmaceutical promise. Despite such diversity, large gaps persist regarding biosynthetic gene clusters, ecological drivers of metabolite expression, and mechanisms of action. The review articles highlights Antarctica as an untapped reservoir of chemically novel fungal metabolites with significant implications for drug discovery, agriculture, biotechnology, and natural product chemistry. Further advance studies such as integrative omics-driven approaches, sustainable sampling strategies, and international collaborations are key to unlock the full potential of Antarctic fungal bioresources.

Considering the widespread use of blood samples in clinical medicine, disease diagnosis, and forensic science, there is an urgent need for the development of Taq DNA polymerases that are resistant to whole blood inhibitors and can maintain high amplification efficiency. This study reveals a dual mechanism by which blood inhibits PCR amplification: first, heme inhibits its activity by binding to DNA polymerase; second, immunoglobulin G binding to template DNA leads to a significant increase in the threshold cycle (Ct) value. Meanwhile, high concentrations of inhibitors generally led to a fluorescence quenching effect of the dye during amplification. Based on protein engineering techniques, this study identified key residues in Taq DNA polymerase that interact with heme. The mutant Taq43(TaqR37A/K314A/P387L), constructed by semi-rational design, had a heme-tolerant concentration (7 µmol/L) that was sevenfold higher than that of the wild type (WT). Further, by deleting the highly flexible 5′-3′ exonuclease structural domain and fusing it with the double-stranded DNA-binding protein Sso7d to form a chimera, the final S-KLTaq43 mutant was obtained to achieve robust PCR amplification in the presence of 50% (v/v) whole blood. This study holds significant practical value for advancing the development of direct testing technologies for blood samples.

Non-Saccharomyces yeasts significantly contribute to aroma complexity in fermented foods and beverages; however, the role of nutrient availability in constraining ester biosynthesis remains poorly understood. Here, an aroma-active non-Saccharomyces yeast, Cyberlindnera fabianii, isolated from a traditional fermented food, was used to investigate how carbon availability and carbon-to-nitrogen (C/N) ratio regulate ester biosynthesis. Five nutritional conditions were examined by integrating gas chromatography–mass spectrometry (GC–MS)–based volatile profiling, transcriptomics, and targeted intracellular metabolomics. High carbon and medium-to-low C/N ratios enhanced ester production, with phenethyl acetate and isoamyl acetate as major discriminant compounds. In contrast, low carbon and high C/N ratios favored organic acid accumulation and off-flavor formation. Multivariate analyses revealed ester-specific metabolic constraints: phenethyl acetate synthesis was limited by aromatic amino acid supply, whereas isoamyl acetate depended on branched-chain amino acid biosynthesis. Overall, these results demonstrate that carbon availability and C/N ratio reposition metabolic bottlenecks in ester formation, providing a nutrition-based framework for aroma modulation.

ε-Poly-L-lysine (ε-PL) is a high-value biopolymer with broad applications in food, pharmaceuticals, and biomaterials. However, the current downstream process remains a major barrier to sustainable large-scale ε-PL biomanufacturing. In this study, a liquid-circulating fluidized bed (LCFB) ion-exchange chromatography using a cationic resin has been developed for the purification of ε-PL for the first time. The adsorption behavior of ε-PL on the resin was well described by the Langmuir isotherm and pseudo-second-order kinetic models. The constructed LCFB process achieved 95.53% recovery and 46.12% purity under the optimized conditions: adsorption at pH 7.0 and desorption with 1.0 mol/L NaOH at 1.5 bed volume per hour. Compared with the fixed-bed mode, the LCFB process demonstrated superior performance, including a 37.94% increase in resin utilization, a 29.28% reduction in alkali consumption and a 27.44% reduction in wastewater generation. The process was successfully scaled-up 30-fold while maintaining operational stability. After integrating LCFB process into our previously developed ε-PL purification scheme, the final ε-PL hydrochloride product exhibited (97.56 ± 0.90)% purity with (81.14 ± 1.18)% recovery. This study highlights the efficiency, scalability, and environmental friendliness of the LCFB process as a promising method for the industrial purification of ε-PL, it also providing a valuable low-emission framework that can be extended to the purification of other bio-based products.

In this study, Enterococcus faecalis was employed as the target microorganism to enhance γ-aminobutyric acid (GABA) production. A high GABA-producing mutant strain, En1203, was obtained through atmospheric and room temperature plasma (ARTP) mutagenesis combined with gradient resistance screening. Subsequently, a microdroplet cultivation (MMC) based adaptive evolution strategy was applied to further improve the tolerance of the mutant strain to high concentrations of monosodium glutamate (MSG), leading to the selection of a genetically stable high-producing strain, EM05. On this basis, key medium components, including substrate composition, carbon sources, nitrogen sources, and sodium succinate, were systematically optimized. The results demonstrated that the use of a mixed substrate consisting of L-glutamic acid and MSG significantly enhanced GABA accumulation efficiency. Under the optimized medium conditions, the maximum GABA concentration reached 60.7 g/L in shake-flask fermentation. Furthermore, scale-up fermentation in a 3 L bioreactor revealed that strain EM05 maintained robust growth and metabolic stability under high substrate loading, resulting in a further increase in GABA production to 64.2 g/L. Overall, this study demonstrates that the combination of mutagenesis breeding and adaptive evolution, together with medium optimization, is an effective strategy for improving the yield and stability of GABA production by lactic acid bacteria, providing valuable theoretical insights and technical support for its application in food fermentation.

RNA helicases are a class of highly conserved RNA-binding proteins that unwind double-stranded RNA by hydrolyzing ATP, playing essential roles in processes such as ribosome biogenesis, pre-mRNA splicing, transport, translation, and decay of pre-mRNA. However, their functional roles in the stress responses of Candida glycerinogenes remain uncharacterized to date. Herein, CgDBP7 (C. glycerinogenes DEAD-box RNA helicase DBP7) and its downstream responsive gene CgMAK5 (C. glycerinogenes ATP-dependent RNA helicase MAK5) were first characterized as dual negative regulators of high-salinity tolerance in C. glycerinogenes. Mechanistically, the enhanced high-salinity tolerance of the engineered strain C. glycerinogenes-antiCgDBP7a-antiCgMAK5 was associated with intracellular reactive oxygen species (ROS) detoxification and glycerol biosynthesis. Glycerol acts as an osmoprotectant to balance osmotic pressure under high salt, while efficient ROS detoxification mitigates cellular oxidative damage, collectively boosting tolerance and metabolite production. Under high-salinity conditions, the stress-resistant engineered strain C. glycerinogenes-antiCgDBP7a-antiCgMAK5 achieved a 13.5% and 16.5% increase in glycerol and ethanol titers, respectively. In undetoxified lignocellulosic hydrolysate, this recombinant strain exhibited a further increase of 45.2% in ethanol titer and 13.4% in glycerol titer. Collectively, these findings demonstrate that CgDBP7 and CgMAK5 form a two-layered repressive circuit and serve as key genetic elements for salinity stress adaptation in C. glycerinogenes, providing programmable targets for engineering robust industrial yeast.

Bioreactors serve as the core of modern bioprocessing, enabling precise control over microbial, enzymatic, and mammalian cell cultures. Yet, their complex dynamics, nonlinear behaviour, and multivariable interactions often limit the effectiveness of traditional modelling and control methods. Recent advancements in artificial intelligence (AI) and machine learning (ML) offer data-driven solutions to enhance modelling accuracy, adaptive control, and real-time optimization. This systematic review investigates the application of AI/ML in bioreactor engineering by examining key bioreactor types, data modalities, algorithmic strategies, control frameworks, and hybrid models. Adhering to PRISMA 2020 guidelines, 58 peer-reviewed articles were selected from five databases—Scopus, IEEE Xplore, ScienceDirect, Scispace, and Google Scholar—using structured Boolean queries. The review highlights the use of algorithms such as artificial neural networks (ANN), support vector machines (SVM), random forests (RF), long short-term memory (LSTM), and reinforcement learning (RL) across batch, fed-batch, continuous, and perfusion systems. Applications include soft sensor development, state estimation, fault diagnosis, and closed-loop control using time-series, sensor, and multi-omics data. Hybrid approaches that fuse mechanistic and AI/ML models—such as grey-box systems, physics-informed neural networks (PINNs), and digital twins—are gaining traction due to their improved generalization and interpretability. Despite these advancements, challenges remain regarding model validation, scalability, and regulatory approval. The review identifies future research directions, including explainable AI, cyber-physical integration, transfer learning, and multi-omics fusion. Overall, AI/ML integration marks a paradigm shift toward intelligent, adaptive, and efficient bioprocess control.

Nitrilase is an important industrial enzyme that catalyzes the one-step conversion of nitriles to carboxylic acids. Due to its diverse origins, heterologous expression of nitrilase in model microbial chassis often leads to the formation of inactive inclusion bodies, which severely hampers its screening, characterization, and industrial application. However, unlike improvements in activity and stability, engineering for enhanced solubility lacks well-defined quantitative metrics, making rational design challenging. Fusion expression with fluorescent proteins provides an effective means to monitor the spatiotemporal characteristics and yield of target proteins. In this study, we established a screening platform for quantitatively assessing the distribution of nitrilase between soluble and insoluble fractions by fusing it with the superfolder green fluorescent protein (sfGFP). Based on this platform, we constructed saturation mutation libraries targeting six solvent-exposed hydrophobic residues on the surface of a previously obtained high-activity mutant PD1, aiming to isolate variants with improved solubility. Four positive single-point mutations (I5S, F25N, V69C, and I201T) were identified, which not only increased nitrilase solubility but also enhanced whole-cell activity and thermostability. Furthermore, by exhaustively exploring all combinatorial patterns of double, triple, and quadruple mutations, we ultimately obtained the optimal triple mutant PD1Mut3 (I5S/V69C/I201T). Compared to PD1, PD1Mut3 demonstrated significantly improved soluble yield, a 5 °C higher optimal temperature (45 °C), a 1.8-fold longer half-life at 50 °C, and superior whole-cell catalytic performance. Molecular dynamics simulations revealed that the introduced mutations reduce structural flexibility, promote a more compact oligomeric assembly, and decrease local solvent exposure, collectively explaining the enhanced thermostability. This work establishes a general integrated strategy combining surface hydrophobic residue engineering and GFP-fusion-based screening to simultaneously improve enzyme solubility and catalytic performance, thereby facilitating further industrial applications.

Astaxanthin, a keto-carotenoid renowned for its strong antioxidant capacity, has become an increasingly preferred target for microbial biosynthesis. Saccharomyces cerevisiae is regarded as a promising host for astaxanthin production because of its native mevalonate pathway. However, the limited catalytic efficiency of the key enzymes β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) remains a major constraint in achieving high yields. In this study, we combined multiple strategies to enhance catalytic performance. First, we performed combinatorial screening of CrtZ and CrtW from diverse sources and identified an enzyme pair that efficiently catalyzed the conversion of β-carotene to astaxanthin. Subsequently, a heterologous ferredoxin-based redox-partner system was introduced and optimized, resulting in a 79.5% increase in astaxanthin production to (63.21 ± 2.74) mg/L. In addition, a dual-compartmentalization strategy targeting both lipid droplets and the endoplasmic reticulum enhanced the spatial proximity between CrtZ/CrtW and the substrate β-carotene while reducing byproduct accumulation, resulting in a 29.9% increase in astaxanthin production to (82.12 ± 2.37) mg/L. Finally, by optimizing induction timing and applying fed-batch fermentation in a 5 L bioreactor, the astaxanthin titer was further elevated to (507.68 ± 27.12) mg/L, representing the highest level reported to date in S. cerevisiae. Collectively, this work establishes an integrated metabolic engineering framework for improving astaxanthin biosynthesis in S. cerevisiae.

Banana peels, an abundant agro-industrial residue rich in carbon and nitrogen, make it a viable low-cost substrate for bacterial growth and cellulase production. The present study employed response surface methodology (RSM) using central composite design (CCD) to systematically optimize key process parameters influencing cellulase production by Bacillus sp. FBT2 via solid-state fermentation (SSF). Five independent variables, namely initial pH, incubation temperature, substrate moisture content, Tween® 80 concentration, and inoculum size, were investigated for their influence on the enzyme production yield. After 72 h of fermentation under optimized conditions (pH 7.0, 30 °C, 60% moisture, 0.2% Tween® 80, and an inoculum size of 10⁸ spores/g) resulted in a maximum cellulase activity of 3.31 U/g, which closely matches the predicted value of 3.29 U/g, confirming the reliability of the statistical model. The current study demonstrates that utilizing banana peels as a solid substrate in SSF, combined with process optimization, offers environmental benefits through waste reduction and potential cost savings in cellulase production.

Fusarium graminearum infects barley easily, causing Fusarium head blight and the subsequent production of mycotoxin deoxynivalenol, which poses significant health risks to both humans and animals. Grains contaminated with deoxynivalenol can only be discarded, resulting in enormous waste. In previous study, carvacrol-loaded chitosan nanoparticles that inhibited the growth of F. graminearum were developed to save the economic losses caused by deoxynivalenol contamination in barley malt. However, its antifungal efficacy and mechanism of action remain unclear, and the present study therefore seeks to elucidate both. The results demonstrated that these nanoparticles effectively inhibited spore germination and germ tube elongation of F. graminearum when the concentrations exceeding 200 µg·mL^− 1, thereby impeding its mycelial growth. Furthermore, the measurement of relative electrical conductivity revealed that treatment with carvacrol-loaded chitosan nanoparticles significantly increased the relative electrical conductivity of F. graminearum cells, which was attributed to severe disruption of cell membrane integrity and consequent leakage of intracellular proteins and nucleic acids. In the barley storage experiment, the content of ergosterol and deoxynivalenol in rehydration spray-treated barley malt was 2.2 times (with 200 mg carvacrol-loaded chitosan nanoparticles addition) and 7.3 times (with 500 mg carvacrol-loaded chitosan nanoparticles addition) higher than that of carvacrol-loaded chitosan nanoparticles treated barley malt. At the same time, they maintained the malt quality parameters (including moisture, extract content, saccharification time, etc.) without significant alteration. These findings carvacrol-loaded chitosan nanoparticles can serve as an environmentally friendly and highly effective antifungal agent, and their large-scale application will help promote global food security and sustainable agricultural development.

Keratinases offer a promising green strategy for valorizing recalcitrant keratin waste, yet their practical application is often hindered by low substrate specificity and limited catalytic efficiency. Herein, we report the successful engineering of a highly specific and robust keratinase variant, G209F/G235S, derived from KerBv via a semi-rational design strategy targeting the active pocket. By integrating homology modeling, molecular docking, and evolutionary conservation analysis, key residues within binding pocket were identified and optimized. The engineered mutant exhibited a doubled catalytic activity of 8,540 U/mL and a significantly enhanced keratin-to-casein hydrolytic ratio (K/C) of 1.64. Comprehensive characterization revealed that G209F/G235S possesses superior thermostability with the optimum reaction temperature of the G209F/G235S mutant shifted from 40 °C (WT) to 45 °C, and maintains robust activity across a broad alkaline pH range (7.0–11.0). Molecular dynamics simulations and structural analyses elucidated the underlying mechanisms: the mutations synergistically expanded the active pocket volume from 561 to 689 ų, and optimized electrostatic complementarity for negatively charged keratin. In practical applications, the mutant achieved over 80% degradation of native feather waste within 2 h, outperforming the WT by 50% degradation ratio. This study not only delivers a potent biocatalyst for the efficient upcycling of keratinous by-products but also provides fundamental insights into the structure–function relationships governing keratinase specificity, establishing a robust strategy for the rational design of high-performance industrial enzymes.

To enhance in-situ bioconversion of coalbed methane by microbial nutrient solution, two long-term operating coalbed methane wells in the Hancheng block of Shaanxi Province, China (up to 2010) were employed as the research objects. The thickness of the main coal seam is approximately 26–115 m, the salinity is appropriate (3,000–12,000 mg/L), and the permeability is low (0.001–3.503 md), which is suitable for large-scale coalbed methane exploration. After nutrient solution (660 m³) was injected, the pH value gradually increased from 6.5 to 7.5 (100th day) and then slowly decreased to 7.0 (300th day) as the accumulated VFAs were rapidly utilized by the inoculated bacteria. The bottom hole pressure recovery rate after injection was 0.37–0.95 MPa (15th day), which was better than 0.7–0.8 MPa (40th day) before injection. The microbial community structure indicated that the bacteria in both wells were more dominant, while the archaea in the test well increased to 6.3%. The relative abundance of Methanobacterium formicicum increased from 0.25% to 4.14%. At the species level, the dominant microorganisms in the control well were Ralstonia pickettii and Variovorax sp., while those in the test well were Stutzerimonas stutzeri and Variovorax paradoxus. The typical denitrifying microorganisms Stutzerimonas stutzeri and Comamonas sp. were significantly increased by 14.29% and 5.53%. The abundance of Ralstonia pickettii decreased by 18.41%, and the methanooxidizing bacteria genus accounted for 15.89%. Gene function enrichment analysis indicated that compared with the phenylpropionate degradation mode of the control group, the metabolic mode after microbial intervention shifted to pyruvate metabolism.

3-dehydroshikimic acid (3-DHS), which is recognized as a key precursor of the shikimate pathway, has been regarded as an important platform compound for the biosynthesis of a wide range of aromatic products. However, microbial production is often constrained by imbalanced pathway expression and the accumulation of by-products, resulting in reduced cellular activity and loss of the target product. In this study, combinatorial metabolic engineering was applied for the production of 3-DHS. The downstream pathway was blocked through the deletion of aroE, after which engineering targets were screened and cumulatively integrated to enhance the basal metabolic flux. Subsequently, the biosynthetic pathway was optimized using a modular design strategy. On this basis, a multidimensional fine-tuning regulation strategy was implemented, by which the accumulation of the by-product gallic acid was reduced by 74.3%. Furthermore, carbon source utilization efficiency was enhanced through the combination of reinforced non-PTS glucose uptake and an optimized feeding strategy. As a result, a 3-DHS titer of 147.5 g/L was achieved in a 5 L bioreactor, with a yield of 0.6 g/g glucose and a productivity of 3.69 g/L/h, all of which represent the highest values reported to date. Overall, this study is expected to provide an important reference for the scalable biomanufacturing of 3-DHS and its high-value derivatives.

This study developed a highly selective and sensitive surface-enhanced Raman scattering (SERS) aptasensor for detecting enrofloxacin (ENR), a persistent fluoroquinolone antibiotic posing environmental concerns. This SERS aptasensor is constructed using flexible carbon cloth (CC) as the substrate, which is oxidized and then modified with titanium dioxide (TiO₂), followed by loading with gold nanoparticles (AuNPs) and functionalization with ENR-specific aptamer (Apt). The flexible nature of CC enables the substrate to adapt to irregular surfaces, providing structural support for potential portable on-site detection applications. It leverages a synergistic signal enhancement effect from localized surface plasmon resonance (LSPR) of AuNPs and the likely charge-transfer contribution of TiO₂. A partial least squares (PLS) regression model correlated full spectral profiles with ENR concentrations, enabling accurate quantification while effectively mitigating spectral complexity and matrix interference. Under optimized conditions, the aptasensor exhibits a wide linear detection range (1–10⁴ nmol/L) with a low detection limit of 0.37 nmol/L. It demonstrates good selectivity against interferents, high reproducibility, with relative standard deviation (RSD) at 1.87%, and excellent stability over 14 days (RSD below 2.83%), and accurate spiked recoveries in real water samples (96.94%–109.47%). These results show the great potential of the proposed SERS aptasensor for reliable and practical monitoring of ENR in environmental applications.

Zaopocu is traditionally produced using Jiuyao as a solid-state starter. However, rapid fermentation processes adopted in recent years often result in reduced aroma intensity and flavor complexity. In this study, a reproducible Jiuyao–Zaopocu fermentation workflow was established to clarify how starter culture affects downstream flavor formation under accelerated conditions. A temperature-controlled strategy was applied during Jiuyao production, and key factors were optimized using Plackett–Burman screening and Box–Behnken response surface methodology. The optimized conditions were identified as a mixing water temperature of 48 °C, Muqu addition of 1.9%, and high-enzyme-activity Jiuyao from Fangxian (FX) addition of 1.6%. Under these conditions, total acid (g/L) and ethanol (%) contents increased by 22.7% and 67.6%, respectively, compared with the basic process. Microbial and flavor analyses revealed a clear ecological transition from a mold-lactic acid bacteria co-dominated community (Rhizopus, Pediococcus) in Jiuyao to a Limosilactobacillus-dominated community during Zaopocu fermentation. Meanwhile, several aroma-active compounds enriched in Jiuyao, including 4-ethyl-2-methoxyphenol, ethyl tetradecanoate, and 3-methylbutyric acid, markedly decreased during rapid liquid fermentation, accompanied by a reduction in overall volatile diversity. Correlation analysis indicated that Rhizopus was positively associated with key aroma compounds, but its contribution weakened after the Jiuyao to Zaopocu transition. These findings suggest that part of the flavor potential generated during Jiuyao preparation is not fully retained under rapid Zaopocu fermentation. Therefore, front-end regulation of starter preparation and targeted retention of key functional microorganisms may help preserve aroma complexity under accelerated processing conditions.

Herein, enhanced PHBV production process by Cupriavidus necator MT-68 was developed through mixotrophic fermentation using low-cost and abundance molasses and various co-substrates. Among nine co-substrates examined, mixture of molasses and propanol provided the best PHBV production performance. By controlling the production of 3HB via feeding of sugars in molasses and 3HV via feeding of propanol in two-stage fed-batch process, the production of PHBV with high 3HV content was obtained and its production efficiency was improved by 2-fold compared to batch process. Under fed-batch cultivation, (2.7 ± 0.2) g/L of PHBV with 3HV molar fractions of 41.9% was achieved. PHBV production, with different 3HV fraction and rearrangement, was also demonstrated through two-stage fed-batch with alternated feeding patterns of molasses and propanol. Overall, this mixotrophic strategy enabled the production of PHBV with a high 3HV content, which could improve bioplastic flexibility and broaden its potential applications.

Anthropogenic carbon dioxide (CO₂) emissions drive global climate change, motivating the development of bioprocesses that improve carbon utilization and enable CO₂ recycling. In this study, we developed a CO₂-fixing Saccharomyces cerevisiae chassis for single-cell protein (SCP) production using xylose derived from cellulosic biomass as a carbon source. A RuBisCO- based CO₂-fixation pathway was previously integrated into a xylose-utilizing strain, enabling the routing of CO₂ into central metabolism. Flux balance analysis combined with ¹³C-based intracellular metabolite analysis verified the assimilation of externally supplied CO₂ into central metabolism, suggesting the potential for assimilation of fermentation-derived CO₂ and improved carbon utilization during SCP production. To enhance SCP production, the PAN2 gene encoding a component of the poly(A)-ribonuclease complex, previously associated with increased global protein production in S. cerevisiae, was truncated in the CO₂-fixing strain. Under anaerobic conditions, the engineered strain exhibited a significant increase in cellular protein content, accompanied by an overall upward trend in amino acid levels relative to the parental strain. Collectively, these results metabolically verify RuBisCO-mediated CO₂-fixation in yeast strain and demonstrate the feasibility of coupling CO₂-fixation to SCP production. This work also provides insights into the potential of integrating CO₂-fixation with renewable carbon metabolism and establishing a proof-of-concept platform for the development of low-carbon yeast bioprocess.

Pleurotus ostreatus is an edible mushroom widely recognized for its nutritional value and high content of bioactive compounds, including phenolics and polysaccharides, which confer potential antidiabetic and anti-obesity effects. These properties are mainly associated with its ability to modulate digestive enzymes involved in carbohydrate and lipid metabolism, as well as its antioxidant activity. This study applied a bioprocess-based approach to produce Amazonian P. ostreatus mycelial biomass via submerged fermentation, followed by the evaluation of its biochemical composition, antioxidant activity, and inhibitory effects on key digestive enzymes. Different combinations of carbon and nitrogen sources were investigated based on mycelial biomass production to determine the most suitable fermentation condition. To evaluate digestive enzyme inhibition, a 2³ factorial experimental design was applied to assess the effects of carbon source, nitrogen source, and extraction solvent, as well as their interactions, on the inhibition of α-amylase, α-glucosidase, and pancreatic lipase. The mycelial biomass obtained under condition T4 (sucrose + peptone) showed strong α-glucosidase inhibition [(96.94 ± 0.49)%], moderate α-amylase inhibition [(76.39 ± 0.36)%] and pancreatic lipase inhibition [(60.32 ± 0.64)%], as well as high antioxidant activity (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS^·+): (90.95 ± 1.52)%; 2,2-difenil-1-picrilhidrazil (DPPH^·): (78.43 ± 0.21)%; chelating ability: 92.32 ± 0.55; reducing power: 0.37 ± 0.00 at 740 nm), particularly in aqueous extracts. These effects were attributed to the combined contribution of phenolic compounds, soluble proteins, and reducing sugars. Overall, Amazonian P. ostreatus mycelial biomass emerges as a promising functional ingredient with potential application in dietary strategies aimed at managing metabolic disorders associated with type 2 diabetes mellitus and obesity.

L-isoleucine, an essential branched-chain amino acid, enjoys rising demand in the pharmaceutical, food, and feed industries. Corynebacterium glutamicum is a major production host for L-isoleucine production, but its native metabolic regulations limit industrial-scale synthesis. In this study, a L-isoleucine producing strain C. glutamicum YW-8 was engineered to efficiently produce L-isoleucine by finely regulating carbon flux toward its biosynthesis. To do this, promoter replacement was firstly performed to upregulate the expression level of the genes (e.g., ilvBN, ilvA) involved in the L-isoleucine biosynthetic pathway. Then, the genes (e.g., alaT, ldh) involved in the byproduct biosynthesise were knocked out to avoid the by-products accumulation, and the ppc gene was overexpressed to augment pyruvate supply. Subsequently, CRISPRi-mediated repression of the dapA gene was employed to dynamically reduce L-lysine diversion. Finally, the lrp-brnFE operon was overexpressed and the brnQ gene was knocked out to optimize L-isoleucine export. The resulted strain I16 produced (18.5 ± 0.9) g/L L-isoleucine in shake flasks, which was a 3.4-fold higher than that of strain YW-8 (i.e., 5.5 g/L). In addition, strain I-16 showed a significantly reduced L-lysine and L-alanine accumulation and the improved fermentation stability. This study provides feasible technical strategies for the systematic reconstruction and dynamic regulation of complex amino acid metabolic networks, and bears important theoretical and practical significance for promoting the rational design of microbial manufacturing processes.

Squalene, a high-value terpenoid, can be sustainably produced by Aurantiochytrium Ch25. This study integrated experimental optimization using Taguchi design with genome-scale metabolic modeling (GEM) to investigate its biosynthesis. The Taguchi L9 orthogonal array identified a promising culture condition (glucose 15 g/L, yeast extract 3 g/L, seawater 20% v/v, agitation 140 r/min), resulting in a squalene titer of 238 mg/L after 98 h. A validated GEM was used to simulate metabolic fluxes and explore potential engineering targets. Flux balance analysis (FBA) indicated that ammonia limitation favors biomass formation, while flux variability analysis (FVA) estimated squalene flux at 0.0014 mmol gDCW⁻¹ h⁻¹. Dynamic FBA simulations aligned well with experimental glucose uptake and biomass profiles. Single-reaction deletion analysis identified 13 essential and 35 semi-essential reactions, with inorganic diphosphatase (PPase) and F-type ATP synthase highlighted as critical for squalene biosynthesis. OptKnock predicted two mutant strains: Mutant A (deletion of 3-Oxoacyl-ACP reductase and Acyl-CoA oxidase) increased simulated squalene flux to 0.00994 mmol gDCW⁻¹ h⁻¹, and Mutant B (additional deletions) further raised it to 0.01274 mmol gDCW⁻¹ h⁻¹. OptGene suggested phosphoenolpyruvate synthase as a key deletion target to redirect flux toward the mevalonate pathway. This integrative approach paves the way for future metabolic engineering efforts and bioprocess optimization in this industrially relevant thraustochytrid.

The current study optimized simultaneous nanoparticle-based saccharification and citric acid (CA) production from pre-treated potato peel waste using Aspergillus brasiliensis. The model gave a high coefficient of determination (R²) (0.929) and predicted optimum pre-treatment conditions of 0.05 wt%, 19.85%, 32.5 °C and 2.03 for ZnO nanoparticle (NP) concentration, solid loading, temperature, and pH respectively. The validated process resulted in A. brasiliensis biomass and CA concentration of 1.54 g/L and 19.85 g/L, respectively. This was 1.19 and 1.38-fold higher compared to the control experiment, respectively. Interestingly, the kinetic assessment also revealed increase (2.67-fold) in maximum specific growth rate (µ_max) and maximum potential CA concentration (P_m) (1.07-fold) in the ZnO nanoparticle-based system. Potential of catalytic micro-environment and steady Zn²⁺ release in the growth medium is the most probable mechanism of ZnO NP triggering of A. brasiliensis for high specific growth rate and CA productivity. Findings from this study could facilitate the implementation of nanoparticle catalysed waste-based CA bioprocessing that might improve waste management and lower CA production cost, in keeping with the waste management, environmental sustainability and food nexus towards developing a circular bioeconomy.

Dynamic metabolic regulation is crucial for optimizing microbial cell factories. To address the limitations of chemical inducers, this study developed a temperature-responsive synthetic biology toolkit for Corynebacterium glutamicum. A high-performance, heat-inducible biosensor was engineered by optimizing the CI⁸⁵⁷ repressor and its cognate promoter, yielding a variant (CI⁸⁵⁷-M3/H1) with a 107-fold dynamic range and minimal background leakage. Additionally, a cold-inducible RNA thermometer was implemented using the Escherichia coli csapA 5’UTR. These components were integrated into a dual-functional genetic circuit enabling bidirectional metabolic control. Finally, the optimized heat-inducible sensor was applied to the production of three secretory proteins with distinct characteristics (AmyE, XylA, and VHH), and the scale-up cultivation of AmyE was successfully achieved in 1 L shake-flasks. This work provides an efficient, inducer-free strategy for precise metabolic regulation, offering a scalable and cost-effective tool for advanced biomanufacturing.

Aspergillus oryzae is an industrial filamentous fungus possessing high-efficiency protein expression and secretion capacity, widely used in enzyme preparation, food fermentation, and other fields. The industrial production strain A. oryzae F1 was selected for this study. Although already used for amylase production, its yield still fails to meet industrial demand. To improve the production of α-amylase, targeted engineering of the strain was performed using CRISPR/Cas9-mediated gene editing combined with high-throughput screening strategies. To enhance the site-specific integration efficiency of exogenous genes in A. oryzae F1, the gene lig4 encoding DNA ligase was knocked out, which increased the efficiency of homology-directed repair (HDR) from 13.4% to 34.7%. On this basis, α-amylase activity was improved by 15.2% through multi-copy gene integration. Furthermore, an excellent mutant strain was obtained by combining ARTP mutagenesis and droplet microfluidic high-throughput screening. In a 50 L fermentation system, the α-amylase activity of this mutant reached 4,052 U g⁻¹, representing a 37.4% increase compared with the parental strain F1. These results not only provide a feasible technical strategy for constructing high-efficiency amylase-producing strains, but also offer technical references for the engineering of filamentous fungal chassis cells.

(−)-α-bisabolol is a sesquiterpene compound found in various plants, with broad applications in pharmaceuticals, cosmetics, and personal care products due to its diverse pharmacological properties. Recombinant Escherichia coli offers an efficient and sustainable platform for its production. To further advance its industrial-scale synthesis, we enhanced the expression and catalytic activity of the key enzyme bisabolol synthase (CcBOS) and optimized the metabolic pathways of the host chassis. In this study, CcBOS from Cynara cardunculus was expressed in a previously engineered sesquiterpene-producing E. coli strain. By adjusting the fermentation temperature to 25 °C, the (−)-α-bisabolol titer reached 1.12 g/L in shake flask cultures. Protein engineering strategies—including optimization of the N-terminal sequence, increasing polar amino acid content, modifying non-conserved residues, and mutating histidine residues near the active site—significantly improved the solubility and catalytic efficiency of CcBOS. The triple mutant T258S/I364N/H479A increased (−)-α-bisabolol production by 154.5%. Metabolic engineering efforts, such as deleting atoDA to reduce fatty acid synthesis and attenuating gltA expression to limit tricarboxylic acid (TCA) cycle competition, further enhanced the titer by 63.1%. Integrating protein and metabolic engineering approaches resulted in a final titer of 3.08 g/L in shake flasks. When scaled up to a 5 L bioreactor, the titer reached 7.41 g/L, representing a high production of (−)-α-bisabolol using glucose as the sole carbon source. This study provides a novel integrated approach for the efficient microbial synthesis of (−)-α-bisabolol.

Rhamnolipids (RLs) are eco-friendly surfactants mainly produced by Pseudomonas aeruginosa. This study explores the sustainable production of RLs using waste cooking oil (WCO) as a carbon source, with NaNO₃ and yeast extract (YE) as nitrogen sources. Response surface methodology (RSM) based on central composite design (CCD) was applied to optimize the concentration of WCO (10–30 g/L), NaNO₃ (0–0.05 mol/L), and YE (0–2 g/L). The optimized conditions were 25.95 g/L WCO, 0.04 mol/L NaNO₃, and 0.41 g/L yeast extract, resulting in 7.93 g/L RLs concentration and 4.92 g/L biomass concentration, representing a 5.6-fold increase in RLs concentration using a similar carbon source and strain. Ten congeners of mono-RLs and di-RLs were detected by LC–MS/Q-TOF. The RLs stability was evaluated at different temperatures (4–121 °C), pH (4–12), and salinity (5%–25% w/v) by measuring the emulsification capacity, where 50% were maintained up to 100 °C, pH 4–8, and salinity up to 25% (w/v), indicating good physicochemical stability towards harsh conditions. Phytotoxicity tests on choy sum, cabbage, and mung bean seeds showed germination index (GI) values above 90% at 1 g/L RLs, indicating strong compatibility with crop growth. Meanwhile, aquatic toxicity test on zebrafish (Danio rerio) embryos showed an LC₅₀ of 67.42 µg/mL RLs, demonstrating lower toxicity compared to the chemical surfactant. These findings highlight the feasibility of high-yield RLs production from WCO through a predictive process modelling, with a stable, highly functional, and low-ecotoxicity profile. The study introduces a resource-efficient strategy to support RLs’ applications in environmental remediation, and green products development.

Traditional offline monitoring of Chinese hamster ovary cell fermentation processes suffers from severe systemic time delays. To achieve real-time monitoring of key physiological and biochemical parameters, this study proposes a non-invasive soft sensing framework based on cellular micro-morphology. Phase-contrast microscopic images were acquired throughout the fed-batch cultivation cycle, systematically extracting multidimensional features at the single-cell level, including geometric dimensions, shape, and internal optical texture. Given the highly non-linear mapping relationship between microscopic phenotypes and macroscopic metabolism revealed by univariate analysis $ \left( {\left| r \right| < 0.5} \right) $, a multivariate predictive model was constructed using the Random Forest (RF) algorithm, which was evaluated in parallel with a Partial Least Squares Regression model. Blind testing using an independent test batch subjected to an abnormal temperature disturbance demonstrated that the RF model effectively overcame the limitations of linear algorithms, achieving high-precision, cross-batch predictions for viable cell density (R² = 0.86), glucose (R² = 0.77), and lactate (R² = 0.90). Furthermore, from the perspective of model interpretability, feature analysis indicated that standard deviation features, representing the morphological heterogeneity of the cell population, showed a higher predictive contribution in characterizing the evolution of metabolic states than mean features. This study suggests the tremendous potential of microscopic population morphological heterogeneity as a digital biomarker for bioprocess monitoring, providing a reliable data-driven strategy for the at-line evaluation of industrial bioreactors.

Salidroside, the key active component of Rhodiola, possesses anti-hypoxia and anti-fatigue properties, making it valuable in medicine, health products, and cosmetics. This study focuses on efficiently synthesizing salidroside using a dual-enzyme cascade system with sucrose synthase AtSUS3 and glycosyltransferase AtUGT85A1 from Arabidopsis thaliana. This system facilitates the regeneration of UDPG and the conversion of tyrosol into salidroside. First, the two key enzymes AtSUS3 and AtUGT85A1 were expressed and purified with optimized His‑tag positions. Solubility enhancement via tag fusion and linker engineering yielded the fusion proteins TSu and ES85 with desirable activity. Subsequently, semi‑rational design on the poorly thermostable ES85 using the FireProt platform generated an S324R mutant with significantly improved thermostability. Ultimately, a recombinant strain TSR85 co‑expressing both enzymes was constructed. A one‑pot system employing this strain achieved 13.61 g/L salidroside with a conversion rate of 90% within 10 h (space‑time yield 1.36 g/L/h). This study thus integrates solubility enhancement, thermostability engineering, and one‑pot catalysis into a balanced, practical, and industrially relevant platform for salidroside biosynthesis.

Traditional Chinese medicines (TCM) represent a valuable resource for drug discovery, with numerous bioactive components identified with diverse pharmacological activities. However, the scarcity of these compounds, particularly those derived from plants, presents significant challenges for their applications. Here, we develop a modular and concise chemoenzymatic platform that features a streamlined enzymatic cascade coupled with a simple chemical module to facilitate the highly cost-effective and sustainable manufacture of bioactive components from Coptidis Rhizoma (Huanglian). After enzyme screening and engineering, as well as the implementation of a “plug-and-play” strategy, we efficiently produce various protoberberine alkaloids, including berberine (44% yield), demethyleneberberine (48% yield), jatrorrhizine (39% yield), berberrubine (31% yield), columbamine (42% yield), and palmatine (44% yield), exhibiting high robustness and industrial potential of this designed chemoenzymatic platform. This approach not only addresses challenges associated with heterologous production in engineered cells and low regioselectivity in chemical synthesis, but also establishes a paradigm for manufacturing other bioactive components derived from TCM, further facilitating the modernization of TCM.

Nitro compounds are indispensable in pharmaceuticals, agrochemicals, and energetic materials; however, their traditional synthesis is often constrained by environmental concerns and high energy demands. Biocatalytic nitration offers a more sustainable alternative, with horseradish peroxidase (HRP) and engineered P450BM3 emerging as two representative and extensively studied catalytic systems. HRP, a classic natural biocatalyst, inherently catalyzes the radical-mediated nitration of aromatic substrates. In contrast, P450BM3 exemplifies the power of enzyme engineering: its scaffold can be repurposed through rational design and the use of double-function small molecules (DFSMs) to enable non-natural transformations, such as alkene nitration. This review provides a comparative analysis of these two systems, examining their catalytic mechanisms, structural features, and performance metrics. By comparing HRP with P450BM3, this work aims to delineate their respective advantages and provide insights to guide the development of more efficient enzymatic nitration strategies.