Biotechnology Journal最新文献

Oxygen supply is critical for mammalian cell culture. Insufficient oxygen supply or oxygen inhomogeneity in large-scale bioreactors often leads to attenuated process performance, such as reduced cell growth, viability, and productivity. However, the understanding of how hypoxia affects process performance by modulating the underlying biological mechanisms of Chinese hamster ovary (CHO) cells remains limited. In this study, a comprehensive multi-omics analysis was performed to investigate the impact of hypoxia on intracellular biology. Systematic analysis of the multi-omics data revealed that moderate hypoxia suppressed mitochondrial respiration function and respiratory electron transport, while enhancing the activity of the glycolytic pathway. The reactive oxygen species (ROS) were also elevated under hypoxia. Correlation analysis revealed that the specific lactate production rate during the late-stage culture correlated significantly with the activity of the biosynthesis pathways for oxidized polyunsaturated fatty acid (PUFA) derivatives and cholesterol. This finding suggests that optimizing the levels of these compounds in the media might improve lactate metabolism and antibody production.

Emerging evidence suggests that disruptions in the gut microbiome may influence autism spectrum disorder (ASD) through altered microbial metabolism and gut-brain communication. However, the specific metabolic impacts of these microbial changes remain unclear. Community-scale metabolic modeling was applied to shotgun metagenomics data from children with ASD and neurotypical controls to predict secretion of host-impacting metabolites. Modeled ASD-associated communities exhibited altered predicted secretion of metabolites related to redox balance and neurotransmission, including increased 2-ketobutyrate and GABA and reduced riboflavin and inositol, with microbiota transfer therapy (MTT) shifting these profiles toward NT. Empirical fecal metabolomics data showed generally consistent directional trends with model predictions. Reductions in autism severity scores following MTT were associated with increased predicted secretion potentials for inositol and arginine. Taxonomic analysis revealed a depletion of beneficial and an enrichment of pro-inflammatory species, such as Escherichia and Flavonifractor, in ASD. Associations between microbial taxa (e.g., Bacteroides, Bifidobacterium) and neuroactive metabolites highlight microbial modulation as a promising therapeutic strategy in ASD. These results emphasize microbial metabolism as a contributor to ASD traits and a target for therapeutic intervention.

Targeted integration (TI) expression system has emerged as an alternative to random integration in cell line development (CLD) involved in biologics development and manufacturing. A key element of a robust TI CLD system is the construction of a high-performance TI host cell line, which significantly influences the productivity, product quality, and cell line stability of expressed biologics. In this study, we implemented a low-copy transfection strategy by optimizing transfection dosages to efficiently achieve over 30% single-copy integrants in a single transfection. This approach dramatically reduces TI host cell screening efforts, enabling rapid isolation of functional TI host cells. In addition, using mRNA transfection of Cre recombinase enabled faster pool recovery and eliminated concerns related to residual Cre coding sequence integration. Under non-optimized fed-batch conditions, this resulting TI system supported monoclonal antibody (mAb) titers of up to 8.03 g/L in 15-L bioreactors.

Esters are vital compounds with wide-ranging applications in food, fragrance, and pharmaceuticals, yet their sustainable supply increasingly relies on microbial biosynthesis. Acyltransferases (ATFs), particularly BAHD family members, catalyze the esterification of alcohols with acyl-CoA donors, a central step in generating structurally diverse esters in vivo. This review highlights recent progress in ATF-mediated microbial biosynthesis of three representative classes: short-chain fatty acid esters (SCFAEs), monoterpenyl esters (MTEs), and aromatic esters (AEs). Advances in enzyme discovery, protein engineering, and metabolic pathway reprogramming have expanded substrate scope, improved catalytic efficiency, and enabled titers from milligram to gram scale. Nonetheless, challenges remain in achieving high activity, selectivity, and stereochemical fidelity under industrial conditions. We summarize strategies including co-culture systems, non-natural precursor biosynthetic pathways, high-throughput screening, and rational enzyme engineering. Future perspectives emphasize developing a precision precursor supply for ATF-mediated esterification, elucidating ATF structure-function relationships, and optimizing downstream processing to achieve efficient, scalable, and sustainable microbial production of diverse natural esters.

Synechocystis sp., a prominent model organism among cyanobacteria, is renowned for its efficient CO₂ utilization to produce valuable metabolites, positioning it as a promising alternative to traditional heterotrophic microbes for sustainable bioproduction. Recent advances in Synechocystis-based cell factories have significantly improved synthetic biology design and the bioproduction of high-value biochemicals. In this review, we provide an updated bioinformatics overview of Synechocystis sp. and comprehensively summarize the latest advancements in its synthetic biology applications. Additionally, we highlight recent progress and challenges in scaling up and industrialization, while proposing future research directions, including the integration of omics strategies. These developments are expected to drive the application of cyanobacteria in industrial biotechnology and contribute to the advancement of sustainable, low-carbon, and high-efficiency bioproduction systems.

Heparosan, a glycosaminoglycan (GAG) derived from bacterial capsular polysaccharide (CPS), shares a similar backbone with both clinically utilized heparin and heparan sulfate (HS). This facilitates its transformation into heparin/HS via chemoenzymatic strategies. Currently, the purification process of heparosan, followed by N-deacetylation and N-sulfation, requires executing several complex steps prone to the unintended loss of polysaccharides and environmental risks. In this study, heparosan extracted from fermentation broth underwent immediate N-deacetylation and N-sulfation before DEAE chromatography purification. The results showed that the recovery of N-deacetylated heparosan increased from less than 40% to 93.6% with negligible contaminants. After sulfation, the overall recovery yield of N-sulfo heparosan was 76.0%. The number average molecular weight (M_n) and weight average molecular weight (M_W) of N-sulfo heparosan were ascertained to be 5.2 and 10.7, respectively, with a polydispersity index (PDI) value of 2.1. The assessment of elemental composition revealed that the efficiency of N-sulfation was 84%, which aligns with that of commercial heparin. The strategy delineated in this investigation avoids the substantial loss of N-deacetylated polysaccharides resulting from the complex procedures. Furthermore, the study avoids using harmful organic solvents in preparing heparosan, thereby promoting the in vitro green synthesis of heparin-like polysaccharides and analogous pharmaceutical compounds.

Virus filtration is a critical step to ensure the safety of antibody-based therapeutics. Due to the stringent performance requirements, commercially available virus filtration membranes are limited, and studies on virus retention behavior across different antibodies remain scarce, partly due to the high cost of these biologics. Herein, we comprehensively evaluated a newly developed virus filtration membrane, UF-Viremoval-Plus, by benchmarking it against leading commercial virus filters. The results demonstrate that UF-Viremoval-Plus exhibits superior antifouling properties and virus retention performance, which are attributed to its less negative charge, higher hydrophilicity, gradient pore structure and funnel-shaped geometry. We further investigated the effects of key process parameters, including pH, ionic strength, antibody type, and concentration, on membrane flux and virus removal efficiency. The membrane maintained stable operation under varying pressures and process disturbances, consistently achieving virus removal levels above 4 log reduction value, with no evidence of virus breakthrough. However, significant shifts in feed solution pH or ionic strength, as well as membrane fouling caused by high protein concentrations, affected virus removal. These observations are governed by complex membrane-protein-virus interactions. This work provides theoretical insights for the rational design of virus filtration membrane microstructures and the optimization of viral clearance processes in biopharmaceutical manufacturing.

Increasing evidence supports the human sodium-coupled citrate transporter (hNaCT) as a potential therapeutic target for metabolic syndrome and early infantile epileptic encephalopathy type 25 (EIEE25). While isotopic tracers remain the reference method for evaluating citrate transport, the need for specialized equipment and regulatory approval restricts their widespread use. Here, we report the development and validation of a robust, fluorescent-based assay to evaluate hNaCT-mediated citrate transport in live cells at both single-cell and high-throughput levels. This method utilizes a baculoviral vector to modify HEK293 cells to co-express a genetically encoded citrate sensor (Citron1) and the hNaCT. This cell-based platform enabled real-time monitoring of citrate transport using fluorescent microscopy and a standard multiwell plate reader. A key strength of this approach is its ability to assess citrate transport in the same cells before and after experimental interventions. Accordingly, this approach enables the functional characterization of hNaCT, including its pharmacological inhibitors and genetic variants with altered activity. Overall, the method provides a reliable assessment of citrate transport and offers a versatile platform suitable for identifying novel lead compounds for the therapeutic modulation of hNaCT.

Polyurethane (PU), as one of the most widely produced and consumed plastics globally, has led to severe environmental pollution and resource wastage due to the substantial amount of solid waste and microplastics it generates. Plastic recycling technologies that employ microorganisms and enzymes as catalysts not only enable efficient depolymerization of PU wastes into monomers while offering notable advantages, such as mild reaction conditions and avoidance of organic solvents. These features position enzymatic and microbial processes as a promising solution for achieving green and low-carbon end-of-life treatment of PU. However, PU biodegradation technologies are still in the early stages of fundamental research and face multiple challenges, including limited biocatalytic resources, low efficiency of enzymatic depolymerization, and difficulties in recovery and reuse. This review systematically summarizes the chemical structures of PU, recent advances in PU-degrading microbes and enzymes. It further discusses the biological metabolic pathways of depolymerized monomers, as well as current resource utilization strategies via closed-loop recycling and upcycling. The aim of this review is to provide theoretical guidance and novel insights into the efficient biodegradation and recovery of PU, thereby supporting the development of a circular plastic economy and contributing to global sustainability and carbon neutrality goals.