The industrial production of nicotinic acid (vitamin B3) primarily relies on chemical synthesis, which suffers from harsh reaction conditions and severe environmental pollution. Although biocatalysis using nitrilase offers a greener alternative, the practical application of free enzymes is constrained by poor operational stability and difficulties in recovery. We developed a biomimetic hybrid immobilization platform that combines a zeolite core with poly(ethylene glycol) (PEG200) crosslinking, and a TEOS-derived silica shell to encapsulate whole cells of Corynebacterium glutamicum engineered to express a mutant nitrilase from Pseudomonas putida. This core–shell architecture is designed to balance mechanical robustness (from the silica shell) with biocompatibility and mass transfer (from PEG200 crosslinking). Systematic optimization of the immobilization conditions (6 g/L zeolite, 50 g/L cell loading, 3 g/L PEG200, 7 g/L TEOS) yielded a molar conversion rate of 88.78
Asparaginase (ASNase), an enzyme that hydrolyzes l-asparagine into l-aspartic acid and ammonia, represents an essential biocatalyst at the interface of biomedicine and food safety. Clinically, ASNase is a cornerstone therapy for acute lymphoblastic leukemia (ALL), while in the food industry it is widely applied to reduce acrylamide formation, thereby lowering neurotoxic and carcinogenic risks without compromising product quality. However, despite separate advancements in using ASNase for cancer therapy and food safety, an integrated optimization strategy is still lacking. Since both applications rely on the same catalytic mechanism, require good biochemical properties (e.g., Km, Kcat, Vmax, and half-life), and share critical production safety concerns, a dual-purpose enzyme could be engineered for both applications. This review bridges this development gap by proposing an integrated framework that unifies structure–function analysis and protein engineering strategies for both applications, enabling cost- and time-efficient workflows starting with bioinformatic evaluation. Ultimately, this review outlines strategies to engineer ASNase variants with enhanced catalytic efficiency, stability, and reusability, alongside reduced glutaminase co-activity and immunogenicity. This review evaluates emerging approaches, including humanized design, rational engineering, AlphaFold 3-based structural and dimerization predictions, and encapsulation, alongside advanced fermentation platforms for cost-effective and large-scale production. By assessing the correlations between enzymatic potency, structure–function relationships, immunogenicity, and industrial viability, this multidisciplinary work integrates bioengineering, computational modeling, and fermentation development to guide future ASNase innovations in healthcare and the food industry. Integrates medical and food ASNase frameworks for cost- and time-effective development. Compiles recent mutational data guiding the rational design of optimized, multi-purpose ASNase variants. Demonstrates the ability of AlphaFold 3 to predict ASNase structural heterogeneity and oligomerization states. Assesses innovative fermentation, humanization, immobilization, and encapsulation of ASNase.
The integrated cyanobacterial biorefineries are emerging as versatile microbial cell factories, where waste management can be coupled with simultaneous resource recovery to align with the circular economy approach. This review emphasizes the use of domestic or industrial wastewaters for cultivation of these photosynthetic prokaryotes due to their ability to simultaneously recover nutrients, capture CO2 and produce multiple high-value bioproducts, including pigments, exopolysaccharides, bioplastics, nanomaterials and/ or biofuels. However, the major technical bottlenecks, such as low biomass productivity, wastewater variability, instability of engineered strains, cost-effective harvesting and downstream processing and insufficient pilot-scale validation remain the critical constraints for commercial success. Therefore, this review emphasizes the use of recent advancements, such as metabolic engineering, CRISPR-based genome editing, metabolic flux analysis, multi-omics strategies to improve productivity. Additionally, the techno-economic and life cycle assessment approaches are also discussed, which may help to evaluate the possible environmental impacts and industrial feasibility of the cyanobacterial biorefineries. Thus, this review proposes transformation of wastewater treatment systems into near zero-waste, scalable and low-cost multiproduct biorefineries aimed at achieving both economic viability and environmental sustainability.
The untapped microbial diversity of the Amazon region represents a strategic frontier for biotechnological applications. This study screened, identified, and evaluated the potential of yeasts isolated from Amazonian environments to bioconvert sugarcane bagasse hydrolysate into ethanol and xylitol. The lignocellulosic hydrolysate was obtained by thermochemical treatment using H2SO4 (0.78
Paddy straw is one of the most abundant lignocellulosic agricultural residues generated worldwide, and its improper disposal through open-field burning contributes significantly to air pollution, greenhouse gas emissions, nutrient loss, and soil degradation. Sustainable management of paddy straw therefore requires efficient and eco-friendly biodegradation approaches. This review highlights the role of lignocellulolytic microorganisms and their enzyme systems in the biodegradation and valorization of paddy straw. The structural complexity of paddy straw, mainly due to cellulose, hemicellulose, and lignin, limits natural degradation and necessitates the synergistic action of specialized microbial communities and extracellular enzymes. The review discusses the contributions of bacteria, fungi, and actinomycetes in producing cellulases, xylanases, pectinases, laccases, manganese peroxidase, and lignin peroxidase involved in lignocellulose breakdown. It also summarizes microbial mechanisms of degradation and the influence of substrate characteristics, environmental conditions, and microbial interactions on enzymatic efficiency. Special emphasis is placed on microbial consortia, pretreatment strategies, and recent advances in consortium engineering for accelerated decomposition and biomass valorization. Overall, the review provides a comprehensive understanding of sustainable paddy straw management through integrated microbial and enzymatic biotechnological approaches.
Glutamate decarboxylase (GAD, EC 4.1.1.15) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the irreversible α-decarboxylation of L-glutamate to produce γ-Aminobutyric acid (GABA). As the rate-limiting enzyme in GABA biosynthesis, GAD is central to research in neuroscience, plant stress physiology, and industrial biotechnology. This review systematically summarizes the structural characteristics, catalytic mechanisms, enzymatic properties, factors affecting the activity, and activity determination methods of GAD in microorganisms, plants, and mammals. Furthermore, from the perspectives of enzyme resource mining and protein engineering, this review presents an evaluation of strategies for enhancing GAD catalytic performance and stability, including site-directed mutagenesis, directed evolution, and semi-rational design. Finally, the applications of engineered GAD in GABA biosynthesis are comprehensively analyzed.
Decomposed wheat straw is a promising seedling substrate but is vulnerable to contamination by filamentous fungi, notably Aspergillus flavus. Multifunctional microbes that combine strong cellulolytic capacity with antifungal activity are needed to enable cleaner bioprocessing and reduce mycotoxin risk. This study sought to identify such a strain and verify its performance from screening to crop-relevant application. From 2,102 Bacillus isolates collected between 2018 and 2023, we selected Bacillus amyloliquefaciens SQ1 exhibiting both cellulolytic activity and antifungal efficacy against A. flavus. During straw decomposition, SQ1 accelerated cellulose and hemicellulose loss, decreased C/N and lignin/N ratios, and rapidly shifted pH toward neutrality. SQ1 suppressed fungal proliferation; Cryo-SEM revealed fractured and collapsed A. flavus hyphae with markedly reduced spore adhesion. Transcriptomic profiling of A. flavus under SQ1 exposure revealed coordinated repression, spanning cell-envelope biogenesis and sterol synthesis (down-regulation of fks1, aflY, erg2, erg11), diminished developmental competence (ligA), and attenuated toxin regulation (aflR). SQ1-amended decomposed straw reduced the relative abundance of Aspergillus and lowered aflatoxin B1 (AFB1, C17H12O6) content. Using the SQ1-amended decomposed straw as a substrate improved peanut seedling survival, plant height, and leaf chlorophyll. SQ1 functions as a dual-purpose biocatalyst, breaking down cellulose while inhibiting fungal growth to enhance straw biotransformation, mitigate mycotoxin risk, and support early crop growth. These findings highlight a sustainable and value-added approach to wheat straw utilization, with practical implications for improved substrate management and cleaner production in crop systems.
Clostridium acetobutylicum ATCC 824 (pCD07239), designated CD07239 in this study, was previously constructed by introducing the Clostridioides difficile CD630_0723–CD630_0729 gene cluster into the parental ATCC 824 strain. The CD07239 strain initiates butanol production during the early growth phase. To investigate the transcriptional basis of this phenotype, we performed RNA-seq-based transcriptome analysis using phenotype-defined RNA samples collected during batch fermentation. At 3 h, CD07239 had already produced approximately 0.1 g/L butanol, whereas butanol was not detected in ATCC 824. At 9 h, both strains produced butanol, but CD07239 accumulated a substantially higher level than ATCC 824. Principal component analysis revealed that the transcriptomes were separated according to fermentation stage and strain background. In ATCC 824, major solventogenic genes, including adhE1, ctfA, ctfB, and adc, were strongly induced at 9 h compared with 3 h. In contrast, early butanol production in CD07239 at 3 h was not accompanied by premature induction of these genes. Instead, adhE2 showed markedly high expression in CD07239, with log2 fold changes of 6.4 at 3 h and 6.6 at 9 h compared with ATCC 824. Additional adhE2 expression in ATCC 824 did not reproduce early butanol production, suggesting that adhE2 alone is insufficient to induce this phenotype. These results indicate that early butanol production in CD07239 is associated with an adhE2-dominant transcriptional state distinct from the conventional solventogenic transition of ATCC 824.
Thermotolerant microbial hosts offer advantages for industrial bioprocesses, yet direct oil-based bioconversion requires efficient coupling of extracellular lipid hydrolysis with intracellular carbon assimilation. In the thermotolerant bacterium Cupriavidus cauae PHS1, direct utilization of triacylglycerol-rich substrates is constrained by the lack of extracellular lipolytic activity and limited glycerol metabolism. Here, the underexplored non-model bacterium C. cauae PHS1 was engineered as a thermotolerant chassis for direct conversion of palm oil (PO) and waste frying oil (WFO) into polyhydroxybutyrate (PHB) at 42 °C. Heterologous expression of a thermostable secretory lipase from C. necator H16 conferred extracellular oil-hydrolysis capability, and adaptive laboratory evolution was applied to improve glycerol metabolism. This combined strategy enabled direct utilization of triacylglycerol-derived carbon and enhanced PHB accumulation from both a model oil feedstock and a complex waste oil feedstock. Under non-optimized culture conditions, the engineered strain produced PHB from 10 g/L PO, reaching 5.11 g/L cell dry weight (CDW) with 64.18
Catalase plays a significant role in improving animal intestinal health and growth performance; however, its industrial application in feed is largely limited by poor thermostability and low acid tolerance. We identified an acid-resistant catalase from Talaromyces pinophilus and improved its thermal stability. We integrated multiple thermal stability design strategies, constructed a mutation library using algorithms from different design platforms, and further refined the library with computational tools. This approach successfully generated the combinatorial mutant M3 (E38Q/S69P/S187A). Its half-life at 80 °C increased by 2.64 times, and two fold increase in half life at 90 ℃, while the specific activity remained unchanged. Molecular dynamics simulations show that this mutational strategy can significantly enhance the thermal stability of catalase through a cascading effect of ‘local rigidity enhancement—global conformational compaction—hydrophobic core stabilization’. Additionally, under simulated high-temperature and gastric acid conditions, the mutant strain demonstrated a high capacity for decomposing H2O2, significantly enhancing its practical value for use in animal feed.
Grapevine trunk diseases threaten sustainable viticulture, while wine lees remain an underexploited biocontrol-relevant by-product within the circular economy framework. An integrated bioprocess was developed to upgrade lees into high-molecular-weight biomolecule mixtures and low-molecular-weight peptide-enriched fractions through a defined sequence of unit operations: fermentation design by using Saccharomyces monocultures and sequential inoculations with non-Saccharomyces strains, ultrasound-assisted autolysis, enzymatic hydrolysis, and 3 kDa ultrafiltration. The antifungal activity of the products was tested in vitro and in vivo against Phaeomoniella chlamydospora and Phaeoacremonium minimum, the two main pathogens that cause Petri disease in grapevine. The results demonstrated that high-molecular-weight autolysates showed no inhibition. The post-hydrolysis fractions reduced mycelial growth of Phaeoacremonium minimum by 45
Stabilizing enzyme functionality during downstream processing remains a central challenge in scalable biomanufacturing. This study presents a process-integrated evaluation of microencapsulation strategies to preserve the activity of a fibrinolytic protease derived from Bacillus tequilensis HSFI-5, with an emphasis on the interplay among the encapsulant matrix, drying method, and enzymatic accessibility. Encapsulation systems based on maltodextrin, Arabic gum, chitosan, carrageenan, and alginate (5
Dilong (earthworm) has been widely used in traditional Chinese medicine (TCM) for centuries to treat conditions such as thrombosis, inflammation, and hypertension. Increasing biochemical and pharmacological evidence indicates that these therapeutic effects are largely attributed to a diverse repertoire of bioactive proteins and peptides. These molecules exhibit a broad spectrum of biological activities, including fibrinolytic, anti-inflammatory, antimicrobial, antioxidant, and neuroprotective functions. With advances in omics technologies and protein engineering, growing attention has been directed toward the discovery, functional characterization, and biotechnological production of these macromolecules. This review summarizes recent progress in earthworm-derived proteins and peptides, with an emphasis on their structural diversity, pharmacological properties, and biomedical applications. We further discuss emerging recombinant production strategies and key challenges related to protein folding, secretion, and post-translational modifications. In addition, the integration of artificial intelligence (AI) with synthetic biology for protein design, structure prediction, and pathway optimization is highlighted. These advances collectively provide a foundation for accelerating the translation of earthworm-derived biomolecules into pharmaceutical, cosmetic, and industrial applications.
β-Xylosidase plays a crucial role in the degradation of xylan and hemicellulose, as well as the hydrolysis of various glycosides. This substrate diversity stems from family-specific structural adaptations: GH3 employs a conserved double-displacement mechanism with a unique pocket for efficient 7-xylosyl-10-deacetyltaxol (XDT) conversion; GH39 uses non-conserved hydrophobic residues to recognize saponin main chains; GH43 exhibits high variability in the β‑hairpin structures of its family members. Beyond hydrolysis, β-xylosidases achieve transglycosylation via a retention mechanism, forming covalent enzyme-xylose intermediates where receptor steric hindrance, polarity, and nucleophilicity determine reaction outcomes. Different families recognize carbohydrate, phenolic, and alcohol receptors through complementary active site topology, enabling green synthesis of alkyl xylosides and bioactive substances. Molecular engineering modifies β-xylosidases by introducing rigid elements, optimizing binding interfaces, and broadening substrate channels. This article reviews recent advances in hydrolysis and transglycosylation activities, explores catalytic mechanisms, and highlights breakthroughs in molecular modification strategies. It is intended to serve as a reference for future research and application of this enzyme family.
The biological sciences, especially biotechnology-based value-added products and services, are key players in sustainable revenue generation and influence the global bioeconomy. The study aims to understand the frameworks for historical advancements, the scope, and the emerging challenges faced by biotechnology-driven industries. Biotechnology-driven therapeutics are gaining attention due to the roles of primary and secondary metabolites, antibiotics, and antiviral vaccines in supporting human health and societal advancement. Drawing on a conceptual outline linking raw biological materials to value-added bioproducts, this study highlights bio-based resources as a cornerstone of contemporary industry and underscores their growing relevance. The review emphasizes the Indian bioeconomy, highlighting the growing significance of biomass, biotechnological innovations, and bio-derived products across multiple sectors, including biofuel production (alcohol, gasohol), enzyme-based cleaning agents, genetically modified (GM) crops, pulp and paper processing, and pharmaceutical manufacturing. This comprehensive study also delineates the economic differentiation of bioresources from conventional resources. It illustrates this with historical examples, such as the important role of India’s jute industry in shaping its national economy during the 1920s and 1930s. Biomanufacturing in India has grown steadily, with strong capabilities in vaccines, biosimilars, and large-scale fermentation-based production. However, while progress is notable, gaps remain in advanced infrastructure, scale-up capacity, and high-end innovation. In conclusion, the emerging biotechnology sectors involved in vaccine, nutraceutical, pharmaceutical, biomolecule, and personalized therapeutic production were examined, offering insights into their roles in value addition and national income generation.
Microbial pigments have been gaining prominence as sustainable alternatives to synthetic dyes, which are often associated with environmental impacts and potential health risks. A wide range of microorganisms, including bacteria, fungi and yeasts, are capable of producing pigments of industrial interest. Despite their potential, large-scale production still faces challenges related to cultivation costs and, in particular, extraction steps, which traditionally rely on the use of organic solvents and may present low yields and limited sustainability. In this context, the use of agro-industrial residues as alternative nutrient sources for microbial growth and pigment biosynthesis emerges as a promising strategy for sustainable valorisation, contributing to reduced production costs, mitigation of environmental impacts and strengthening of the circular bioeconomy. However, the recovery of microbial pigments remains a technological obstacle, as many of these compounds are intracellular or associated with cellular structures, making extraction and purification challenging and affecting industrial feasibility. To overcome these limitations, recent advances have focused on improving extraction techniques in order to increase yield, purity and sustainability. This review addresses the main microbial pigments and their producers, highlighting the potential of agro-industrial residues for sustainable production. In addition, it synthesises recent advances in extraction strategies, emphasising approaches that combine efficiency, reduced environmental impact and economic viability. In an integrated perspective, the discussion demonstrates that the combination of low-cost substrates and more sustainable extraction technologies represents a promising pathway to expand production and meet the growing demand for natural colourants in the food, cosmetic and pharmaceutical industries.
Escherichia coli Nissle 1917 (EcN), owing to its proven safety and robust intestinal colonization capacity, has emerged as a highly promising microbial chassis for metabolic engineering and live biotherapeutic applications. However, its genetic manipulation has long been constrained by low editing efficiency, instability of a foreign plasmid, and the lack of robust and tightly controllable expression systems. In this study, we establish a rapid and scalable genome engineering platform for EcN based on an enhanced and fast iterative Ampicillin-Chloramphenicol-Spectinomycin-CRISPR–Cas9 system (ACS-CRISPR-Cas9). By integrating a streamlined dual-sgRNA design with an antibiotic-cycling-driven ACS-CRISPR-Cas9 iterative editing workflow, together with a chromosomally integrated T7 expression system, efficient and inducible gene expression was achieved. Using homologous recombination, dual-sgRNA plasmids were rapidly constructed and enabled precise deletion of 11 gene loci as well as large genomic fragments ranging from 17 to 45 kb, with a maximal editing efficiency of 97.9
Lactoferrin exerts a variety of physiological functions, including iron metabolism regulation, antibacterial activity and antiviral activity, indicating its considerable application potential in the feeding of weaned young animals. As porcine lactoferrin (PLF) is scarce in source, bovine lactoferrin and other substitutes are generally utilized in related studies on piglet feeding. Trichoderma reesei possesses efficient protein secretion ability and eukaryotic post-translational modification capacity, and has been successfully used for the efficient expression of various food and feed enzyme preparations. This study aimed to construct an engineered T. reesei strain capable of efficiently expressing porcine lactoferrin (PLF), and to conduct a preliminary analysis of the recombinant protein. Using T. reesei RUT-C30Δpyr4Δtku70 as the parental strain, the protease activation factor gene (pea1) and cellobiohydrolase II gene (cbh2) were sequentially knocked out via CRISPR/Cas9 technology, resulting in the construction of recombinant expression host strains C30Δpyr4Δtku70Δpea1 and C30Δpyr4Δtku70Δpea1Δcbh2, respectively. In these constructed host strains, the expression of the porcine lactoferrin gene (plf) was driven by the strong inducible promoter Pcbh1. Shake-flask fermentation assays demonstrated that knockout of the pea1 gene significantly enhanced the yield and stability of PLF in the fermentation broth. In a 30-L fermenter, the recombinant strain C30Δpyr4Δtku70Δpea1Δcbh2Δcbh1::plf achieved a maximum PLF yield of 2.37 g/L. The purified PLF protein was also subjected to iron-removal and iron-binding assays, with evident color changes observed. Iron saturation analysis confirmed that PLF could bind iron ions in iron-rich solutions, thereby increasing its iron saturation. In contrast, in iron-removal solutions, iron-saturated PLF released iron ions, leading to a reduction in the protein’s iron saturation level. This study is the first to achieve the heterologous expression of PLF in T. reesei. The recombinant PLF exhibited intact iron-binding and iron-release functions, laying a key foundation for further investigations into its application in piglet nutrition.
L-2-aminobutyric acid (L-ABA) is a non-proteinogenic chiral α-amino acid and, as an important chemical feedstock and pharmaceutical intermediate, has broad application potential in the pharmaceutical and fine chemical industries. In this study, Escherichia coli TWF106 was used as the chassis strain for L-ABA production. In strain TWF106, we individually deleted rhtA, rhtC, ptsG and gabP to optimize the metabolic flux toward L-ABA production, and constructed multiple single-deletion and double-deletion strains. The reductive amination of 2-ketobutyric acid (2-KB) to L-ABA catalyzed by leucine dehydrogenase (LeuDH) is considered a potential bottleneck in this pathway; therefore, we engineered LeuDH to improve the L-ABA titer. By substituting the key residue Ser331 in LeuDH with different amino acids, 19 mutants were constructed and tested for L-ABA production. Two mutants, LeuDHS331R and LeuDHS331C, showed the greatest improvement in L-ABA production, the L-ABA titers of TWF106-331R and TWF106-331 C reached 5.46 g/L and 5.45 g/L respectively. Molecular docking and molecular dynamics simulations revealed that mutations at Ser331 altered the static binding mode and modulated the dynamic binding mechanism of the protein–ligand complex. Subsequently, by combining metabolic flux optimization with site-directed mutagenesis, TWF1604-331 C was constructed through deletion of gabP in TWF106 coupled with introduction of plasmid pB-AmDHS331C. TWF1604-331 C could produce 6.47 g/L L-ABA. Finally, fed-batch fermentation of TWF1604-331 C was conducted in a 2-L bioreactor, achieving an L-ABA titer of 16.58 g/L.
Fusarium venenatum is an important mycoprotein-producing fungus, but the contribution of mannitol metabolism to carbon allocation and fermentation performance remains insufficiently characterized. In this study, single-, double-, and triple-deletion mutants targeting three mannitol metabolism-related genes, the mannitol dehydrogenase gene (FvMDH), the mannitol-1-phosphate dehydrogenase gene (FvM1PDH), and the mannitol metabolism-associated dehydrogenase gene (FvMTDH), were constructed and compared in terms of phenotypes, mannitol accumulation, fermentation performance, and transcriptional responses. FvM1PDH deletion was the main genetic factor associated with reduced mannitol accumulation. The TB6050ΔFvM1PDH strain, hereafter referred to as ΔP, showed 97.60
