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Journal of Integrative Plant Biology Jul 2024Aporphine alkaloids have diverse pharmacological activities; however, our understanding of their biosynthesis is relatively limited. Previous studies have classified...
Aporphine alkaloids have diverse pharmacological activities; however, our understanding of their biosynthesis is relatively limited. Previous studies have classified aporphine alkaloids into two categories based on the configuration and number of substituents of the D-ring and have proposed preliminary biosynthetic pathways for each category. In this study, we identified two specific cytochrome P450 enzymes (CYP80G6 and CYP80Q5) with distinct activities toward (S)-configured and (R)-configured substrates from the herbaceous perennial vine Stephania tetrandra, shedding light on the biosynthetic mechanisms and stereochemical features of these two aporphine alkaloid categories. Additionally, we characterized two CYP719C enzymes (CYP719C3 and CYP719C4) that catalyzed the formation of the methylenedioxy bridge, an essential pharmacophoric group, on the A- and D-rings, respectively, of aporphine alkaloids. Leveraging the functional characterization of these crucial cytochrome P450 enzymes, we reconstructed the biosynthetic pathways for the two types of aporphine alkaloids in budding yeast (Saccharomyces cerevisiae) for the de novo production of compounds such as (R)-glaziovine, (S)-glaziovine, and magnoflorine. This study provides key insight into the biosynthesis of aporphine alkaloids and lays a foundation for producing these valuable compounds through synthetic biology.
PubMed: 38953746
DOI: 10.1111/jipb.13724 -
Food & Function Jul 2024Obesity is often accompanied by low-grade chronic inflammation and metabolic syndrome. It has been established that microbiota influences many physiological processes,...
Obesity is often accompanied by low-grade chronic inflammation and metabolic syndrome. It has been established that microbiota influences many physiological processes, including the development of obesity, and dysbiosis has been observed in obese individuals. In this study, we aimed to evaluate the impact of a new probiotic formulation, containing two probiotic strains and the bioactive compound octacosanol, on body weight, metabolic parameters, and concentrations of certain adipocytokines and appetite-regulating hormones in obese women. This double blind placebo-controlled supplementary intervention study included twenty-five women in the intervention group and twenty-three in the placebo group, and it lasted 12 weeks. Daily oral supplementation included 7 × 10 CFU of 299v (DSM9843), 5 × 10 CFU of var. (DBVPG6763), and 40 mg of octacosanol or placebo. Body weight, metabolic parameters, adipocytokines, and appetite-regulating hormones were assessed before (T0) and after the intervention (T1). After the intervention, significantly lower median concentrations of CRP ( = 0.005) and IL-6 ( = 0.012) were measured in the intervention group than the baseline, while the median concentrations of ghrelin ( = 0.026) and HDL-cholesterol ( = 0.03) were significantly increased. The intervention group had lower CRP levels ( = 0.023) and higher ghrelin levels ( = 0.006) than the placebo group. Significant changes in BMI between groups were not observed. In summary, although the new probiotic formulation showed beneficial effects on IL-6, CRP, HDL, and ghrelin levels, its potential effects on regulating triglyceride, insulin, and glucose levels require further studies before the novel dietary intervention could be considered a useful adjuvant therapy and an effective strategy for the management of obesity and obesity-associated comorbidities.
PubMed: 38953736
DOI: 10.1039/d4fo01269k -
MBio Jul 2024Phytopathogens secrete numerous molecules into the environment to establish a microbial niche and facilitate host infection. The phytopathogenic fungus which causes...
UNLABELLED
Phytopathogens secrete numerous molecules into the environment to establish a microbial niche and facilitate host infection. The phytopathogenic fungus which causes pear anthracnose, can colonize different plant tissues like leaves and fruits, which are occupied by a diversity of microbes. We speculate that this fungus produces antimicrobial effectors to outcompete host-associated competitive microorganisms. Herein, we identified two secreted ribonucleases, CfRibo1 and CfRibo2, from the secretome. The two ribonucleases both possess ribonuclease activity and showed cytotoxicity in without triggering immunity in an enzymatic activity-dependent manner. CfRibo1 and CfRibo2 recombinant proteins exhibited toxicity against , , and, importantly, the phyllosphere microorganisms isolated from the pear host. Among these isolated microbial strains, is a pathogenic bacterium causing pear soft rot. Strikingly, CfRibo1 and CfRibo2 were found to directly antagonize to facilitate infection. More importantly, CfRibo1 and CfRibo2 functioned as essential virulence factors of in the presence of host-associated microorganisms. Further analysis revealed these two ribonucleases are widely distributed in fungi and are undergoing purifying selection. Our results provide the first evidence of antimicrobial effectors in fungi and extend the functional diversity of fungal ribonucleases in plant-pest-environment interactions.
IMPORTANCE
is emerging as a devastating pathogenic fungus causing anthracnose in various crops in agriculture, and understanding how this fungus establishes successful infection is of great significance for anthracnose disease management. Fungi are known to produce secreted effectors as weapons to promote virulence. Considerable progress has been made in elucidating how effectors manipulate plant immunity; however, their importance in modulating environmental microbes is frequently neglected. The present study identified two secreted ribonucleases, CfRibo1 and CfRibo2, as antimicrobial effectors of . These two proteins both possess toxicity to pear phyllosphere microorganisms, and they efficiently antagonize competitive microbes to facilitate the infection of pear hosts. This study represents the first evidence of antimicrobial effectors in fungi, and we consider that CfRibo1 and CfRibo2 could be targeted for anthracnose disease management in diverse crops in the future.
PubMed: 38953357
DOI: 10.1128/mbio.01053-24 -
Microbiology Spectrum Jul 2024The genes, and , encode for 2-deoxyglucose-6-phosphate phosphatases. These enzymes of the haloacid dehalogenase superfamily are known to utilize the non-natural...
UNLABELLED
The genes, and , encode for 2-deoxyglucose-6-phosphate phosphatases. These enzymes of the haloacid dehalogenase superfamily are known to utilize the non-natural 2-deoxyglucose-6-phosphate as their substrate. However, their physiological substrate and hence their biological role remain elusive. In this study, we investigated their potential role as enzymes in biosynthesizing glycerol through an alternative pathway, which involves the dephosphorylation of dihydroxyacetone phosphate into dihydroxyacetone, as opposed to the classical pathway which utilizes glycerol 3-phosphate. Overexpression of or rescued the osmotic and ionic stress-sensitive phenotype of or mutants, both affected in the production of glycerol. While small amounts of glycerol were observed in the overexpression strains in the background, no glycerol was detected in the mutant background. This indicates that overexpression of the enzymes can rescue the osmosensitive phenotype of the mutant independent of glycerol production. We also did not observe a drop in glycerol levels in the as compared to the mutant, indicating that the Dog enzymes are not involved in glycerol biosynthesis. This indicates that Dog enzymes have a distinct substrate and their function within the cell remains undiscovered.
IMPORTANCE
Yeast stress tolerance is an important characteristic that is studied widely, not only regarding its fundamental insights but also for its applications within the biotechnological industry. Here, we investigated the function of two phosphatase encoding genes, and , which are induced as part of the general stress response pathway, but their natural substrate in the cells remains unclear. They are known to dephosphorylate the non-natural substrate 2-deoxyglucose-6-phosphate. Here, we show that overexpression of these genes overcomes the osmosensitive phenotype of mutants that are unable to produce glycerol. However, in these overexpression strains, very little glycerol is produced indicating that the Dog enzymes do not seem to be involved in a previously predicted alternative pathway for glycerol production. Our work shows that overexpression of the DOG genes may improve osmotic and ionic stress tolerance in yeast.
PubMed: 38953324
DOI: 10.1128/spectrum.00136-24 -
Frontiers in Microbiology 2024starter serves as the saccharifying and fermenting agent in the production of Cantonese soybean-flavor (Chi-flavor) , and the complex microbial communities determine...
starter serves as the saccharifying and fermenting agent in the production of Cantonese soybean-flavor (Chi-flavor) , and the complex microbial communities determine the flavor and quality of the product. Round-Koji-mechanical starter (produced by using an automated starter-making disk machine) is advantageous as it decreases operator influence, labor costs, and fermentation time, but the product quality is lower compared to traditional starter. Thus, two types of starters (traditional and Round-Koji-mechanical starter) from a Cantonese factory were compared in a metagenomic analysis to investigate the differences in microbial community composition and core microbes. The results showed that several core microbes related to carbohydrate metabolism, amino acid metabolism and lipid metabolism, were differentially enriched in the traditional starter. and were significantly positively correlated with the three key metabolic pathways. and were significantly positively correlated with two of the three key metabolic pathways. The results of this study provide a basis for understanding the differential core microbes in traditional and Round-Koji-mechanical starters of Chi-flavor , and they also provide guidance for improving Round-Koji-mechanical starter.
PubMed: 38952453
DOI: 10.3389/fmicb.2024.1390899 -
Nature Communications Jun 2024Co-immobilization of cells and enzymes is often essential for the cascade biocatalytic processes of industrial-scale feasibility but remains a vast challenge. Herein, we...
Co-immobilization of cells and enzymes is often essential for the cascade biocatalytic processes of industrial-scale feasibility but remains a vast challenge. Herein, we create a facile co-immobilization platform integrating enzymes and cells in covalent organic frameworks (COFs) to realize the highly efficient cascade of inulinase and E. coli for bioconversion of natural products. Enzymes can be uniformly immobilized in the COF armor, which coats on the cell surface to produce cascade biocatalysts with high efficiency, stability and recyclability. Furthermore, this one-pot in situ synthesis process facilitates a gram-scale fabrication of enzyme-cell biocatalysts, which can generate a continuous-flow device conversing inulin to D-allulose, achieving space-time yield of 161.28 g L d and high stability (remaining >90% initial catalytic efficiency after 7 days of continuous reaction). The created platform is applied for various cells (e.g., E. coli, Yeast) and enzymes, demonstrating excellent universality. This study paves a pathway to break the bottleneck of extra- and intracellular catalysis, creates a high-performance and customizable platform for enzyme-cell cascade biomanufacturing, and expands the scope of biocatalysis process intensification.
Topics: Biocatalysis; Escherichia coli; Enzymes, Immobilized; Glycoside Hydrolases; Cells, Immobilized; Metal-Organic Frameworks; Saccharomyces cerevisiae
PubMed: 38951487
DOI: 10.1038/s41467-024-49831-8 -
Current Genetics Jul 2024The ability to regulate the expression of genes is a central tool for the characterization of fungal genes. This is of particular interest to study genes required for...
The ability to regulate the expression of genes is a central tool for the characterization of fungal genes. This is of particular interest to study genes required for specific processes or the effect of genes expressed only under specific conditions. Saccharomycopsis species show a unique property of necrotrophic mycoparasitism that is activated upon starvation. Here we describe the use of the MET17 promoter of S. schoenii as a tool to regulate gene expression based on the availability of methionine. Conditional expression was tested using lacZ and GFP reporter genes. Gene expression could be strongly down-regulated by the addition of methionine or cysteine to the growth medium and upregulated by starvation for methionine. We used X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) to detect lacZ-expression in plate assays and ONPG (ortho-nitrophenyl-β-galactopyranoside) as a substrate for β-galactosidase in liquid-phase assays. For in vivo expression analyses we used fluorescence microscopy for the detection and localization of a MET17-driven histone H4-GFP reporter gene. With these assays we demonstrated the usefulness of the MET17 promoter to regulate expression of genes based on methionine availability. In silico analyses revealed similar promoter motifs as found in MET3 genes of Saccharomyces cerevisiae and Ashbya gossypii. This suggests a regulation of the MET17 promoter by CBF1 and MET31/MET32 in conjunction with the transcriptional activator MET4, which were also identified in the S. schoenii genome.
Topics: Promoter Regions, Genetic; Gene Expression Regulation, Fungal; Methionine; Genes, Reporter; Fungal Proteins; beta-Galactosidase; Green Fluorescent Proteins
PubMed: 38951203
DOI: 10.1007/s00294-024-01294-6 -
Archives of Microbiology Jul 2024Ionic liquids (ILs) are interesting chemical compounds that have a wide range of industrial and scientific applications. They have extraordinary properties, such as the...
Ionic liquids (ILs) are interesting chemical compounds that have a wide range of industrial and scientific applications. They have extraordinary properties, such as the tunability of many of their physical properties and, accordingly, their activities; and the ease of synthesis methods. Hence, they became important building blocks in catalysis, extraction, electrochemistry, analytics, biotechnology, etc. This study determined antifungal activities of various imidazolium-based ionic liquids against yeast Saccharomyces cerevisiae via minimum inhibitory concentration (MIC) estimation method. Increasing the length of the alkyl group attached to the imidazolium cation, enhanced the antifungal activity of the ILs, as well as their ability of the disruption of the cell membrane integrity. FTIR studies performed on the S. cerevisiae cells treated with the ILs revealed alterations in the biochemical composition of these cells. Interestingly, the alterations in fatty acid content occurred in parallel with the increase in the activity of the molecules upon the increase in the length of the attached alkyl group. This trend was confirmed by statistical analysis and machine learning methodology. The classification of antifungal activities based on FTIR spectra of S. cerevisiae cells yielded a prediction accuracy of 83%, indicating the pharmacy and medicine industries could benefit from machine learning methodology. Furthermore, synthesized ionic compounds exhibit significant potential for pharmaceutical and medical applications.
Topics: Saccharomyces cerevisiae; Ionic Liquids; Imidazoles; Antifungal Agents; Cell Membrane; Microbial Sensitivity Tests; Spectroscopy, Fourier Transform Infrared
PubMed: 38951200
DOI: 10.1007/s00203-024-04043-y -
PLoS Biology Jul 2024The fitness effects of new mutations determine key properties of evolutionary processes. Beneficial mutations drive evolution, yet selection is also shaped by the...
The fitness effects of new mutations determine key properties of evolutionary processes. Beneficial mutations drive evolution, yet selection is also shaped by the frequency of small-effect deleterious mutations, whose combined effect can burden otherwise adaptive lineages and alter evolutionary trajectories and outcomes in clonally evolving organisms such as viruses, microbes, and tumors. The small effect sizes of these important mutations have made accurate measurements of their rates difficult. In microbes, assessing the effect of mutations on growth can be especially instructive, as this complex phenotype is closely linked to fitness in clonally evolving organisms. Here, we perform high-throughput time-lapse microscopy on cells from mutation-accumulation strains to precisely infer the distribution of mutational effects on growth rate in the budding yeast, Saccharomyces cerevisiae. We show that mutational effects on growth rate are overwhelmingly negative, highly skewed towards very small effect sizes, and frequent enough to suggest that deleterious hitchhikers may impose a significant burden on evolving lineages. By using lines that accumulated mutations in either wild-type or slippage repair-defective backgrounds, we further disentangle the effects of 2 common types of mutations, single-nucleotide substitutions and simple sequence repeat indels, and show that they have distinct effects on yeast growth rate. Although the average effect of a simple sequence repeat mutation is very small (approximately 0.3%), many do alter growth rate, implying that this class of frequent mutations has an important evolutionary impact.
PubMed: 38950062
DOI: 10.1371/journal.pbio.3002698 -
MLife Jun 2024The silencing information regulator (SIR) complex contains up to four proteins, namely Sir1, Sir2, Sir3, and Sir4. While Sir2 encodes a NAD-dependent histone... (Review)
Review
The silencing information regulator (SIR) complex contains up to four proteins, namely Sir1, Sir2, Sir3, and Sir4. While Sir2 encodes a NAD-dependent histone deacetylase, other SIR proteins mainly function as structural and scaffold components through physical interaction with various proteins. The SIR complex displays different conformation and composition, including Sir2 homotrimer, Sir1-4 heterotetramer, Sir2-4 heterotrimer, and their derivatives, which recycle and relocate to different chromosomal regions. Major activities of the SIR complex are transcriptional silencing through chromosomal remodeling and modulation of DNA double-strand-break repair pathways. These activities allow the SIR complex to be involved in mating-type maintenance and switching, telomere and subtelomere gene silencing, promotion of nonhomologous end joining, and inhibition of homologous recombination, as well as control of cell aging. This review explores the potential link between epigenetic regulation and DNA damage response conferred by the SIR complex under various conditions aiming at understanding its roles in balancing cell survival and genomic stability in response to internal and environmental stresses. As core activities of the SIR complex are highly conserved in eukaryotes from yeast to humans, knowledge obtained in the yeast may apply to mammalian Sirtuin homologs and related diseases.
PubMed: 38948145
DOI: 10.1002/mlf2.12108