Monascus red pigment is a natural pigment produced by Monascus purpureus, known for its high medicinal and nutritional value. To enhance the yield of Monascus red pigment in liquid fermentation, ARTP mutagenesis was applied iteratively to M. purpureus LBBE. A high-throughput screening method was developed based on double-dye fluorescent labelling, flow cytometry sorting, microplate solid-state culture, and other advanced techniques to improve the screening efficiency. Through ten rounds of iterative mutagenesis and high-throughput screening, a total of 24,000 mutants were analyzed, ultimately leading to the identification of the high-yield strain LBBE-29. Shake flask fermentation demonstrated that the Monascus red pigment color value of LBBE-29 reached 1,117.6 U·mL^− 1, which was 2.03 times higher than that of the starting strain while maintaining strong genetic stability. This high-throughput screening method not only provided an effective approach for the mutagenesis breeding of M. purpureus strains with enhanced pigment production but also served as a theoretical reference for high-throughput mutagenesis breeding of other filamentous fungi.

Komagataella phaffii (formerly Pichia pastoris) is a widely used host for heterologous protein expression and biotransformation, and simplifying its cell wall polysaccharides is a promising strategy for designing advanced chassis strains. Previously, we constructed two superior chassis hosts by inactivating the β-glucan biosynthesis genes PAS_chr1-3_0225 and PAS_chr1-3_0661. In this study, PAS_chr2-1_0263 gene responsible for β-glucan synthase was inactivated to investigate the impact of β-glucan deficiency on sophorolipid (SL) biosynthesis. Furthermore, we systematically evaluated the combined effects of this mutation and faa1 inactivation on SL production and host performance. Firstly, the SL biosynthesis-related genes (comprising cyp52M1, ugtA1, ugtB1, sble, at, and mdr) were overexpressed for the first time, demonstrating that P_GAP-driven K. phaffii GS115 can synthesize seven structural types of SLs with a total titer of 4.09 g/L. Subsequently, to facilitate subsequent gene editing, the DNA repair-related genes ku70 and mph1 were deleted; this deletion did not depress SL production. PAS_chr2-1_0263 was then knocked out to obtain a novel chassis host, and the SL productivity, oil-to-SLs ratio, and precursor UDP-glucose level were systematically investigated. The results showed that knocking out PAS_chr2-1_0263 reduced glucan content by 24.21% while increasing total SLs to 7.69 g/L, a 43.8% increase, and improving the conversion ratios of both oil and glucose to SLs. Moreover, the combined deletion of PAS_chr2-1_0263 and faa1, which encodes fatty acid acyl-CoA synthase, further elevated the SL titer to 11.53 g/L and achieved even higher glucose-to-SLs and oil-to-SLs conversion rates. These findings indicate that weakening 1,3-β-glucan synthesis not only improves the utilization ratio of glucose but also increases intracellular UDP-glucose levels, both of which contribute to enhanced SL biosynthesis. This study demonstrates for the first time that attenuating 1,3-β-glucan synthesis in K. phaffii is an effective strategy to boost SL biosynthesis and improve host performance, and this novel chassis possesses excellent potential for the biotransformation of other glycolipids.

p-Coumaric acid (p-CA) is a valuable phenolic compound widely applied in food, pharmaceutical, and cosmetic industries. While the chemolithoautotrophic Cupriavidus necator H16 is a potent host for converting CO₂ into biochemicals, its potential for synthesizing aromatic-derived compounds remains to be fully explored. In this study, p-CA was selected as a model compound to systematically engineer the metabolic network of C. necator H16 for aromatic biosynthesis from fructose and CO₂. We first established a tyrosine-derived pathway and subsequently enhanced the metabolic flux by identifying and overexpressing key pathway genes—aroG1, aroQ1, and aroC. Then, the carbon flux was redirected towards tyrosine by replacing the native prephenate dehydratase (PheA) with Escherichia coli prephenate dehydrogenase (TyrA). Furthermore, we introduced the E. coli nicotinamide nucleotide transhydrogenase to increase cofactor availability and optimized the process by substituting ammonium chloride with urea. These systematic modifications resulted in an engineered strain producing 25.4 mg/L of p-CA from fructose, a 1,593.3% increase compared to the initial strain. Significantly, under autotrophic conditions, the strain enabled de novo synthesis of p-CA from CO₂, reaching 3.1 mg/L. This work not only demonstrates the first light-independent p-CA biosynthesis from CO₂ but also validates the feasibility of using C. necator H16 as a sustainable platform for the production of aromatic chemicals.

Spray drying is a scalable method for producing dry microbial inoculants, but Gram-negative bacteria often exhibit poor survival under desiccation stress. We employed a directed-evolution approach to improve the survivability of diazotroph Klebsiella michiganensis M5al during spray-drying encapsulation in cross-linked alginate microcapsules (CLAMs). Wildtype K. michiganensis M5al was serially passaged through the CLAMs spray-drying process under incrementally increasing selection pressure. Viable cell density in powders improved over 1000-fold compared to baseline, reaching 3.07 × 10¹⁰ CFU/g over the course of the directed evolution experiment. Paired trials confirmed a statistically significant increase in survival of evolved isolates relative to the parental strain (mean difference of 1.39 log₁₀, P < 0.05). Whole-genome sequencing and analysis using breseq identified candidate mutations associated with desiccation tolerance, including a single point mutation in the phosphoenolpyruvate carboxylase gene (pepC), suggesting a potential link between central carbon metabolism and stress adaptation. These results demonstrate that genetic adaptation can rapidly enhance microbial fitness under industrial drying conditions without modifying formulation, providing a practical strategy for improving the manufacturability and deployment of Gram-negative microbial biostimulants and related products.

2′-Fucosyllactose (2′-FL) is the most abundant human milk oligosaccharide (HMO), playing vital roles in promoting infant gut health, enhancing immunity, and defending against pathogens. However, conventional 2′-FL biosynthesis typically relies on exogenous supplementation of lactose or fucose precursors, leading to high costs and process complexity. In this study, we report a streamlined de novo biosynthesis process for 2′-FL using glucose as the sole carbon source, achieved through systematic engineering of endogenous precursor pathways in E. coli. First, the complete biosynthetic route from glucose to lactose and then to 2′-FL was reconstructed in a chassis strain capable of producing fucose. Unlike previous studies that primarily focused on the GDP-L-fucose supply module, this work systematically balanced the metabolic flux between phosphorylated and non-phosphorylated glucose pools to coordinate the supply of precursors for both lactose and GDP-L-fucose synthesis. This balance was achieved by reconstructing the glucose uptake system, modulating the expression of key enzymes at critical metabolic nodes, and eliminating competing pathways. Subsequently, the endogenous lactose synthesis pathway was further enhanced through coordinated overexpression of pgm, galU, and galE, and the pentose phosphate pathway and purine salvage pathway were optimized to enhance the supply of NADPH and GTP. Using this strategy, an engineered E. coli strain for efficient 2′-FL production was successfully constructed. The engineered strain produced 7.11 g/L 2′-FL in shake-flask fermentation and achieved a titer of 43.2 g/L in a 3-L bioreactor after 58 h, representing the highest reported titer for de novo 2′-FL biosynthesis in E. coli using glucose as the exclusive carbon source without exogenous lactose supplementation.

The aqueous esterification of isoamyl alcohol represents an environmentally friendly synthetic route. However, its industrial implementation is hindered by the scarcity of efficient catalysts, and conventional enzyme-directed evolution methods are often cumbersome and costly. To circumvent these limitations, this study employs the advanced protein design algorithm LigandMPNN for the rational design of lipase mutants. Using the aqueous esterification of isoamyl alcohol as a model reaction, we employed LigandMPNN to design mutations targeting the substrate-binding region. A key variant, Y181V, was created. Under optimized conditions, the mutant achieved a high yield of 217 mg/L isoamyl caproate. The Y181V variant exhibited 37% higher catalytic activity in aqueous phase than the wild-type. This mutation optimized the hydrophobic microenvironment of the active center, favoring the accommodation and transformation of hydrophobic substrates. This study successfully developed an improved lipase mutant for esterification, thereby validating the efficacy of LigandMPNN in protein rational design. Our work provides novel insights and a promising candidate for the creation of industrial-grade biocatalysts.

Nitrile hydratase (NHase) is a promising biocatalyst for the industrial synthesis of high value amides, yet its practical application is often limited by conformational instability and activity loss under high product concentrations. In this study, we employed a rational engineering strategy guided by molecular dynamics simulations in high amide concentrations to elucidate the structural dynamics of NHase acrylamide/water mixed solvents and identify key residues governing its stability. Through systematic mutagenesis, we developed a double mutant, βV4I/βI177K (designated M1), which exhibited a 2.2-fold increase in specific activity toward acrylonitrile, a 1.7-fold improvement in catalytic efficiency, and a 9.6-minute extension (corresponding to a 37% increase in enzyme durability) in thermal inactivation half-life compared to the wild-type enzyme. Under simulated industrial conditions, the superior catalytic stability and conversion efficiency of M1 resulted in a 1.3-fold increase in acrylamide yield. Furthermore, the mutant displayed broadened substrate adaptability, with activities toward 1-naphthonitrile and cinnamonitrile enhanced by 2.98- and 3.34-fold, respectively. These findings not only reveal the molecular mechanism underlying NHase inactivation acrylamide/water mixed solvents but also establish a robust engineering framework for developing highly efficient and stable biocatalysts for industrial amide synthesis.

25-Hydroxyvitamin D₃ (25-OH-VD₃) is the main active form of Vitamin D₃ and has broad applications in clinical treatment and agriculture. Although current industrial production predominantly relies on chemical synthesis, the growing demand for green and efficient manufacturing has accelerated the development of microbial enzymatic conversion methods. Due to its high reaction specificity, the unspecific peroxygenase from Coprinopsis cinerea (CciUPO) represents a promising biocatalyst for the synthesis of 25-OH-VD₃. However, its low catalytic efficiency limits further application in the biosynthesis of 25-OH-VD₃. To address this, semi-rational design was employed to modify the substrate-binding pocket and non-conserved residues of CciUPO. The resulting triple mutant I73M/P108K/G245A increased the 25-OH-VD₃ concentration to (87.83 ± 3.47) mg/L, representing a 41.18% increase over the wild type [(62.21 ± 3.02) mg/L]. The mechanism for enhanced catalytic efficiency was elucidated through analysis of the substrate-binding pocket, enzyme-substrate interactions, and molecular dynamics simulations. Subsequently, the fermentation conditions and multi-enzyme cascade reaction were optimized. Under optimized conditions with a substrate VD₃ concentration of 0.5 g/L, the 25-OH-VD₃ concentration further increased to (152.50 ± 1.95) mg/L. The combination of semi-rational engineering and process optimization of CciUPO offers a feasible, green and efficient strategy for the biosynthesis of 25-OH-VD₃.

High-temperature fermentation (HTF) can reduce cooling requirements and contamination risks in industrial production, especially for producing bioethanol from lignocellulosic biomass. DAP1 regulates ergosterol biosynthesis and determines thermotolerance, and amino acid position 39 is the key site for its thermostability. Here, we explored and modified the 39th amino acid of Dap1 (valine) via CRISPR/Cas9-assisted precise genome editing technology to enhance the HTF performance of industrial Saccharomyces cerevisiae CEN.PK2-1C. The results showed that converting valine to aspartic acid (V39D) or glutamine (V39Q) increased the growth of the mutants by 12.33% and 12.82%, respectively, at 42 °C. Correspondingly, ethanol yields of the DAP1-V39D and DAP1-V39Q mutants increased by 10.98% and 10.82%, respectively, compared with the wild-type. In addition, overexpressing DAP1-V39Q with the strong promoter pTDH3 in the DAP1-V39Q mutant (DAP1-V39Q-OE) further increased high-temperature growth ability and ethanol production by 3.10% and 0.91%, respectively, compared with the DAP1-V39Q mutant. Finally, from the predicted protein model, we found significant changes in the protein structures of the DAP1-V39D and DAP1-V39Q mutants. Our findings would provide guidance for developing more robust yeast for the industrial production of ethanol at high temperature.

The current search for green alternatives to substitute fossil-based compounds has increased interest in renewable biomass sources. In this context, corn (Zea mays) has been extensively explored for first-generation (1G) bioethanol production. Over the past 20 years, the production of this biofuel has increased from 4.5 to 28.5 billion gallons, led mainly by the US and Brazil. The synthesis of corn-bioethanol in biorefineries is a prime example of the circular economy principles, as there is co-generation of different bioproducts: corn oil, animal feed, and CO₂. Although 1G bioethanol production from corn is well-known and has been extensively studied, its processing chain can be further explored, promoting advances in already existing biorefinery systems. Beyond animal feed and corn oil, novel biomolecules can be produced in such industrial platforms—advanced fuels, biopolymers, and enzymes, for example. Therefore, this review article aims to present and discuss up-to-date literature reports on the topic, providing insights into new technologies that could be explored in the corn biorefinery context. New advances in this field, as well as challenges and strategies to overcome them, will be described and explored.

The world is facing the fastest-growing crisis of electronic-waste (e-waste), which is projected to surpass 75 million tonnes by 2030, posing a major environmental threat due to heavy metal toxicity from Pb, Cd, Hg and the other plastic components of the electrical wires and the circuit boards etc. Initially categorised as solid waste, all conventional recycling methods for e-waste, like incineration and landfilling, are either energy-intensive or unsustainable in practice. Microorganisms, being the eco-innovators, have the potential to detoxify and recover the important metals (i.e., Au, Ag, Cu, Pd) from these e-waste through various microbial processes like biosorption, bioleaching, and other enzymatic degradation methods. These microbial processes have been established internationally for their bioremedial and resource generation outcomes, like biogenic nanoparticle (NP) (i.e. AgNP. AuNP) generation, pure metal recovery, biosurfactants (i.e. Rhamnolipids, Sophorolipids), organic acids, and bioplastic (Polyhydroxyalkanoates) generation, etc. However, the challenges lie in scaling up these processes as well as ascertaining the product quality and market acceptance. Most importantly, the shortcomings of the regulatory framework for promoting the biotech-based recycling process for these e-waste. In this review, we critically analyse the wondrous role of microbes in e-waste management. A broad spectrum of bacteria like Acidithiobacillus ferrooxidans, Bacillus sp., Pseudomonas putida and fungi like Aspergillus niger, Candida bombicola participate in sustainable product recovery in numerous ways. It highlights the current research gaps in conjoining the attainable soil microbial ecology with environmental pollution surveillance and remediation. It also brings attention to various e-waste management strategies to combat the threat of toxic waste driven climate change and biodiversity loss.

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a stable derivative of L-ascorbic acid widely used in food, cosmetic, and pharmaceutical industries; however, its enzymatic production remains limited by high enzyme consumption and low catalytic efficiency. In this study, a marine-derived cyclodextrin glycosyltransferase (CGTase) was engineered using a semi-rational design strategy to enhance AA-2G synthesis with maltodextrin as a cost-effective glycosyl donor. Based on sequence alignment, two residues were selected for saturation mutagenesis, and beneficial variants (Y260F and A236P) were identified from mutant libraries through activity-based screening. Subsequently, the double mutant Y260F/A236P was constructed and characterized. Compared with the wild-type enzyme, the double mutant achieved an AA-2G concentration of 30.2 g/L, corresponding to an 11.8% increase in yield. Kinetic analysis revealed a 20.8% decrease in K_m and a 1.48-fold increase in k_cat/K_m toward L-ascorbic acid. Under optimized conditions, the engineered system achieved high AA-2G yield with significantly reduced enzyme loading (120 U/g substrate), and the double mutant enabled rapid conversion, reaching high yield within 12 h. In addition, A236 was identified as a novel mutagenesis site in CGTases. These results provide an efficient and cost-effective strategy for AA-2G production and offer new insights into enzyme engineering.

Hexadic tank system represents an extension of quadruple tank system for controlling non-growth-associated product dynamics in bioprocess industries, including two stage continuous fermentations, multiple distillation columns, pharmaceutical, and food processing applications. This study presents a comprehensive analysis encompassing theoretical foundations, simulation frameworks, hardware implementation, and experimental validation of three control algorithms: LQR, Linear MPC, and Robust MPC, evaluated under disturbance and non-disturbance conditions. Among the three control algorithms, Linear MPC with disturbances ($\hbox {LMPC}_{\text {D}}$) achieves superior performance with the lowest mean error (1.67), maximum error (2.00), control variance (3.47), and overall sensitivity (2.52), with high settling times. $\hbox {RMPC}_{\text {D}}$ shows the fastest minimum response (1.93 s) but exhibits higher mean error (2.5) and maximum error (5.0), and overall sensitivity (3.94). LQR controllers exhibit poor performance, with high sensitivity (94.08–226.47), large errors, and longer settling times (especially for $\hbox {LQR}_{\text {D}}$), rendering them unsuitable for practical implementation. All controllers maintain zero steady-state error with stable eigenvalues ($-6.76\times 10^{-3}$ to $-4.34\times 10^{-19}$). This confirms that the model predictive control strategies are optimal for tracking precision, disturbance rejection, and parameter insensitivity in bioprocess applications.

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% and 59.62% reductions in cellular and supernatant mannitol, respectively, compared with TB6050. During 5-L bioreactor fermentation, ΔP produced 9.18% more biomass, achieved 10.91% higher glucose conversion efficiency, and increased total crude protein yield per liter by 7.1%, although crude protein content was not significantly changed. Transcriptome analysis revealed changes in central metabolism, including upregulation of the glyoxylate shunt and branched-chain amino acid biosynthesis pathways. Expression of ICL and MS was significantly higher in ΔP than in TB6050 (p_adj < 0.001). In addition, ilvD expression was 2.88-fold higher in ΔP, whereas ACO expression was 2.13-fold lower. Several genes related to oxidative phosphorylation and basal heat response also showed reduced expression. These results support FvM1PDH as a major contributor to mannitol accumulation in F. venenatum and suggest that its deletion is associated with carbon allocation-related metabolic adjustments during glucose-based fermentation.

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, LeuDH^S331R and LeuDH^S331C, 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-AmDH^S331C. 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.

Plastic waste is increasingly recognized not only as an environmental burden but also as a carbon feedstock for plastic waste biorefineries. This concept is supported by recent advances in the discovery and engineering of polyester hydrolases, as well as the development of microbial hosts capable of metabolizing depolymerization products. Among the major plastic classes, polyesters provide the most practical biological entry point because their hydrolyzable ester bonds enable enzymatic depolymerization into soluble monomers that can subsequently be taken up and assimilated by microorganisms. Consolidated bioprocessing (CBP) has been proposed as a promising strategy for plastic waste biorefineries. CBP integrates upstream biological depolymerization with downstream microbial utilization of the released monomers. Achieving this process integration depends on the coordination of hydrolase expression and localization, polymer depolymerization, monomer uptake, assimilation, and product formation within a single biological system. However, practical implementation remains limited by the lack of robust host strains capable of performing these functions, together with the challenge of establishing compatible process configurations. In this review, we summarize recent advances in the biological modules required for CBP of polyesters and discuss current integration architectures, key engineering bottlenecks, and design priorities for developing CBP-ready strains for economically viable plastic waste biorefineries.

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 P_cbh1. 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.

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%. Through T7-driven sfGFP integration at 11 distinct chromosomal loci, three high-expression neutral sites—wecB, nagAB, and manXYZ—were identified as suitable targets for modular pathway integration. Within 30 days, 12 competing metabolic pathways were sequentially eliminated and nine N-acetylneuraminic acid (NeuAc) biosynthetic modules were integrated in the genome, yielding a total of 13 iteratively engineered strains in 30 days. The final strain, EcNSA13, achieved a NeuAc titer of 35.78 g/L with a productivity of 0.61 g/L/h in a 3-L bioreactor. In addition, the toolkit enabled high-level, antibiotic-free production of ovalbumin (OVA), reaching a titer of 202.91 mg/L based on the T7 expression system. Collectively, the ACS-CRISPR-Cas9 platform combined with the T7 expression system markedly accelerates genome editing and modular engineering in EcN, providing a versatile strategy for constructing high-performance probiotic cell factories and establishing a technical foundation for the industrial-scale production of NeuAc and other high-value bioproducts.

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.

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.