Microbial cell factory is a powerful biological tool for synthesizing value-added molecules due to its unmatched sustainability and selectivity. However, the inherent catalytic specificity of natural enzymes limits product diversity. Although photobiocatalysis has expanded enzyme catalytic capabilities, challenges such as laborious enzyme purification, incompatibility with in vivo conditions, and complex unnatural substrate synthesis hinder photobiomanufacturing efforts. Herein, we combine a microbial cell factory with a new-to-nature photobiocatalytic transformation via a modular design that combines aerobic fermentation (Module I) and anaerobic photocatalysis (Module II) to achieve de novo biosynthesis of D-homotryptophan. In Module I, we engineer Escherichia coli equipped with a biosynthetic gene cluster to produce indole-3-acetic acid (IAA) via modifying metabolic flux and multi-copy genetic amplification strategies. In Module II, we develop an efficient synergistic photoredox/enzymatic synthesis of D-homotryptophan from L-serine and IAA by a pyridoxal phosphate (PLP)-dependent tryptophan synthase beta-subunit (TrpB) variant. This work synergistically merges emerging photobiocatalytic reactivity with natural biosynthesis, demonstrating a platform with potential for future biomanufacturing of non-natural products.
Occupational hazards in the cement industry significantly increase the risk of oral diseases. This study aimed to determine the prevalence and factors associated with tooth wear among 800 cement factory workers in Durg, Chhattisgarh, to inform the planning of preventive oral health services. A cross-sectional study was conducted among workers with at least 1 year of experience. Data on demographics, work history, and department were collected. Tooth wear was assessed using the tooth wear index (TWI), with statistical analysis performed via Kruskal-Wallis H and Mann-Whitney U tests. The mean age of participants was 32.71 ± 8.33 years. Cement mill workers had the highest prevalence of tooth wear with a mean TWI score for Score 1 (8.00 ± 3.57). Those with over 10 years of experience had significantly higher total TWI scores than those with less than 10 years for all categories of workers ( P < 0.001). Mean tooth wear scores were significantly lower among protective mask users than non-users, particularly with Score 1 and 2 ( P < 0.001). Prolonged occupational exposure is associated with more severe grades of tooth wear, indicating the accumulative risk for poor dental health in cement factories. These results underline the importance of appropriate improved occupational safety and health measures such as routine dental examinations and the provision of personal protective equipment such as masks in the workplace for the well-being of employees. The results would be able to provide evidence for the decision-making and prevention programs toward raising awareness of occupational oral health among workers.
This study evaluated the performance and safety of a 15-day real-time continuous glucose monitoring (rtCGM) system in Chinese adults with diabetes, focusing on its accuracy and clinical utility for long-term glucose management. A clinical evaluation was conducted using four rtCGM sensors per participant, with placement sites on both the upper arm and the abdomen. The system's accuracy was assessed using a factory-calibrated model. Primary outcomes included the mean absolute relative difference (MARD), the 20/20% agreement rate, and the proportions of Clarke and Consensus Error Grid in zones A+B. A total of 74 participants were screened. The MARD values were 8.44% for the upper arm and 8.91% for the abdomen under factory-calibrated model. The 20/20% agreement rates were 95.78% for the upper arm and 94.41% for the abdomen under factory-calibrated model. The Clarke and Consensus Error Grid A+B proportions were 99.53% and 99.96% for the upper arm, and 99.46% and 99.82% for the abdomen under factory-calibrated model, respectively. The CGM system showed high accuracy, robust alert performance, stable repeatability, and favorable safety over 15 days in Chinese adults with diabetes, supporting its clinical utility for glucose monitoring in these adult patients.
A dataset of two spectral lighting simulation reference models - one office and one factory hall - is presented. It aims to demonstrate and support full-spectral daylight and electric lighting simulations and facilitate evaluation of non-visual effects of light. The dataset includes Rhino CAD geometry, comprehensive spectral material and light source data and window system BSDF data. Example implementations in the two software tools, Radiance and OWL, enable reproducible workflows and support adoption in other software. The dataset is openly available on Zenodo. The office model reproduces Room 518 at the University of Innsbruck, including a west-facing façade and interior furnishings. The factory hall model follows the proposed geometry in the European standard 15193 for building energy performance. Interior reflectances in the office were measured in-situ using a handheld spectrometer. Exterior spectra and factory hall materials matching specified reflectances were obtained from an online spectral materials database. Glazing transmittance was derived from IGDB data using LBNL Optics/WINDOW. BSDFs for venetian blinds at various tilt angles, and for a diffusing pane adapted from the Complex Glazing Database, were generated in WINDOW. Luminaires in both models are specified with photometric files (Eulumdat/IES) and lamp spectra (Fluorescent 840, 4000 K LED). The provided example implementations (Radiance, OWL) include prepared input data and scripts to run first spectral simulations; example results are also included. The dataset is prepared to support reuse by researchers, designers and software developers for method validation, software engineering and comparison, and development of spectral metrics and controls.
Government regulation is an important factor influencing corporate investment and financing decisions. However, little research has examined the role of voluntary regulation. This paper focuses on how voluntary environmental regulation shapes this process by investigating the impact of Green Factory Certification on corporate financialization. Using a multi-period difference-in-differences model and data on China's A-share listed manufacturing firms from 2012 to 2023, we find that Green Factory Certification significantly curbs corporate financialization. This inhibitory effect is more pronounced in companies with stronger financing constraints, better corporate governance quality, and cities with stricter financial and environmental regulations. Furthermore, the mechanism analysis reveals that voluntary environmental regulation alleviates the "reservoir" motive via "green signal transmission" (improving financing conditions and securing government subsidies) and weakens the "investment substitution" motive through "voluntary commitment and reputation maintenance" (proactively boosting fixed investments and green innovation). This study enriches research on voluntary environmental regulation's economic effects and provides empirical support for optimizing green industrial policies.
This study aimed to develop and evaluate a novel method for customizing CPAP nasal mask cushions to improve patient comfort, mask fit, and adherence to therapy for individuals with Obstructive Sleep Apnea (OSA). A customized CPAP nasal mask cushion design was developed using a combination of 3D facial data analysis and a replaceable rapid mold system. Twenty moderate OSA patients were enrolled for facial scanning and design analysis, and five representative participants were selected for validation testing using a within-subject comparison between their original factory-fitted cushions and the customized versions. Principal Component Analysis (PCA) to identify key measurement points influencing mask design. A pocket-style mold system with interchangeable cores enabled the efficient fabrication of patient-specific cushions. Objective assessments and patient-reported outcomes were used to compare the customized cushions with conventional designs in terms of comfort, strap tension, facial pressure, and air leakage. Customized nasal mask cushions demonstrated significant improvements over factory-fitted designs. Patient-reported comfort scores increased from 6.2 ± 0.8 to 8.8 ± 0.6 (p < 0.01), while scores for strap tension and facial pressure improved from 6.5 ± 0.9 to 9.0 ± 0.7 (p < 0.01). Patients also reported a substantial reduction in air leakage and a preference for the customized designs for long-term use. The findings indicate that customized CPAP nasal mask cushions effectively enhance fit and comfort, addressing key barriers to CPAP adherence. This method provides a scalable and cost-effective solution leveraging 3D scanning and rapid mold technologies. Further research involving larger sample sizes, longer durations, and objective performance metrics will be necessary to assess clinical applicability and long-term adherence outcomes.
Monoterpene Indole Alkaloids (MIAs) are a diverse family of plant natural products with various medicinal applications. Although MIAs, such as vinblastine and reserpine, are clinically validated, sourcing of MIAs for clinical use or drug discovery from natural resources or via chemical synthesis is hampered due to their scarcity and chemical complexity. Refactoring MIA biosynthesis pathways in microbial cell factories could offer an alternative, more stable and potentially sustainable manufacturing route for alkaloid medicines and novel therapies. However, reaching commercially attractive titers, rates and yields remains challenging owing to the length and complexity of these metabolic pathways. One critical bottleneck is the low screening throughput and very high cost of the analytical methods used to quantify MIA for optimizing production. In this study, we evolved RamR, a promiscuous bacterial transcription factor to respond to five different MIAs, resulting in highly sensitive and selective sensor variants (EC50<10 μM). X-ray crystallography and computational modeling provided insight into the MIA binding of the evolved biosensor variants. The RamR biosensing platform was functionalized in yeast and subsequently applied in a cost-effective semi-throughput screening campaign of a 188-gene overexpression library to identify high-performing cell factory designs for strictosidine, the common precursor for all MIAs. The fluorescent biosensor signal correlated with HPLC quantification (r2 = 0.932) allowing identification of single metabolic engineering hits which when combined yielded a maximum titer of >220 mg/L strictosidine, 3-fold higher than the parental reference strain. This study demonstrates the development of selective biosensors for MIAs and the cost-effective identification of novel metabolic engineering hits for optimizing MIA production in microbial cell factories.
We describe a streamlined set-up for Lloyd's-mirror-based laser interference lithography (LM-LIL), using only a fibre-coupled single-frequency laser (SFL), a plane mirror and an optional half-wave plate as the optical components. Using an SFL with a factory-set, pigtailed single mode fibre as the LM-LIL light-source greatly simplifies experimental implementation by removing the usual need to manually align a free-space laser-beam to a pinhole-based spatial filter or external fibre. The resulting system is inexpensive, robust and easy to use, allowing for the straightforward preparation of one- and two-dimensional arrays with pitches down to the sub-220-nm level when using a 405-nm laser. Full details of the experimental set-up are provided, together with a step-by-step description of sample preparation, exposure and subsequent processing.
The textile industry is a major contributor to water pollution, releasing effluents containing 10-15% unused synthetic dyes. These dyes have complex aromatic structures that resist biodegradation, posing serious risks to aquatic ecosystems and human health. This study evaluated the physicochemical properties of wastewater from the Maa Garment Textile Factory and investigated the bioremediation potential of indigenous bacterial isolates. Effluent samples collected from different treatment points were analyzed for pH, temperature, total suspended solids (TSS), total dissolved solids (TDS), biological oxygen demand (BOD), and chemical oxygen demand (COD). The wastewater exhibited a neutral to slightly alkaline pH (7.2-7.5), favorable for microbial activity, but had elevated temperatures (up to 38 °C) and high TDS (2633-2866 mg/L), indicating thermal and chemical pollution. A total of 30 bacterial isolates were screened for their ability to biodegrade reactive red, blue, and yellow dyes under varying conditions of temperature (25-40 °C), pH (5-9), and dye concentration (50-150 mg/L). Sixteen isolates demonstrated biodegradation capacity, with optimal performance (mean 89.4%) observed at 37 °C, pH 7, and 50 mg/L dye concentration, and significant variation across tested conditions (p < 0.001). The effective isolates included Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Shigella species, and Salmonella Typhimurium. Among them, P. aeruginosa strains (H5P, C2P, and C4P) showed the highest efficiency, achieving up to 90% biodegradation and maintaining strong performance under alkaline conditions (pH 9) and higher dye concentrations (150 mg/L). These findings highlight the adaptability of indigenous bacteria for dye degradation and identify P. aeruginosa as a promising candidate for sustainable, cost-effective industrial bioremediation, although further field validation across diverse dye systems is recommended.
Ergothioneine (EGT), salidroside and gadusol are high-value cosmetic ingredients valued for their moisturizing, skin-brightening and ultraviolet (UV)-protective properties. Leveraging Saccharomyces cerevisia, a GRAS organism already widely used in cosmetics, we engineered a single strain for their simultaneous co-production. We first identified the 5-histidylcysteine sulfoxide synthase domain reaction as rate-limiting and addressed the poor solubility of ergothioneine biosynthesis protein 1 (EGT1) by screening heterologous homologs and optimizing EGT1 through solubility-enhancing fusions and structure-guided stabilization, increasing EGT titer in shake flask from 34 to 132 mg L-1. Multicopy integration of the optimized pathway into the ribosomal DNA (rDNA) locus further boosted EGT production to 690 mg L-1 in a 5-L bioreactor. Subsequently, biosynthetic pathways for salidroside and gadusol were introduced, yielding a tri-product strain that achieved final titers of 1329 mg L-1 EGT, 224 mg L-1 salidroside and 556 mg L-1 gadusol in fed-batch fermentation. This work establishes a multifunctional yeast cell factory capable of simultaneous co-production of multiple premium cosmetic actives, offering a scalable and sustainable platform for next-generation ingredient development.
β-Alanine is one of the most valuable three-carbon compounds. In this work, for enhanced β-alanine biosynthesis by probiotic Escherichia coli Nissle 1917 (EcN), multilevel engineering strategies were employed, including the introduction of heterologous key enzymes, dual-pathway reinforcement of the precursor L-aspartate (L-Asp) supply, dynamic modulation of the competing pathways, rewiring of the central metabolism, and engineering the transport systems. A final antibiotic-free strain was constructed, which exhibited robust biosynthesis throughout the fermentation process and produced 60.55 g/L β-alanine with a yield of 0.30 g/g glucose in a 5-L bioreactor, representing the highest level of β-alanine production achieved to date using probiotic E. coli. Notably, it is the first time the function of TdcC (proton-driven threonine/serine symporter), SstT (sodium-driven serine/threonine symporter), SdaC (proton-driven serine symporter), and RhaT (proton-driven rhamnose symporter) has been examined in β-alanine accumulation. This study demonstrated the practicality of EcN as a microbial cell factory for β-alanine production and provided an effective synthetic biology strategy for the production of other L-Asp-derived compounds.
Rhodotorula toruloides is an emerging oleaginous yeast with strong potential as a microbial cell factory for the production of acetyl-CoA-derived bioproducts. However, engineering of this organism has been limited by the absence of a functional episomal plasmid system, a foundational genetic tool for rapid gene expression, pathway testing, and CRISPR-based genome engineering. Here, we report the first episomal plasmid system for R. toruloides. Through systematic screening of candidate autonomously replicating sequences (ARSs) from diverse sources, we identified multiple functional ARS elements and selected C63F4, a fragment derived from Contig 63 of R. toruloides CBS14, because of its stable performance. The resulting pC63F4 plasmid was maintained episomally, supported GFP reporter expression, exhibited a copy number of 2.39 ± 0.13, and showed good stability during long term cultivation. To overcome poor transformation efficiency, we developed a Cre-loxP-mediated in vivo re-circularization strategy that enabled reliable delivery of the episomal plasmid. Using this improved system, we demonstrated functional episomal expression of metabolic engineering genes and multi-gene pathways for the production of triacetic acid lactone, fatty alcohols, and limonene. Finally, we leveraged this platform to establish a redesigned CRISPR system that enables seamless genome editing in R. toruloides for the first time, while also simplifying marker recycling. Together, this work establishes a long-needed episomal plasmid platform and associated CRISPR toolkit that will accelerate metabolic engineering, synthetic biology, and fundamental studies in R. toruloides.
The aim of the study Conducting a comparative analysis of the strength characteristics of model samples of modern polymer materials used for the manufacture of temporary orthopedic structures. The materials used were Dental Sand Pro (HARZ Labs, Russia) and Huge PMMA Monolayer (Huge, China). Dental SAND PRO material is positioned as a ceramic-filled polymer material designed for 3D printing of temporary crowns and bridge-like structures for long-term wear. As part of the experimental part of the work, models were developed and manufactured for conducting a series of comparative compression tests. Two modern production technologies are compared: milling from ready-made factory blocks (CAD/CAM) and 3D printing. Tests have shown that structures made of HUGE PMMA Monolayer material using CAD/CAM technology have higher mechanical strength compared to crowns made of HARZ Labs "Dental Sand Pro" material made by 3D printing. The destructive load of milled samples from HUGE PMMA is 2-4 times higher. Model molar crowns also had higher values for milled samples. Bridge-like prostheses made by milling could withstand about 1600 N, and 3D printing - about 500 N (p<0.05). Temporary structures made by milling from ready-made polymer blocks have a sufficient margin of safety for use in all areas of the dentition. In turn, 3D-printed structures, due to their lower strength characteristics, should be used mainly in the area of frontal teeth and premolars, where the load is significantly. Сравнительный анализ прочностных характеристик модельных образцов современных полимерных материалов, применяемых для изготовления временных ортопедических конструкций. Исследовали материалы Dental Sand Pro («HARZ Labs», Россия) и Huge PMMA Monolayer («Huge», Китай). Материал Dental SAND PRO позиционируется как керамонаполненный полимерный материал, предназначенный для 3D-печати временных коронок и мостовидных конструкций длительного ношения. В рамках экспериментальной части работы разработаны и изготовлены модели для проведения серии сравнительных тестов на сжатие. Проведено сравнение двух современных технологий производства: фрезерования из готовых заводских блоков (CAD/CAM) и 3D-печати. Испытания показали, что конструкции, изготовленные из материала HUGE PMMA Monolayer по технологии CAD/CAM, обладают более высокой механической прочностью по сравнению с коронками из материала HARZ Labs «Dental Sand Pro», изготовленными методом 3D-печати. Разрушающая нагрузка у фрезерованных образцов из HUGE PMMA в 2—4 раза выше. Модельные коронки моляров также имели более высокие показатели у фрезерованных образцов. Мостовидные протезы, изготовленные методом фрезерования, выдерживали около 1600 Н, а изготовленные методом 3D-печати — около 500 Н (p<0,05). Временные конструкции, изготовленные методом фрезерования из готовых полимерных блоков, обладают достаточным запасом прочности для применения во всех зонах зубного ряда. В свою очередь конструкции, выполненные методом 3D-печати, ввиду их более низких прочностных показателей целесообразно использовать преимущественно в области фронтальных зубов и премоляров, где нагрузка существенно ниже.
Heterologous expression of enzymes from higher organisms limits the construction of microbial cell factories for natural product biosynthesis. Here we develop a ProteinMPNN-based sequence redesign strategy, guided by essential structural features and evolutionary conservation, to improve the bacterial expression of heterologous enzymes. Applied to two plant glycosyltransferases, TOGT and UGT84A56, for esculetin glycosylation, this strategy generates extensively redesigned variants with markedly enhanced soluble expression and catalytic activity in Escherichia coli. In vitro enzyme assays and in vivo whole-cell conversions show consistent improvements, outperforming conventional solubility-enhancing strategies. Molecular simulations suggest that the improved performance arises from global optimization of hydrophobic and hydrophilic residue exposure while preserving productive substrate-binding interactions. Fed-batch fermentation achieved titers of 2.11 g/L cichoriin and 4.05 g/L aesculin. This work establishes a generalizable route for converting difficult-to-express eukaryotic enzymes into efficiently expressible and functional variants, thereby expanding the enzymatic toolbox for microbial cell factory construction.
Komagataella phaffii is a promising cell factory that can use CO2 derived methanol, a sustainable carbon (C1) source, for chemical production. Introducing the ß-alanine biosynthetic pathway alongside a NADP+-dependent formate dehydrogenase (FDH) in K. phaffii enables the production of the platform chemical 3-hydroxypropionic acid (3-HP). Co-feeding of formate and methanol (MeOH) was systematically explored to enhance cellular reducing power and improve 3-HP biosynthesis. Implementing a pulsed formate strategy alongside MeOH resulted in up to a 20.7% increase in 3-HP per gram of MeOH (Yg3-HP gMeOH-1) compared to MeOH alone in shake flask cultivations. This co-feeding strategy likely enhanced NADPH availability through formate oxidation via the introduced NADP⁺-dependent FDH, thereby improving redox balance, as supported by simulation studies based on the K. phaffii genome-scale metabolic model. Similar improvements were demonstrated in repeated batch cultivations at 1-L bioreactor scale, where a formate pulse every 4-hour led to a 25.8% increase in Yg3-HP gMeOH-1 and a 41% increase in volumetric productivity over the control without formate (only MeOH). In addition to the feeding strategy, pH regulation also played a crucial mechanistic role: strict pH control at 5 inhibited growth, due to the predominance of undissociated formic acid, whereas allowing the pH to rise to 6-7 favoured dissociation and supported higher productivity. These findings elucidate the pH dependent nature of formate assimilation and highlight the potential of coupling MeOH and formate co-utilization with dynamic feeding and pH strategies to enhance bioproduction in K. phaffii.
To address the issue of 2D laser-guided automated guided vehicles (AGVs) in industrial intelligent material handling scenarios being susceptible to interference from changes in lighting and complex obstacles, leading to abnormal positioning and mapping and frequent false stops, this paper designs a lightweight, multi-dimensional perception and anti-false-stop YOLOv8 anomaly recognition network, achieving accurate identification of various interferences in complex environments. An adaptive decision-making fault-tolerant control algorithm is proposed, introducing a temporal logic verification and dynamic threshold adjustment mechanism to achieve real-time dynamic switching of obstacle avoidance levels, ensuring efficient coordination between perception decision-making and control execution. An AGV anomaly detection sample set suitable for complex industrial scenarios is constructed, providing reliable data support for model optimization and accuracy evaluation. Finally, real-world deployment verification in a real electronics factory environment shows that this method reduces the vehicle false-stop rate and improves task handling efficiency. This research effectively solves the robust perception problem of AGVs in complex industrial environments and has significant engineering application value.
Although the oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory, its application remains constrained by inefficient homology-directed repair (HDR) and a lack of precise genomic integration tools. To address these limitations, we developed a comprehensive genetic toolkit featuring three synergistic advancements. First, we systematically identified 55 neutral integration sites with tunable expression profiles, enabling stable, position-independent gene integration with predictable transcriptional output across a 12.88-fold dynamic range. Second, we established a dual-readout high-throughput screening platform combining colony morphology analysis with hrGFP fluorescence. This approach accurately measures locus-specific homologous recombination (HR) efficiency while eliminating false positives by dominant non-homologous end joining (NHEJ). Third, we engineered a transient HR enhancement system by fusing the Sae2 exonuclease to Cas9 via a flexible (GGGGS)3 linker. This fusion significantly boosts HR efficiency and surpasses the cleavage activity of unmodified Cas9 without introducing permanent genomic modifications or compromising cellular fitness. Finally, HR efficiency for single-gene integration was increased from 46.5% to 77.5% while the dual-locus editing efficiency reached 64.1% when using 500-bp homology arms, and the engineered strains demonstrated improved genetic stability compared to those with constitutive HR enhancement.
Covering: up to December 2025The incorporation of distinctive structures such as uncommon functional groups, distinct molecular scaffolds, unusual modifications and other characteristic structural features can significantly enhance metabolic stability, bioactivity, and pharmacokinetic properties of drug molecules, offering ways to optimize the design and synthesis of novel bioactive molecules in synthetic biology. Streptomyces, a highly diverse and widespread bacterial genus, produces an extraordinary array of secondary metabolites, exhibiting remarkable structural variety. This structural ingenuity lies at the heart of functional innovation, establishing Streptomyces as a prolific source for drug discovery. The vast and diverse structural repertoire of these natural products offers valuable inspiration for novel structural designs in medicinal chemistry. Moreover, the vast array of natural enzymes provides a versatile toolkit for the site-specific modifications of complex scaffolds, facilitating the development of novel drug molecules that bypass the structural limitations of traditional chemical synthesis. This review highlights Streptomyces natural products with rare functional groups, unique scaffolds, and atypical modifications, examining the enzymatic mechanisms to link biosynthetic diversity with synthetic biology applications and efficient cell factory design.
Unicellular cyanobacteria due to their faster growth and ease of cultivation can be used as potential cell factories for different biotechnologically important metabolites. In the present study, methanolic extracts of twenty different terrestrial unicellular cyanobacteria were evaluated for antioxidant properties. DPPH activity ranged between 9.28 and 37.77% while ABTS activity was in a range of 48.22-100.00%. Fe+ 2 chelating activity ranged from 39.62 to 98.66% and deoxyribose protection (DRP) activities ranged between 41.18 and 71.53%. Total phenolic content (TPC) and total flavonoid content (TFC) were found to be in the range of 17.87-81.67 µg GAEs g- 1 FW and 0.30-7.46 mg QEs g- 1 FW respectively. Aphanothece sp. K93 and Chroococcidiopsis sp. K63 had significant ABTS and DPPH radical scavenging activity. The ferrous ion chelating activity for these two isolates was recorded as 98.66 ± 0.53% and 90.846 ± 0.27% respectively, while the DRP was noted as 64.36 ± 0.23% and 62.482 ± 0.25% respectively. Aphanothece sp. K93 was recorded with the highest TPC (81.67 ± 0.84 µg GAE g- 1 FW) whereas Cyanobacterium aponinum K17 showed the highest TFC (7.46 ± 0.12 mg QE g- 1 FW). TPC was significantly positively correlated with DPPH, and ferrous ion chelating activity while TFC showed negative correlation with ABTS activity. Untargeted metabolite profiling of Aphanothece sp. K93 via LC-MS analysis, showed the abundance of bis(4-ethylbenzylidene)sorbitol; cetrimonium; ethyl-N-[(3alpha,7alpha,12alpha)-3,7,12-trihydroxy-24-oxocholan-24-yl]glycinate and myristoyl glutamic acid with known bioactive properties. Rare bioactive compounds like artemotil and militarinone A were also putatively annotated from the MS analyses. Due to the presence of high phenolics, radical scavenging activity and bioactive metabolites in unicellular cyanobacteria (like Aphanothece sp. K93), it can be further explored as potential cell factory for bioactive phenolic compounds for applications in pharmaceutical industries.
Indigoidine, a novel blue microbial pigment with excellent dyeing performance, emerges as a sustainable alternative to synthetic dyes. However, prevailing plasmid-based production systems suffer from genetic instability and reliance on antibiotic selection, rendering them incompatible with green industrial manufacturing. In this study, we developed a genetically stable, plasmid-free Escherichia coli biocatalyst to drive the green synthesis of indigoidine utilizing glycerol, a renewable and abundant feedstock. First, CRISPR-based multicopy integration was employed to enhance expression of the key enzymes including indigoidine synthase and its phosphopantetheinyl transferase. Furthermore, to improve precursor supply, functionally analogous enzymes from different microbial sources were screened. Subsequently, to address the challenge of modifying the TCA cycle, a protein degradation tag system was introduced and the strength of specific genomic promoters was attenuated, two strategies that acted synergistically. Engineering of the NADPH regeneration pathway effectively elevated energy precursor pools, while knockout of acetate pathways alleviated metabolic stress. Ultimately, the optimal strain MIG41 achieved indigoidine titers of 6.09 g/L in shake flasks and 35.21 g/L in a 5 L fed-batch fermentation, with a productivity of 0.73 g/L/h. Moreover, the purified indigoidine exhibited excellent dyeing performance. Ultimately, this work provides a robust, scalable microbial cell factory platform for the sustainable, high-yield production of eco-friendly dyes.