O-Succinyl-L-homoserine (OSH) plays a pivotal role in L-methionine biosynthesis. Microbial cell factories for high-yield OSH production have been progressively optimized, achieving substantial improvements in fermentation titers. In this study, a multi-step progressive optimization strategy was adopted to construct a high-yield OSH-producing strain. First, the feedback inhibition of the key enzyme HST was relieved, and the key genes involved in the byproduct metabolic pathways were knocked out. This modification enabled engineered strain to produce 9.77 ± 0.27 g/L OSH in shake-flask fermentation. Second, ribosome binding site (RBS) engineering, promoter engineering, and dynamic metabolic regulation were integrated to strengthen and balance the intracellular supply of the two core precursors, L-homoserine and succinyl-CoA. These strategies greatly increased the OSH titer to 18.54 ± 0.03 g/L. Finally, global optimization of cofactor and energy optimization was carried out to further enhance strain performance, and the engineered strain OSHM40 achieved the OSH titer of 20.15 ± 0.21 g/L via shake-flask cultivation, and 104.09 ± 2.06 g/L in a 5-L bioreactor under fed-batch fermentation, with a sugar-acid conversion rate of 64.99% and a volumetric productivity of 1.43 g/L/h. Notably, the OSH titer and sugar-acid conversion rate of this strain represent the highest levels reported to date among all plasmid-free OSH-producing strains. The plasmid-free system constructed in this study effectively avoids the plasmid-induced metabolic burden and genetic instability. This work demonstrates the prominent advantages and great application potential of plasmid-free modular engineering for the efficient biosynthesis of OSH and other high-value amino acids.
Marine phototrophic bacteria represent promising platforms for sustainable microbial biopolymer production. Here, we report the screening and functional characterization of MAR44, a Mediterranean isolate identified as the aerobic anoxygenic phototrophic bacterium (AAPB) Roseibium alexandrii. This strain carries a complete set of genes for polyhydroxyalkanoate (PHA) metabolism, including phaC, phaAB, phaR, phaZ and multiple phaP homologues. Phenotypic screening confirmed substantial intracellular PHA accumulation, and gravimetric determination revealed intracellular polymer contents of up to approximately 50% of cell dry weight. The purified polymer exhibited the characteristic FTIR absorption bands and 1H NMR signals associated with PHAs. A key finding is that illumination significantly (p < 0.05) increased glucose-induced intracellular PHA accumulation. Under continuous illumination and long-day photoperiods, MAR44 showed higher mean intracellular PHA accumulation levels, with increases of up to approximately two-fold compared with short-day conditions. These results suggest an association between illumination and enhanced intracellular PHA accumulation in this AAPB, although the underlying mechanisms remain to be elucidated. This study also introduces MAR44 as a promising candidate for further research into light-supported biopolymer production, expanding the current set of organisms beyond traditional soil-derived producers such as Cupriavidus necator.
Plant-based systems have proved to be a versatile platform for recombinant vaccine production. Numerous species of model and agronomic edible plants have been engineered to express antigens targeting diverse pathogens. Leaves, fruits, tubers, and seeds have served as expression tissues, while subcellularly, the endoplasmic reticulum, chloroplasts, and vacuoles have been targeted to enhance antigen yield and stability. Furthermore, most of the studies have demonstrated the immunogenicity of vaccines produced and administered within plant tissue. Interestingly, certain studies of wild-type (near-isogenic and non-transgenic) plant tissues used as experimental controls consistently exhibit intrinsic immunostimulatory properties. These effects, often overlooked, include enhanced antibody production, cytokine modulation, and partial protection during pathogen challenge. Bioactive compounds such as alkaloids, flavonoids, terpenoids, glycoalkaloids, polysaccharides, glucosinolates, carotenoids, and phenolics may contribute to these responses, varying across species. Evidence from multiple animal models shows that WT plant matrices can influence immune outcomes, sometimes outperforming inert controls. Recognizing the immunomodulatory contribution of plant-derived compounds is essential for accurately evaluating plant-made vaccines and for optimizing future formulations in both human and veterinary applications.
Erythritol is a natural zero-calorie sweetener with potential for sustainable healthy diets. While yeasts can convert biodiesel-derived glycerol into erythritol, the low production and by-product accumulation limit its industrial commercialization. Here, we isolated different morphologically Yarrowia lipolytica mutants with higher erythritol production through adaptive laboratory evolution under hyperosmotic stress, in which mutant Z exhibited superior growth performance and membrane-related genetic variants compared to the parent strain. We then systematically engineered strain Z to optimize the flux towards erythritol by improving glycerol utilization, reducing the synthesis of competing sugar alcohols, complementing auxotrophic markers, and boosting precursor supply. After fermentation condition optimization and two-stage fed-batch fermentation in a 5-L bioreactor, the final engineered strain Z12 produced 250.76 g/L erythritol from pure glycerol and 232 g/L erythritol from crude glycerol, which is the highest reported titers for both feedstocks. This study demonstrates the effective integration of adaptive evolution with metabolic remodeling for efficient erythritol biosynthesis.
While perfusion culture for Chinese hamster ovary (CHO) cells offers advantages such as continuous operation and flexibility, it suffers from product loss through cell bleeding and difficulties in reaching high productivity due to sustained rapid cell growth. Growth inhibitory strategies are widely used to enhance productivity in fed‑batch processes; however, their practical implementation and comparative effectiveness in perfusion processes remain insufficiently explored. Meanwhile, process development often relies on costly trial‑and‑error approaches. Here, we systematically compared three growth inhibitory strategies in perfusion culture-low cell‑specific perfusion rate (CSPR), sodium butyrate, and mild hypothermia-with respect to cell growth, metabolism, productivity, and product quality. Genome‑scale metabolic flux sampling analysis revealed that low‑CSPR and sodium butyrate induce a convergent up‑regulation of energy metabolism, correlating with greater gains in specific productivity (qp). Building on this insight, we developed a growth‑kinetic model for the combined low‑CSPR + butyrate strategy, incorporating parameter uncertainty. This model‑guided framework enabled the rational design of two distinct high‑productivity perfusion processes: a sustained mode that achieved robust long‑term stability alongside substantial productivity gains, and a high‑intensity mode that pushed qp and daily volumetric titer to their maxima, with increases of up to 108.94% and 190.36%, respectively, in a model CHO cell line with a moderate baseline productivity. Our study provides a proof‑of‑concept framework for perfusion intensification, from strategy selection to rational process design.
Cysteine is an essential amino acid for Chinese hamster ovary (CHO) cell culture, yet its poor stability and solubility at neutral pH complicate media and feed formulation, particularly in fed-batch and intensified processes. Thiazolidines, formed by condensation of cysteine with α-ketoacids, offer enhanced chemical stability; however, there are no published applications of their use as cysteine sources. This may be due to limitations by their slow reaction kinetics and poor compatibility with industrial feed preparation. Here, we reported a rapid, high-yield, and manufacturing-compatible cysteine-to-thiazolidine condensation method using α-ketoacids naturally present in mammalian metabolism. By optimizing reaction pH, substrate concentration, and molar ratios, complete conversion was achieved within 1-2 h under near-neutral conditions, as indicated by depletion of free thiols to negative control levels. This enabled the preparation of highly concentrated and storage-stable thiazolidine solutions suitable for direct incorporation into single-feed formulations. Using this optimized approach, thiazolidines were deployed as the sole cysteine source in CHO cell culture feeds and supported cell growth, productivity and product quality comparable to established cysteine delivery approaches across multiple CHO cell lines in both standard and intensified fed-batch processes. Mechanistically, cysteine-condensed thiazolidines function as reversible reservoirs that release cysteine through non-enzymatic equilibrium, maintain low free thiol levels while ensuring sufficient intracellular cysteine availability. Together, these results demonstrate that optimized cysteine-condensed thiazolidines provide a manufacturing-compatible alternative for single-feed cysteine delivery in CHO cell culture processes.
High-temperature fermentation is a strategy to increase the fermentation rates and process stability while reducing cooling costs; however, metabolic heat generated during large-scale cultivations can impair cell growth and product formation. To address this bottleneck, we sought high-temperature L-glutamic acid fermentation processes using naturally thermotolerant Corynebacterium strains as alternatives to experimentally evolved strains. We isolated Corynebacterium glutamicum PP80 from soil in Thailand and evaluated its performance in penicillin-triggered L-glutamic acid fermentation at elevated temperatures. Compared with the type strain C. glutamicum KY9002 (ATCC 13032) and other isolates, including the thermotolerant species Corynebacterium suranareeae N24, PP80 maintained higher fermentation performance at 37-39°C, producing more than 40 g/L L-glutamic acid in jar fermentor cultures. PP80 also retained higher cell viability than KY9002 after penicillin treatment. Although penicillin induced membrane vesicle production in both strains, PP80 showed a smaller penicillin-associated increase in phospholipid-rich membrane vesicles than KY9002. A focused comparison of proteins associated with stress responses, cell-envelope biogenesis, and L-glutamic acid metabolism and export showed broad conservation of the canonical pathways but identified candidate differences, including an additional MscCG2-like protein in PP80. The results identify PP80 as a candidate strain for further evaluation in elevated-temperature L-glutamic acid fermentation.
The chelating interactions amongst chitosan (CS) and Mg²⁺ were exploited to fabricate CS beads, with glutaraldehyde (GA) incorporated into the gelling solution to introduce the functionalities necessary for β-galactosidase (βGAL) covalent binding and also to provide additional cross-linking. The fabrication parameters of the GA/Mg-CS beads were systematically optimized by varying the concentrations of MgSO₄, GA, CS and acetic acid (AA). It was disclosed that the most efficient gelation solution comprised a mixture of 0.5% MgSO4-1% GA, and that it was optimal to utilize a 2% CS solution in 0.5% AA. Structural, morphological and elemental characteristics of the beads were assessed using FTIR, SEM and EDX, respectively. The mechanical durability of the GA/Mg-CS beads was also corroborated. The GA/Mg-CS beads successfully immobilized βGAL, achieving immobilization efficiencies of up to 59.78%. Following immobilization, the βGAL optimal pH shifted from 5.3-4.9 to 4.2, whereas its optimal temperature was unchanged within the range of 56-60°C. The immobilized βGAL (iβGAL) demonstrated higher tolerance to solvents compared to its free homologue. Furthermore, the iβGAL preserved 75.25% of its initial enzymatic activity following 15 consecutive assay cycles. It also maintained 109.14% activity by the 50th storage day. Finally, the applicability of the iβGAL was demonstrated in 24 h whey-permeate lactose degradation experiments at 45 °C.
Enzyme immobilization is pivotal for industrial biocatalysis yet often suffers from activity loss, structural distortion, operational instability, and high production costs. This study presents a magnetic bead-T4 capsid biocarrier (MBTCB) platform for efficient immobilization of an engineered tryptophan synthase Pf0A9 to enable sustainable synthesis of high-value tryptophan analogs. Soc-fused Pf0A9 was immobilized onto T4 phage capsids via high-affinity Soc-capsid binding under mild physiological conditions, preserving native enzyme conformation, and combining with magnetic bead conjugation for rapid separation. The immobilized form of the enzyme, Pf0A9@MBTCB, retained near-native enzyme activity and efficiently synthesized diverse analogs. The system demonstrated exceptional stability with full activity retention over five consecutive catalytic cycles and 84.5% activity after 9-day storage at 4°C, attributed to the robust T4 capsid scaffold, spatially ordered enzyme array preventing aggregation, and ultra-stable Soc-capsid anchoring. Economically, the process utilized unpurified crude lysate, eliminated centrifugation/filtration steps via magnetic recovery, and enabled reuse of unbound enzymes and capsids. In scaled biocatalysis, Pf0A9@MBTCB produced 443 mg of L-6-Cl-tryptophan over three cycles, which was a 2.75-fold yield enhancement versus single-use free enzyme. This platform merges high catalytic efficiency, operational robustness, and cost accessibility, offering a scalable solution for green manufacturing. The modular MBTCB strategy holds broad potential for multi-enzyme cascades and continuous-flow bioreactors, advancing sustainable biocatalysis for chiral chemical synthesis.
In this work, different strategies for the stabilization of commercial Alcalase based on glutaraldehyde modification were investigated. First, the free Alcalase was modified with 0.01, 0.1, or 1% (v/v) glutaraldehyde, which led to a significant increase in thermal stability without loss of enzyme activity. Subsequently, two immobilization strategies on aspartic-functionalized agarose (Asp-agarose) were evaluated: (i) immobilization of the glutaraldehyde-modified enzyme, and (ii) glutaraldehyde modification of Alcalase after its cation exchange onto the support. In strategy (i), full immobilization was achieved, but was accompanied by a reduction in expressed activity (∼40%), attributed to intense intermolecular crosslinking on the support as confirmed by SDS-PAGE. In strategy (ii), only ∼60% of the enzyme activity could be immobilized on the support, yet glutaraldehyde modification post-immobilization produced no activity loss and yielded the most stable biocatalyst. In both cases, SDS-PAGE analysis of the supernatants after boiling in SDS showed a marked reduction in protein, indicating extensive intermolecular crosslinking of the immobilized enzyme molecules. These results demonstrate that glutaraldehyde-based chemical modification is an effective approach for Alcalase stabilization, both in free and immobilized forms. The post-immobilization modification strategy offers the best balance between stability and retained activity.
We have created a new data-analysis pipeline for the discovery of host-specific candidate DNA biomarkers derived from sequencing data of cell-free blood. Unlike approaches that rely on specific molecular or genetic signatures, our method leverages the coverage distribution of cell-free DNA sequences mapped to a reference genome, applying statistical analyses to identify informative short genomic regions for biomarker discovery. The pipeline is applicable to diverse diseases and can be used to analyze cell-free DNA sequences from plasma or serum to identify candidate biomarkers that are characteristic of disease states in mammals. Core functionalities were developed in Java and integrated with open-source software tools for the preprocessing of raw sequencing data, complemented by Python scripts for the machine-learning analysis and statistical validation. The pipeline is designed for HPC use and users can access the pipeline through a Galaxy workflow, which offers a user-friendly web interface for input selection prior to execution and analysis progress monitoring. Performance tests, carried out using duplicate sets of COVID-19 samples and controls, showed linear scalability of execution time with an increasing dataset size, as well as a substantial reduction in execution time through parallelized computation, whereby each HPC node is used to process the data of one chromosome. Further statistical tests confirmed the quality of the pipeline's results by showing that the set of identified candidate biomarkers remained stable across varying dataset sizes.
Vanadium-dependent haloperoxidases are enzymes found in bacteria, fungi and red or brown macroalgae. They are used as a defense system for the latter by producing bromoform. The intermediate product, HOBr or HOCl, is also a microbicidal compound that can be exploited in many domains as medical tools or disinfectant sprays for example. We show that the mutation identified in Ohshiro’s work, which alters substrate specificity toward chloride, is transferable to the homologous enzyme from Chondrus crispus. We then applied genetic and enzymatic engineering to design a chimera with glucose oxidase, thereby enhancing antimicrobial properties by providing a local source of H2O2. We used the SpyTag/SpyCatcher technology to form the chimera and obtained homogenous objects, indicating that one oligomeric form is favored. We demonstrated the release of HOBr and HOCl thanks to NADH that reacts spontaneously with it outside the active site of the enzyme. We also measured the steady-state kinetic parameters of the wild-type or modified enzymes. The formation of the chimera increased the specificity of ccVHPO1 inside the chimera, towards KBr or H2O2, compared to the enzyme alone with the SpyCatcher. Finally, we showed a significant increase in the microbicidal effect between a coupled enzymatic system with the glucose oxidase (the two enzymes are free in solution) compared to the chimera system, which is completely bactericidal at concentrations around 20 nM.
The bioplastics field has oriented around industrial compostability as the primary biodegradation target. We argue this constitutes a regime mismatch: most plastic waste enters landfills - anaerobic, ambient-temperature environments in which poly(lactic acid) (PLA) and the majority of certified compostable bioplastics exhibit negligible degradation. Accepting landfill-active biodegradability as the correct constraint immediately reorders the candidate space. We conduct a systematic analysis across molecule class, production organism, processing architecture, and degradation mechanism, identifying three primary microbial production architectures with credible paths to the $1-2/kg commodity cost target: (1) Halomonas bluephagenesis Next-Generation Industrial Biotechnology (NGIB) for polyhydroxyalkanoate (PHA) - the most industrially-validated architecture, with demonstrated 149 g/L cell dry weight at 82% PHA in 5,000-L continuous non-sterile fermentation; (2) poly(γ-glutamic acid) (γ-PGA) / chitosan polyelectrolyte composites - extracellular production from Bacillus subtilis, water-phase processing without organic solvents, thermoplastic behavior when dry, and rapid protease-mediated landfill degradation, a combination not previously proposed as an integrated commodity plastic production strategy; and (3) a beetle cuticle-mimetic composite produced entirely from microbial sources - presented as a biomimetic materials hypothesis pending experimental validation. Two orthogonal strategies - bacterial cellulose pellicle production and viral capsid protein nanofillers - are evaluated as secondary approaches. A claim-status framework distinguishing evidence levels across all architectures is provided.
Lignocellulosic biomass is an attractive renewable feedstock for sustainable biomanufacturing, but inhibitors generated during pretreatment and saccharification severely limit microbial growth and productivity. Among various strategies to overcome this, we proposed a novel strategy to control a morphology-related transcriptional regulator and compared the wild-type Cupriavidus necator H16 with its mraZ deletion mutant H16 ΔmraZ, in which mraZ functions as a transcriptional regulator influencing cell size, nutrient utilization, and polyhydroxybutyrate (PHB) synthesis under lignocellulose-derived inhibitors such as furfural, vanillin, acetate, and formate. The H16 ΔmraZ strain mostly exhibited higher growth and PHB production than the wild type across all tested inhibitors. Scanning electron microscopy (SEM) revealed that ΔmraZ maintained cell morphology and length after furfural treatment, whereas the wild type displayed significantly decreased cell size. Consistent with these observations, viability and IC50 analyses demonstrated a 3.5-fold increase in viability and a 1.7-fold increase in IC50 in H16 ΔmraZ. When the xylA and xylB genes from Bacillus subtilis 168 were introduced into both H16 and H16 ΔmraZ for cultivation with barley straw- and pine-derived hydrolysates, H16 ΔmraZ showed 1.18-fold higher biomass accumulation and 1.41-fold higher PHB synthesis than the wild type. H16 ΔmraZ showed a higher cyclopropane index in phospholipid fatty acid analysis and increased cfa and H16_A0706 (groEL) expression under furfural stress, suggesting that membrane fatty acid remodeling and chaperone-associated stress responses contributed to improved tolerance. These findings indicate that deletion of the transcriptional regulator mraZ is an effective strategy to enhance stress tolerance and improve bioproduction.
Bacteriophytochromes (BphPs) are a class of widely distributed biliverdin (BV) bound bacterial photoreceptor proteins that can respond to red or far-red light. BphPs have been employed as photoresponsive module in red/far-red light control of physiological processes, and the photoresponsive properties of BphPs can be modulated through various engineering strategies, such as addition, deletion and substitution. Therefore, BphPs have been the focus of a variety of researches. Recently, progress has been made in both the engineering of BphPs and their subsequent applications across diverse biomedical fields, paving the way for their use as versatile biotechnological tools. However, there has not yet been a comprehensive review that systematically summarizes the recent advancements of engineering and diverse biomedical applications of BphPs. Here, the structural characteristics and photochemical properties of BphPs, the advancements of engineering approaches and various biomedical applications of BphPs, the comparative analyses of the engineering and applications of phytochromes from bacteria and plants, as well as the research challenges and future research directions of this field are systematically summarized and analyzed, aiming to provide a basis for further in-depth research and the expansion of the application fields of BphPs.
Human lactoferrin (hLF) is a multifunctional glycoprotein of the transferrin family derived from milk and mucosal secretions, which exhibits antibacterial, anti-tumor, and immunomodulatory functions, and is an important component of infant formula. Conventional methods for lactoferrin expression are often inefficient, primarily due to inadequate protein synthesis capabilities and poor stability within microbial hosts. Herein, a Komagataella phaffii yeast strain capable of high-level secretory expression of hLF was constructed by reprogramming the endoplasmic reticulum (ER) and vacuole using CRISPR/Cas9 technology. A dual-expression cassette containing the AOX1 promoter, an α-secretion signal peptide, the hLF gene, and a terminator was integrated into three different sites of the K. phaffii genome. The stepwise strategy combining expansion of the ER membrane involved in protein synthesis with knockout of vacuolar proteases further enhanced hLF production. Subsequently, 0.1 g/L FeCl₃ was added to the medium to reduce the toxicity of hLF and improve its stability. After high-density cultivation of K. phaffii through optimization of cultivation conditions in shake flasks and a 5 L bioreactor, the secretory intact hLF titer reached 2214 mg/L, representing a 76.3-fold increase achieved through these engineering strategies. In addition, antibacterial experiments demonstrated that this secretory hLF had a significant inhibitory effect on Escherichia coli, Staphylococcus aureus, and yeast. Overall, the developed K. phaffii protein expression platform enabled efficient production of lactoferrin, demonstrating its potential for expressing other lactoproteins.
Pentostatin is a potent adenosine deaminase inhibitor, yet its industrial application is hindered by low extraction yields and complex chemical synthesis. Here, we report an efficient de novo biosynthesis platform for pentostatin in Saccharomyces cerevisiae. Starting with the heterologous expression of cns3 from Cordyceps militaris, we optimized the cell factory via promoter engineering, multicopy integration, and AAH1 knockout. This integration strain achieved a maximum pentostatin titer of 16.28 mg/L in shake-flask cultivation, representing a 19.38-fold improvement over our initial production. Separately, to alleviate severe product toxicity, we implemented flux balance analysis (FBA)-guided transporter engineering; the engineered strain expressing the episomal efflux pump Cns4 yielded a titer of 8.27 mg/L while significantly accelerating the production process. Molecular docking revealed a distinct binding cavity where key residues (e.g., Asp296, Ala292) capture pentostatin via specific hydrogen bonds and hydrophobic interactions. Furthermore, transcriptomics demonstrated that Cns4 globally reprograms carbon and energy metabolism to boost precursor supply and cellular robustness. This work integrates structural insights with systems metabolic engineering, providing a generalizable paradigm for biosynthesizing toxic nucleoside natural products.
The long maturation period and strong environmental reliance of Tuber borchii's fruiting bodies limit its commercial and medicinal utilization, leading to unstable yield and limited supply of bioactive compounds. Developing a controlled cultivation strategy for consistent production of truffle-derived metabolites, therefore, remains a critical challenge. In this study, grain-based solid-state fermentation was investigated as an alternative platform for mycelial biomass generation and triterpenoid production under controlled conditions. During the main solid-state cultivation phase, adlay was identified as the most effective substrate, yielding the highest biomass (45.32 ± 1.58 mg/g substrate) and triterpenoid content (1.00 ± 0.13 mg/g substrate) on day 28. Optimization of fermentation parameters was conducted by evaluating initial pH, moisture content, and inoculum age. The optimal conditions were found to be pH 7, 50% moisture content, and an inoculum age of 21 days, balancing both mycelial growth and triterpenoid accumulation. Regarding nutrient supplementation, 3% (w/v) glucose was identified as the most effective carbon source, resulting in the highest biomass and triterpenoid yield per gram of substrate (1.37 ± 0.20 mg/g substrate), while 1% (w/v) yeast extract proved to be the most effective nitrogen source, yielding 88.42 ± 1.77 mg/g substrate mycelial biomass and 1.91 ± 0.12 mg/g substrate triterpenoid content. Furthermore, co-culturing with 1 g Quercus glauca somatic embryos increased the mycelial biomass and triterpenoid content to 100.72 ± 1.98 mg/g substrate and 2.39 ± 0.14 mg/g substrate, respectively. Overall, the study demonstrated the potential of solid-state fermentation as a sustainable method for the production of T. borchii mycelia and triterpenoids under controlled conditions.
Thermoresponsive elastin-like polypeptide (ELP) assemblies are promising platforms for functional nanoparticles, but the elevated temperatures required for self-assembly can inactivate heat-labile proteins of interest (POIs). To address this issue, we introduced a genetically encoded SnoopTag/SnoopCatcher cyclization module into an ELP-poly(aspartic acid) (ELP-D) scaffold. Using Renilla luciferase (RLuc) as a model POI, tail-to-side-chain cyclization increased both melting temperature and half-inactivation temperature by ∼6°C, indicating enhanced thermal stability. This stabilization was preserved upon fusion to ELP-D without affecting nanoparticle size or morphology. ELP-D-cRLuc nanoparticles retained ∼70% activity after heating to 42°C, compared with ∼30% for non-cyclized constructs. These results demonstrate that genetically encoded cyclization effectively protects POIs during heat-triggered nanoparticle assembly and enables activity retention at elevated temperatures.
