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.

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.

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.

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.

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.

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.

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.