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PubMed2025

Breeding of yeast strains with intracellular amino acid accumulation for value-added alcoholic beverages.

The yeast Saccharomyces cerevisiae converts amino acids into volatile compounds with fruity and floral aromas during fermentation. These amino acid-derived aroma compounds play a critical role in defining the taste and flavor of alcoholic beverages such as sake, beer, and wine. The productivity of amino acid-derived aroma compounds depends on the intracellular availability of their precursor amino acids. Therefore, breeding yeast strains that accumulate amino acids provides a practical approach to developing alcoholic beverages with more unique and attractive sensory characteristics. In this minireview, we describe the isolation of yeast strains that overproduce branched-chain amino acids and phenylalanine, obtained through conventional mutagenesis of industrial brewing yeasts. We also discuss the mechanisms responsible for the increased production of these amino acids in the mutant strains, including altered feedback regulation and transcriptional control of key enzymes involved in their biosynthesis. In addition, we briefly introduce a plasmid-free genome editing system that enables precise modification of metabolic pathways without the integration of foreign DNA, allowing the construction of strains that are not classified as genetically modified organisms. This method represents a promising tool that allows flexible and fine-tuned engineering of yeast metabolic pathways, including the development of strains with tailored aroma profiles.

Saccharomyces cerevisiae↗
PubMed1979

Isolation of UV-sensitive clones from a haploid frog cell line.

An isolation procedure has been developed which yielded five clones of haploid frog cells which are sensitive to ultraviolet light. This procedure employed a conventional mutagenesis, followed by time for phenotypic expression and then an enrichment for UV-sensitive mutants. The enrichment relies upon the uptake of bromodeoxyuridine (BrdU) by repairing cells following UV-induced damage, rendering repair-proficient cells differentially sensitive to photolysis by black light. The photolysis is potentiated by use of the bisbenzimidazole dye Hoechst 33258. The enriched population was screened for radiation-sensitive isolates resulting in 5 sensitives from 96 tested. No mutants were obtained from 300 isolates tested from a population which had not undergone enrichment.

Animals↗
PubMed2026

Innovations in microbial physical mutagenesis for food fermentation: An overview from traditional to emerging technologies.

Microbial strains serve as an important factor affecting fermentation efficiency and product quality. To obtain superior strains, mutation breeding is a classic strategy. Compared to chemical mutagenesis, physical mutagenesis directly induces genomic changes, providing notable advantages such as the elimination of chemical residues and environmental sustainability, hence rendering it a favored method for enhancing food-grade microorganisms. Conventional physical mutagenesis mostly depends on UV, rays, high pressure, or space radiation. As physical technologies advance, emerging methods such as ion implantation, plasma, microwave, ultrasound, and pulsed light are widely utilized for genetic modification. Mutagenesis technologies are progressively transitioning from single-effect to multi-effect synergy. Recent evaluations indicate that emerging technologies can enhance microbial mutation efficiency at the application level relative to established technologies. Nonetheless, the systematic clarification and comparative analysis at the mechanistic level remain inadequate, hindering intuitive comprehension of the qualities and distinctions across techniques. Furthermore, physical mutagenesis encounters several significant obstacles, such as cellular damage, limited rates of advantageous mutations, and laborious screening processes. This review carefully elucidates the mechanisms and properties of physical mutagenesis technology and delineates the distinctions among approaches through comparative analysis. Simultaneously, solutions for optimizing mutagenesis are presented to tackle the principal challenges mentioned above. This review aims to offer a theoretical foundation and practical guidance for the enhanced application of physical mutagenesis technologies in microbial breeding.

Mutagenesis↗
PubMed2026

Yeast Strain Development and Process Intensification in High-Gravity Fermentation.

High- and very-high-gravity (HG/VHG) fermentation increases substrate loading and product titers, thereby improving fermenter utilisation and potentially reducing water use and downstream processing requirements. Initially developed for brewing and fuel ethanol production, these approaches are now applied more broadly in food, beverage, and bioproduct manufacturing. This MiniReview summarises operational definitions and industrial drivers of HG/VHG fermentation and examines the associated constraints in rheology, mass and heat transfer, osmotic and ethanol stress, nutrient availability, and oxidative damage. Yeast improvement strategies are reviewed, including adaptive laboratory evolution, mutagenesis, genome shuffling, multiplex genome editing, non-conventional yeasts, and multi-omics-guided selection. Process developments such as no-cook simultaneous liquefaction, saccharification and fermentation (SLSF), enzyme formulation, nutrient management, and in situ product recovery are considered together with applications in alcoholic beverages, organic acids, microbial lipids, and other value-added products. The review also discusses coproduct valorisation and the need to integrate strain development with process design. Current evidence supports HG/VHG fermentation as a useful process-intensification platform, although performance and sustainability depend strongly on feedstock, operating conditions, product requirements, and the basis used to report fermentation outcomes.

circular bioeconomy↗
PubMed2026

Single-cell vector copy number analysis of phenotypically defined long-term hematopoietic stem cells for gene therapy safety assessment.

Hematopoietic stem cell (HSC)-based gene therapy has emerged as a transformative approach for the treatment of genetic diseases; however, accurate evaluation of vector copy number (VCN) remains critical for ensuring safety. Conventional bulk VCN assays, including quantitative PCR (qPCR) and droplet digital PCR (ddPCR), do not resolve clonal heterogeneity and cannot identify rare high-VCN cells that may contribute disproportionately to insertional mutagenesis risk. Here, we developed an accessible single-cell VCN profiling method by combining fluorescence-activated cell sorting (FACS) of phenotypically defined long-term HSCs (Lineage- CD34+ CD38- CD90+ CD45RA- cells) with whole-genome amplification followed by conventional qPCR. This approach enabled resolution of VCN distributions at single-cell level using standard laboratory techniques. Notably, single-cell analysis revealed a high VCN tail that bulk VCN analysis could not resolve. Furthermore, in a humanized mouse transplantation model, single-cell VCN profiling demonstrated that overall VCN distributions could be analyzed after engraftment, although inter-donor and inter-mouse variability was observed. Collectively, this method provides a rapid, cost-effective, and phenotypically resolved strategy for assessing VCN heterogeneity in gene-modified HSCs. Single-cell VCN profiling offers complementary insights beyond conventional bulk assays and may enhance preclinical safety evaluation of gene and cell therapy products.

lentiviral vector↗
PubMed1978

Relationship between somatic mutation and neoplastic transformation.

Somatic mutation and neoplastic transformation of diploid Syrian hamster embryo cells were examined concomitantly. Mutations induced by benzo[a]pyrene and N-methyl-N'-nitro-N-nitrosoguanidine were quantitated at the hypoxanthine phosphoribosyltransferase and Na(+)/K(+) ATPase loci and compared to phenotypic transformations measured by changes in cellular morphology and colony formation in agar. Both cellular transformations had characteristics distinct from the somatic mutations observed at the two loci. Morphological transformation was observed after a time comparable to that of somatic mutation but at a frequency that was 25- to 540-fold higher. Transformants capable of colony formation in agar were detected at a frequency of 10(-5)-10(-6), but not until 32-75 population doublings after carcinogen treatment. Although this frequency of transformation is comparable to that of somatic mutation, the detection time required is much longer than the optimal expression time of conventionally studied somatic mutations. Neoplastic transformation of hamster embryo cells has been described as a multistep, progressive process. Various phenotypic transformations of cells after carcinogen treatment may represent different stages in this progressive transformation. The results are discussed in this context and the role of mutagenesis in the transition between various stages is considered. Neoplastic transformation may be initiated by a mutational change, but it cannot be described completely by a single gene mutational event involving a dominant, codominant, or X-linked recessive locus. Neoplastic transformation induced by chemical carcinogens is more complex than a single gene mutational process. Thus, this comparative study does not give experimental support to predictions of the carcinogenic potential of chemicals based on a simple extrapolation of the results obtained from conventional somatic mutation assays.

Adenosine Triphosphatases↗
PubMed2025

Comprehensive profiling of activity and specificity of RNA-guided transposons reveals opportunities to engineer improved variants.

Recently discovered CRISPR-associated transposons (CASTs) are natural RNA-guided DNA transposition systems capable of single-step genomic integration of large DNA cargo. Wild-type CASTs exhibit low integration activity in heterologous systems; therefore, engineering efforts are required to develop therapeutically relevant tools. Here we developed a high-throughput dual genetic screen capable of accurately quantifying the relative activity and specificity of a large pool of CAST variants. Under the conditions of our screen, we discovered that the wild-type V-K CAST system can consistently achieve between 88% and 95% on-site targeting specificity. We used site-saturation mutagenesis of the conserved core transposition machinery (TnsB, TnsC, and TniQ) to reveal novel mechanistic insights into the function of these transposon proteins. Furthermore, we found that different components have varying trade-offs between activity and specificity, a critical aspect overlooked in conventional screening pipelines. These findings provide clear engineering principles for further optimization of CASTs. Finally, we identified several mutations that, together, enhance CAST activity up to four-fold while minimally impacting targeting specificity. These methods are a powerful tool to characterize the sequence-function landscape across multiple functional parameters while also providing a robust platform for developing enhanced genome-editing tools.

DNA Transposable Elements↗
PubMed2026

Narasin used as a feed additive in conventional rearing of broilers can co-select for vancomycin-resistant Enterococcus faecium through the NarAB ionophore resistance mechanisms.

OBJECTIVES: To investigate the role of the NarAB resistance mechanism in the selection of vancomycin-resistant Enterococcus faecium (VREfm) and assess the impact of ionophore feed additives, particularly narasin, on the emergence of VREfm in broiler chickens. MATERIALS AND METHODS: Three isogenic E. faecium strains with different antimicrobial resistance determinants were created by mutagenesis and conjugation and used in a controlled animal experiment. Ross 308 broiler chickens were inoculated with either a rifampicin-resistant, a rifampicin- and vancomycin-resistant or a rifampicin-, vancomycin- and narasin-resistant strain and fed diets supplemented with selected ionophores. Bacterial populations were analysed on selective Slanetz and Bartley agar to determine the presence and selection of VREfm and other vancomycin-resistant species. Bacterial inoculation strains and isolates were whole genome sequenced for species identification and to identify genetic resistance mechanisms. RESULTS: Narasin was shown to select for VREfm in broilers, with NarAB being essential for co-selection. Intrinsically vancomycin-resistant Pediococcus acidilactici and Enterococcus gallinarum were identified as part of the broilers' vancomycin-resistant resident microbiota. Notably, among the P. acidilactici isolates that were susceptibility tested, strains resistant to both vancomycin and narasin were only found in broilers fed narasin, supporting that narasin promotes the growth of narasin-resistant populations. CONCLUSION: Narasin use in broiler feed can co-select for vancomycin-resistant bacteria, including VREfm, through the NarAB mechanism. These findings emphasize the concerns associated with the use of particular ionophores in poultry and suggest that vancomycin and narasin resistance may be more widespread in the broiler microbiota than previously recognized. Further research is needed to understand the implications for antimicrobial resistance and human health.

Animals↗
PubMed1977

Mutagenesis by 4-nitrobenzofurazans and furoxans.

15 nitrobenzofurazans and 10 nitrobenzofuroxans synthesized primarily for testing as potential anti-rheumatic drugs were also tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100. The method used involved placing each compound in a "well" cut out of a plate of selective medium previously seeded with the appropriate tester strain, and then adding a rat liver microsome/cofactor mixture to one of the two wells on each plate. This method is considerably cheaper and more convenient than the conventional agar overlay technique, but in the present series of experiments failed to detect 4 compounds which could be detected by the overlay technique. Using a combination of the two techniques, 21 of the 25 compounds tested were found to be mutagenic. All 10 benzofuroxans and 9 of the 15 benzofurazans were detected as direct-acting mutagens with at least one of the two tester strains. Two of the benzofurazans gave positive results only in the presence of rat liver microsomes, and hence are pro-mutagens. One of the 4 benzofurazans which gave negative results for mutagenicity in these tests was found to be an efficient inhibitor of a neutral protease activity found in rheumatic synovial fluid, and may therefore have some potential as an anti-rheumatic drug.

Animals↗
PubMed1978

[Practical aspects of mutagenesis--today and later].

The struggle of medicine and biology with the phenomenon mutation is discussed from a philosophical (C. BRESCH: "Zwischenstufe Leben") and a practical point of view. In the beginning age of the intellectual evolution mutations have become a pruely negative relict of the declining phase of the biological evolution. Effective means of medical genetics suitable in reducing the number of manifest mutants are: prenatal genetic diagnoses in as many pregnancies as possible; protection from exogenous mutagenic sources such as chemical mutagens and consequent protection from ionizing radiations, especially in the field of medicine. Possibly also effective is conventional genetic counselling which, however, should be supported for purely humanitarian reasons. The author denies the necessity for extraordinary efforts in the field of "genetic engineering" for various reasons, without casting doubt on the purely scientific aspects of molecular genetics.

Biological Evolution↗
PubMed2026

Protocol for Duplex Sequencing of Mitochondrial DNA in Single Human Oocytes.

Oocytes are densely packed with mitochondria, the energy-producing organelles that contain their own genome, mitochondrial DNA (mtDNA). Each cell contains multiple copies of mtDNA, with copy number varying among tissue types. Oocytes possess the highest mtDNA copy number, containing hundreds of thousands of mtDNA molecules per cell. Because mitochondria are inherited exclusively through the maternal lineage, accurate detection of mtDNA variants is essential for studies of inheritance, aging, and disease. The presence of multiple mtDNA copies allows wild-type and mutant molecules to coexist within the same cell, a condition known as heteroplasmy, in which low-frequency and de novo variants may occur at frequencies below 1%. Conventional next-generation sequencing (NGS) lacks sufficient accuracy to reliably distinguish these rare variants from errors introduced during library preparation and sequencing. Here, we present a protocol for enriching mtDNA from single human oocytes using Exonuclease V to remove linear DNA, followed by duplex sequencing library preparation for highly accurate mtDNA analysis. This workflow enables error-corrected sequencing of individual oocytes, facilitating reliable detection of low-frequency mtDNA variants and analysis of heteroplasmy and de novo mutagenesis. The protocol provides a reproducible approach for investigating mitochondrial genome variation in single oocytes using Illumina-compatible sequencing platforms.

Humans↗
PubMed2026

Selective Inhibition of DNA Polymerase Proofreading: A Metabolic-Fidelity Mechanism Explains Agent Orange-Associated Myelodysplasia.

We performed a focused review to better understand the pathogenesis of Agent Orange (AO)-associated myelodysplastic syndrome (MDS). We first examined the mechanisms underlying conventional (de novo) MDS, a clonal hematopoietic neoplasm that typically develops in later life, and integrated these findings with our recent analysis of obesity-associated carcinogenesis. Accordingly, we propose that genomic instability in de novo MDS results from selective inhibition of the DNA polymerase proofreading exonuclease. In obesity-associated carcinogenesis, impaired AMP-activated protein kinase (AMPK) activity disrupts mitochondrial ATP production, increasing intracellular AMP concentrations. Elevated AMP selectively inhibits the proofreading exonuclease while preserving polymerase activity, allowing replication errors to escape correction and become fixed as somatic mutations. Molecular studies demonstrate that AO-associated MDS exhibits essentially the same mutational profile as de novo disease despite arising after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in young, otherwise healthy military personnel. Because TCDD is highly lipophilic, it accumulates in adipose tissue and is released slowly over decades, producing sustained mitochondrial dysfunction, reduced ATP synthesis, and chronic elevation of intracellular AMP. We propose that this metabolic disturbance converges on the same endpoint-selective inhibition of the proofreading exonuclease-thereby promoting mutagenesis and clonal evolution. Recent studies further strengthen the central role of proofreading by demonstrating that many mutations, including many found in MDS, previously attributed to spontaneous cytosine deamination, instead arise from DNA polymerase misincorporation of thymidine opposite cytosine, particularly at CpG dinucleotides, emphasizing the critical importance of fully active proofreading in preventing such misincorporations from accumulating as mutations in the genome of the cell.

AMP↗
PubMed2026

Genetic Analysis of Genomic and Methylomic Variation and Identification of Multi-Trait Mutants in Rice Carried on Chang'e-5.

Global food security is facing challenges from population growth to diminishing arable land. Space mutation breeding holds promise for overcoming the variation limitations in conventional breeding; however, the mutagenic effects of the deep-space environment on rice and the transgenerational inheritance patterns of induced variations remain unclear. In this study, rice seeds carried by the Chang'e-5 spacecraft were used as materials. Whole-genome sequencing and whole-genome bisulfite sequencing were performed on the first (SP1) and second generations (SP2) of space-mutagenized plants after their return to Earth. The results showed that the number of genomic variants in the SP2 generation increased significantly compared with SP1, and SNPs, homozygous sites, and variants in coding regions were more heritable. The genome-wide methylation level was elevated in the SP2 generation, and among differentially methylated cytosines, those in the CG context exhibited the highest heritability. Furthermore, large-scale screening for nitrogen efficiency, tolerance to PEG-induced stress, and germination-stage cold resistant mutants was conducted in the SP2 generation, and phenotypic validation was performed in the third generation (SP3). By integrating multi-omics analyses of representative mutants to mine candidate genes, a number of heritable elite mutants were obtained, and seven candidate genes for key traits were identified. This study systematically elucidates the transgenerational inheritance patterns of deep-space-induced variation in rice. The multi-trait mutants obtained provide valuable germplasm resources for gene cloning and breeding applications in rice.

DNA methylation↗