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.