Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “biomanufacturing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

12 recordsLinked to original sources
PubMed2025

Engineering of xylose metabolic pathways in Rhodotorula toruloides for sustainable biomanufacturing.

The oleaginous yeast Rhodotorula toruloides is a promising microbial cell factory for the sustainable production of biofuels and value-added chemicals from renewable carbon sources. Unlike the conventional yeast Saccharomyces cerevisiae, R. toruloides can naturally metabolize xylose, the second most abundant sugar in lignocellulosic hydrolysates. However, its native xylose metabolism is inefficient, characterized by slow xylose uptake and accumulation of D-arabitol. Moreover, despite its phenotype, research on the enzymes involved in xylose metabolism has yet to reach a consensus. Therefore, this review provides a comprehensive analysis of the non-canonical xylose metabolism in R. toruloides, focusing on the properties of key enzymes involved in xylose metabolism. Native xylose reductase and xylitol dehydrogenase exhibit broad substrate promiscuity compared to their counterparts in the xylose-fermenting Scheffersomyces stipitis. Additionally, the absence of xylulokinase expression under xylose-utilizing conditions redirects metabolism toward D-arabitol accumulation. Consequently, D-arabitol dehydrogenases and ribulokinase play essential roles in the xylose metabolism of R. toruloides. These findings highlight the fundamental differences between R. toruloides xylose metabolism and the oxidoreductase pathways observed in other xylose-fermenting yeast, providing insights for metabolic engineering strategies to improve xylose utilization and enhance bioconversion of cellulosic hydrolysates to different bioproducts by R. toruloides.

Xylose↗
PubMed2026

Recombinant bovine chymosin expression in microalgae Chlamydomonas reinhardtii chloroplast: A step towards algal biomanufacturing of dairy enzymes.

Chymosin is the major proteolytic enzyme for cheese manufacture, where it plays an important role in the co-precipitation of milk casein. Traditional extraction of chymosin from the abomasum of young ruminants is associated with high limitations, including low yield, high production cost, and ethical issues of animal slaughter. In this study, we report on a recombinant strategy towards the production of bioactive Bos taurus chymosin in the chloroplasts of the microalga Chlamydomonas reinhardtii. The cym gene encoding preprocymosin was inserted into the chloroplast genome by the glass bead-mediated DNA transformation procedure. Successful integration and expression of the transgene were confirmed by spot test analysis, polymerase chain reaction (PCR), western blot, and enzyme-linked immunosorbent assay (ELISA). The functional activities of the recombinant enzyme were checked by the standard milk clotting assay. The engineered microalgal strains produced chymosin with an average concentration of 90 mg/kg fresh weight, i.e., 1.6% of the total soluble protein. These results show that chloroplast-engineered C. reinhardtii is a promising, sustainable, and animal-free platform for the efficient production of the industrially relevant chymosin.

Animals↗
PubMed2025

Comparison of stress tolerance mechanisms between Saccharomyces cerevisiae and the multistress-tolerant Pichia kudriavzevii.

Yeasts play a vital role in both research and industrial biomanufacturing. Saccharomyces cerevisiae has been extensively utilized as a model system. However, its application is often constrained by limited tolerance to the diverse stress conditions encountered in bioprocesses. These challenges have driven increasing interest in nonconventional, multistress-tolerant yeasts as alternative biomanufacturing hosts. This review highlights Pichia kudriavzevii as a promising nonconventional yeast for industrial applications. Unlike S. cerevisiae, P. kudriavzevii exhibits exceptional tolerance to high temperatures, elevated concentrations of furanic and phenolic inhibitors, osmotic stress, salinity, and extreme pH. These traits make it an attractive candidate for industrial processes without requiring extensive genetic modifications to enhance stress resistance. As a result, P. kudriavzevii has emerged as a flagship species for advancing bioeconomy. Despite its industrial potential, the molecular mechanisms underlying P. kudriavzevii's superior stress tolerance remain poorly understood. This review compiles current knowledge on P. kudriavzevii and compares its stress tolerance mechanisms with those of S. cerevisiae, providing insights into its innate resilience. By expanding our understanding of nonconventional yeasts, this review aims to facilitate their broader adoption as robust microbial platforms for industrial biomanufacturing.

Saccharomyces cerevisiae↗
PubMed2026

A CHO-Derived Matrix Attachment Region Enhances Transgene Dosage, SATB1 Recruitment, and Monoclonal Antibody Expression in a Dual-Promoter Vector System.

The production of monoclonal antibodies (mAbs) in Chinese hamster ovary (CHO) cells is often affected by position-effect variegation and the gradual loss of transgene expression over time. Hence, we have designed a dual-promoter IgG expression vector and compared versions that either contained or lacked a CHO-derived matrix-attachment region (MAR). Stable CHO-S pools, cultured in serum-free conditions, revealed that the MAR-containing construct produced higher and more consistent antibody levels across ten passages, as confirmed by Western blot and Protein A Octet analysis. Product-quality analysis by size-exclusion chromatography and reducing SDS-PAGE confirmed formation of properly assembled, mainly monomeric antibodies in both cases. Quantitative PCR indicated greater transgene copy numbers in MAR pools (+ 48% for the light chain and + 71% for the heavy chain), and RT-qPCR showed roughly fourfold higher transcript levels for both chains relative to controls. Bioinformatic analysis revealed several SATB1 binding motifs within the MAR sequence, and ChIP-qPCR demonstrated SATB1 association with the MAR-linked transgene locus. Overall, the data suggested that a CHO-native MAR could enhance transgene dosage and transcriptional activity, while preserving product integrity, possibly through SATB1-mediated chromatin organization. Ongoing work includes chromatin-mark profiling and process-level productivity measurements to better define the impact of MAR-based vector design on biomanufacturing performance.

Animals↗
PubMed2026

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems↗
PubMed2026

Directing fratricide within T cell products using an anti-uPAR chimeric antigen receptor to drive the production of potent therapeutic cells.

Cell therapy manufacturing of primary T cells often results in heterogeneous cell populations in the final product, with many cells lacking desired receptor expression or exhausted and other dysfunctional phenotypes. Here, we design a novel cell-intrinsic strategy to genetically reprogram primary human T cells to autonomously detect and eliminate dysfunctional cells. This integrated detection and elimination process, known as directed fratricide, is programmed via non-viral CRISPR genome editing to eliminate the T cell receptor (TCR) alpha chain (TRAC gene knockout) and integrate a chimeric antigen receptor (CAR) against the urokinase-type plasminogen activator receptor (uPAR), also known as CD87. In these cell products, strong T cell stimulation or activation during manufacturing causes a small subset of cells to express uPAR, which triggers CAR-mediated killing within the product. This fratricide induces proliferation in the desired cells and destroys undesired cells, a process that could be modeled computationally and controlled robustly via supplements to the culture media. The strategy enabled enrichment of anti-uPAR and anti-disialoganglioside (GD2) CAR-T cell products up to ≥99% CAR+/TCR-, favoring a memory-like phenotype. Understanding growth dynamics among T cell subsets and reprogramming them via CRISPR could accelerate the biomanufacturing of potent cell products without extensive selection methods.

Humans↗
PubMed2026

Identification of a Novel Thermal Promoter and Its Application in Glutamate Decarboxylase Protein Expression in Bacillus licheniformis.

As a thermotolerant bacterium, Bacillus licheniformis is an attractive chassis for high-temperature biomanufacturing. Here, we identified a novel temperature-responsive promoter, PycgM, which maintained strong transcriptional activity at 37-52 °C. In a promoter-mCherry reporter system, PycgM exhibited 2287.3-fold higher activity than P2 at 52 °C, demonstrating excellent compatibility with a thermotolerant host. Truncation analysis identified a 150-bp core functional region responsible for optimal activity under induction and heat stress. When applied to drive glutamate decarboxylase expression at 50 °C, PycgM enabled γ-aminobutyric acid production of 391.67 g/L with a 98.69% conversion rate, representing a 275% increase over 37 °C fermentation. The whole-cell biocatalyst retained 86% activity after five reuse cycles, and SEM analysis indicated acceptable structural stability despite moderate morphological changes. These results demonstrate that PycgM is a robust, high-temperature genetic element for efficient enzyme and metabolite production in thermotolerant hosts.

Bacillus licheniformis↗
PubMed2026

A sequence motif enables widespread use of noncanonical redox cofactors in natural enzymes.

Noncanonical redox cofactors (NRCs) are low-cost alternatives to the natural redox cofactors nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) for biomanufacturing, offering exquisite electron-delivery control, yet their adoption is limited by the scarcity of compatible enzymes. Screening the aldehyde dehydrogenase (ALDH) family, we identified a conserved RH/QxxR motif that enables widespread NRC activity among natural enzymes. Bos taurus ALDH3a1 exhibits unprecedented turnover with nicotinamide mononucleotide (NMN+), with kcat values exceeding NAD+ and surpassing most engineered NRC-active enzymes by 10-105-fold. Structural analyses reveal that this motif reinforces cofactor positioning and preorganizes the active site independently of the NAD+ adenosine monophosphate moiety. This motif supports activity across simple-synthetic NRCs such as 1-(2-carbamoylmethyl)nicotinamide and, when introduced into diverse ALDH scaffolds, enhances NMN+ activity up to 60-fold. These findings elucidate nature's solution to engineering NRC-active enzymes and offer a blueprint to mine latent evolutionary plasticity in natural enzymes that serve as superior engineering starting points.

Journal Article↗
PubMed2025

A Sequence Motif Enables Widespread Use of Non-Canonical Redox Cofactors in Natural Enzymes.

Non-canonical redox cofactors (NRCs) are promising alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) for biomanufacturing due to low cost and exquisite electron delivery control, yet their adoption is limited by the scarcity of compatible enzymes. Here, we screened the aldehyde dehydrogenase (ALDH) protein family and identified a conserved RH/QxxR sequence motif that enables widespread NRC activity among natural enzymes. Bos taurus ALDH3a1 and Pseudanabaena biceps ALDH exhibit unprecedented turnover with nicotinamide mononucleotide (NMN+), with kcat values matching or exceeding that of NAD+ and surpassing most engineered NRC-active enzymes by 10 to 105-fold, based on the relative NRC to native activity. Structural and dynamic analyses reveal this motif reinforces cofactor positioning and pre-organizes the active site without dependence on the adenosine monophosphate moiety of NAD+. When introduced into diverse ALDH scaffolds, the RH/QxxR motif enhances NMN+ activity up to 60-fold. In addition to NMN+, this motif also supports activity across multiple non-nucleotide, simple synthetic NRCs such as 1-(2-carbamoylmethyl)nicotinamide (AmNA+). These findings elucidate Nature's solution to the engineering challenge of obtaining NRC-active enzymes and offers a blueprint to mine latent evolutionary plasticity in natural enzymes that serve as superior engineering starting points.

Active site pre-organization↗
PubMed2026

Rewiring Carbon Metabolism in Bacillus methanolicus via Heterologous Phosphoketolase Expression Enhances Biomass Yield From Methanol and Reduces CO2 Loss.

Methylotrophic microbes are attractive alternatives to traditional heterotrophic production platforms, yet their efficiency is constrained by carbon loss through pyruvate decarboxylation and the oxidative branch of the RuMP cycle. The phosphoketolase (PKT) pathway provides a carbon-conserving alternative by cleaving fructose-6-phosphate and/or xylulose-5-phosphate into acetyl-phosphate, which can subsequently be converted to acetyl-coA without pyruvate decarboxylation. The remaining carbon intermediates are recycled through central metabolism to regenerate RuMP cycle intermediates without direct CO2 release. Here, we engineered this strategy in Bacillus methanolicus, a thermophilic methylotroph with strong industrial potential. We first established a versatile expression toolkit comprising inducible and constitutive promoters, benchmarked using an sfGFP reporter. Leveraging this system, we heterologously expressed the phosphoketolase B (pktB) gene from Methylotuvimicrobium buryatense 5GB1C which increased methanol-to-biomass yields by 18%-24% relative to controls and reduced biogenic CO2 production by 9%-12%. Chromosomal integration of pktB preserved these gains, demonstrating stability without reliance on plasmid-based expression. Together, these results show that PKT-driven metabolic rewiring enhances substrate yields in B. methanolicus and provides a scalable strategy to improve methylotrophic bioprocesses. This work expands the metabolic engineering toolbox for methylotrophs and highlights carbon-conserving pathway design as a key lever for advancing single carbon (C1) biomanufacturing.

Bacillus↗
PubMed2026

Improving recombinant protein productivity in CHO cells via multi-omics data integration.

Chinese hamster ovary (CHO) cells represent the dominant host system for the production of recombinant therapeutic proteins. In recent decades, extensive research has focused on process/media optimization and cell line engineering to improve both the productivity and quality of biopharmaceutical proteins produced in CHO cells. Nevertheless, the inherent complexity of biological pathways and the heterogeneous cellular responses to different environmental conditions have posed substantial challenges to traditional methodologies. Recent advances in omics technologies have enabled comprehensive characterization of CHO cell physiology, providing multidimensional molecular and phenotypic insights that facilitate the enhancement of recombinant protein production. This review first summarizes the methodologies and advances in CHO omics research, including genomics, transcriptomics, proteomics, metabolomics, and epigenomics. It then examines contemporary approaches to integrate and analyze multi-omics data in CHO cells. The review further elucidates how these multi-omics datasets can be strategically applied across various developmental stages, including cell line selection, genetic engineering, expression vector design, and bioprocess optimization. Finally, we explore the transformative potential of integrating multi-omics with artificial intelligence and discuss promising future research directions in CHO cell studies. These emerging paradigms offer novel opportunities for data-driven cell engineering and bioprocess optimization in CHO-based biomanufacturing.

Bioprocessing↗
PubMed2026

Engineering Bacillus Subtilis for Efficient Biosynthesis of Riboflavin: Current Knowledge and Future Perspectives.

Riboflavin is an essential water-soluble vitamin that serves as a precursor for the biosynthesis of the flavin cofactors FMN and FAD, which play pivotal roles in numerous redox and energy metabolism reactions. With the growing global demand for sustainable vitamin production, microbial fermentation has become an attractive alternative to chemical synthesis due to its environmental and economic advantages. Among microbial hosts, Bacillus subtilis has emerged as a leading cell factory for riboflavin production owing to its GRAS status, well-characterized genetics, and efficient protein secretion system. This review provides a comprehensive overview of recent advances in metabolic engineering strategies to enhance riboflavin biosynthesis in B. subtilis. Key topics include strengthening biosynthetic and precursor pathways, relieving feedback inhibition, balancing metabolic flux and cell growth, employing adaptive laboratory evolution, and utilizing omics-guided optimization and 13C metabolic flux analysis. Moreover, the integration of synthetic biology tools such as riboswitch engineering, regulatory element design, and high-throughput screening has significantly accelerated strain improvement. Despite remarkable progress, challenges remain in achieving precise regulatory control, optimizing multi-gene expression, and enhancing genome integration efficiency. Future research combining multi-omics data, synthetic regulatory design, and machine learning-driven predictive modeling is expected to further advance the development of intelligent B. subtilis cell factories. However, the practical implementation of these systems remains constrained by the metabolic burden of overproduction and the lack of universal regulatory models that can predict strain performance across varying industrial scales.

Bacillus subtilis↗