Production Of Secondary Metabolites Research Articles

Influence of Silver Nanoparticles on the Metabolites of Two Transgenic Soybean Varieties: An NMR-Based Metabolomics Approach.

This study investigated the effect of AgNPs and AgNO3, at concentrations equivalent, on the production of primary and secondary metabolites on transgenic soybean plants through an NMR-based metabolomics. The plants were cultivated in a germination chamber following three different treatments: T0 (addition of water), T1 (addition of AgNPs), and T2 (addition of AgNO3). Physiological characteristics, anatomical analyses through microscopic structures, and metabolic profile studies were carried out to establish the effect of abiotic stress on these parameters in soybean plants. Analysis of the 1H NMR spectra revealed the presence of amino acids, organic acids, sugars, and polyphenols. The metabolic profiles of plants with AgNP and AgNO3 were qualitatively similar to the metabolic profile of the control group, suggesting that the application of silver does not affect secondary metabolites. From the PCA, it was possible to differentiate the three treatments applied, mainly based on the content of fatty acids, pinitol, choline, and betaine.

Screening and genetic engineering of marine-derived Aspergillus terreus for high-efficient production of lovastatin

BackgroundLovastatin has widespread applications thanks to its multiple pharmacological effects. Fermentation by filamentous fungi represents the major way of lovastatin production. However, the current lovastatin productivity by fungal fermentation is limited and needs to be improved.ResultsIn this study, the lovastatin-producing strains of Aspergillus terreus from marine environment were screened, and their lovastatin productions were further improved by genetic engineering. Five strains of A. terreus were isolated from various marine environments. Their secondary metabolites were profiled by metabolomics analysis using Ultra Performance Liquid Chromatography–Mass spectrometry (UPLC–MS) with Global Natural Products Social Molecular Networking (GNPS), revealing that the production of secondary metabolites was variable among different strains. Remarkably, the strain of A. terreus MJ106 could principally biosynthesize the target drug lovastatin, which was confirmed by High Performance Liquid Chromatography (HPLC) and gene expression analysis. By one-factor experiment, lactose was found to be the best carbon source for A. terreus MJ106 to produce lovastatin. To improve the lovastatin titer in A. terreus MJ106, genetic engineering was applied to this strain. Firstly, a series of strong promoters was identified by transcriptomic and green fluorescent protein reporter analysis. Then, three selected strong promoters were used to overexpress the transcription factor gene lovE encoding the major transactivator for lov gene cluster expression. The results revealed that compared to A. terreus MJ106, all lovE over-expression mutants exhibited significantly more production of lovastatin and higher gene expression. One of them, LovE-b19, showed the highest lovastatin productivity at a titer of 1512 mg/L, which represents the highest production level reported in A. terreus.ConclusionOur data suggested that combination of strain screen and genetic engineering represents a powerful tool for improving the productivity of fungal secondary metabolites, which could be adopted for large-scale production of lovastatin in marine-derived A. terreus.

Integrated metabolomic and metagenomic strategies shed light on interactions among planting environments, rhizosphere microbiota, and metabolites of tobacco in Yunnan, China

Changes in climatic factors and rhizosphere microbiota led plants to adjust their metabolic strategies for survival under adverse environmental conditions. Changes in plant metabolites can mediate crop growth and development and interact with rhizosphere microbiota of the plant rhizosphere. To understand the interactions among environmental factors, rhizosphere microbiota, and metabolites of tobacco, a study was conducted by using integrated metagenomic and metabolomic strategies at four typical representative tobacco planting sites in Yunnan, China. The results showed that the agronomical and biochemical traits were significantly affected by temperature, precipitation (PREP), soil pH, and altitude. Correlation analyses revealed a significant positive correlation of temperature with length, width, and area of the leaf, while PREP correlated with plant height and effective leaf numbers. Furthermore, total sugar and reducing sugar contents of baked leaves were significantly higher, while the total nitrogen and total alkaloid levels were lower in tobacco leaves at site with low PREP. A total of 770 metabolites were detected with the highest number of different abundant metabolites (DMs) at Chuxiong (CX) with low PREP as compared to the other three sites, in which secondary metabolites were more abundant in both leaves and roots of tobacco. A total of 8,479 species, belonging to 2,094 genera with 420 individual bins (including 13 higher-quality bins) harboring 851,209 CDSs were detected. The phyla levels of microorganisms such as Euryarchaeota, Myxococcota, and Deinococcota were significantly enriched at the CX site, while Pseudomonadota was enriched at the high-temperature site with good PREP. The correlation analyses showed that the metabolic compounds in low-PREP site samples were positively correlated with Diaminobutyricimonas, Nissabacter, Alloactinosynnema, and Catellatospora and negatively correlated with Amniculibacterium, Nordella, Noviherbaspirillum, and Limnobacter, suggesting that the recruitment of Diaminobutyricimonas, Nissabacter, Alloactinosynnema, and Catellatospora in the rhizosphere induces the production and accumulation of secondary metabolites (SMs) (e.g., nitrogen compounds, terpenoids, and phenolics) for increasing drought tolerance with an unknown mechanism. The results of this study may promote the production and application of microbial fertilizers and agents such as Diaminobutyricimonas and Alloactinosynnema to assemble synthetic microbiota community or using their gene resources for better cultivation of tobacco as well as other crops in drought environments.

Isolation and Structure Determination of New Pyrones from Dictyostelium spp. Cellular Slime Molds Coincubated with Pseudomonas spp.

Cellular slime molds are excellent model organisms in the field of cell and developmental biology because of their simple developmental patterns. During our studies on the identification of bioactive molecules from secondary metabolites of cellular slime molds toward the development of novel pharmaceuticals, we revealed the structural diversity of secondary metabolites. Cellular slime molds grow by feeding on bacteria, such as Klebsiella aerogenes and Escherichia coli, without using medium components. Although changing the feeding bacteria is expected to affect dramatically the secondary metabolite production, the effect of the feeding bacteria on the production of secondary metabolites is not known. Herein, we report the isolation and structure elucidation of clavapyrone (1) from Dictyostelium clavatum, intermedipyrone (2) from D. magnum, and magnumiol (3) from D. intermedium. These compounds are not obtained from usual cultural conditions with Klebsiella aerogenes but obtained from coincubated conditions with Pseudomonas spp. The results demonstrate the diversity of the secondary metabolites of cellular slime molds and suggest that widening the range of feeding bacteria for cellular slime molds would increase their application potential in drug discovery.

Impact of silver nanoparticles on secondary metabolite composition and toxicity in anise (Pimpinella anisum L.) callus culture

BackgroundThere are numerous challenges associated with producing desired amounts of secondary metabolites (SMs), which are mostly unique and cannot be chemically synthesized. Many studies indicate that nanoparticles (NPs) can boost the production of SMs. Still, the precise manner in which NPs induce metabolic changes remains unidentified. This study examines the influence of eco-friendly silver NPs (AgNPs) on the chemical makeup and toxicity of Pimpinella anisum L. (anise).ResultsAgNPs were introduced into anise callus cultures at different concentrations (0, 1.0, 5.0, 10, and 20 mg/L). The induced oxidative stress was tracked over intervals of 7, 14, 28, and 35 days. Chemical composition evaluations were carried out on the 35th day. Within the first 14 days, plant stress was evident, though the plant adapted to the stress later on. Notably, the plant showed high tolerance at 1 mg/L and 5 mg/L concentrations despite increased toxicity levels. However, relatively high toxicity levels were identified at 10 and 20 mg/L. The AgNP-induced stress significantly impacted anise SMs, particularly affecting fatty acid content. In the 10 and 20 mg/L AgNP groups, essential metabolites, including palmitic and linoleic acid, showed a significant increase. Polyunsaturated (omega) and monounsaturated fatty acids, vital for the food and pharmaceutical industries, saw substantial growth in the 1 and 5 mg/L AgNP groups. For the first time, vanillyl alcohol and 4-Hydroxybenzoic acid were detected along with various phenolic compounds, such as t-anethole, Salicylic acid, and Thiamazole.ConclusionAgNPs can function as an elicitor to efficiently generate essential SMs such as omegas and phenolic compounds in anise callus culture. This study explores the application of AgNPs as plant elicitors in anise SM production, offering invaluable insight into potential uses.

Influence of Cold Stress on Physiological and Phytochemical Characteristics and Secondary Metabolite Accumulation in Microclones of Juglans regia L.

The current study investigated the impact of cold stress on the morphological, physiological, and phytochemical properties of Juglans regia L. (J. regia) using in vitro microclone cultures. The study revealed significant stress-induced changes in the production of secondary antioxidant metabolites. According to gas chromatography-mass spectrometry (GC-MS) analyses, the stress conditions profoundly altered the metabolism of J. regia microclones. Although the overall spectrum of metabolites was reduced, the production of key secondary antioxidant metabolites significantly increased. Notably, there was a sevenfold (7×) increase in juglone concentration. These findings are crucial for advancing walnut metabolomics and enhancing our understanding of plant responses to abiotic stress factors. Additionally, study results aid in identifying the role of individual metabolites in these processes, which is essential for developing strategies to improve plant resilience and tolerance to adverse conditions.

Protective Role of Plant Growth Promoting Rhizobacteria Inoculation in the Development of Drought Tolerance in Shallot: Effects on Hydroxygen Peroxide Production, Lipid Peroxidation, and Secondary Metabolite Production

Protective Role of Plant Growth Promoting Rhizobacteria Inoculation in the Development of Drought Tolerance in Shallot: Effects on Hydroxygen Peroxide Production, Lipid Peroxidation, and Secondary Metabolite Production

Influence of seasonality and habitat on chemical composition, cytotoxicity and antimicrobial properties of the Libidibia ferrea

Libidibia ferrea Mart, belonging to the Fabacee family, is a medicinal plant known for its biological properties and production of phenolic compounds. Previous studies reveal the biological activity of its phenolic constituents, making it very promising for the development of new medicines. Seasonality and geographic distribution of species can modify the production of secondary metabolites in Fabaceae species in terms of the preferentially activated metabolic pathways and, consequently, interfere with the medicinal properties of these species. Studying the influence of seasonality on the production of phenolic constituents is essential to establish conditions for “cultivation,” species collection, standardization, production, and safety in traditional medicine. This unprecedented study proposed to evaluate the influence of seasonal variations and habitat on the production of phenolic compounds and biological properties of the ethanolic extracts of the stem bark from L. ferrea, whose specimens were collected from the Caatinga and the Atlantic Forest, biomes of Brazil. Antimicrobial activity was determined by broth microdilution. Cytotoxicity was evaluated through a colorimetric assay using MTT. ABTS and DPPH radical reduction methods estimated antioxidant capacities. Folin-Ciocalteu and AlCl3 spectrophotometric methods quantified total phenolics and flavonoids, respectively. In turn, radial diffusion quantified tannin content. PCA score plot and HCA dendogram were obtained by multivariate analysis of 1H NMR data. The cytotoxicity against C6 glioma cells was observed only for Atlantic Forest extracts (EC50 = 0.13–0.5 mg mL−1). These extracts also showed selectivity against Gram-positive bacteria Bacillus subtilis (ATCC 6633) [MICs 500–2000 μg mL−1], B. cereus CCT 0096) [MIC = 250 μg mL−1], Staphylococcus aureus (ATCC 6538) [MICs = 250–500 μg mL−1], S. epidermidis (ATCC 12228) [62.5–1000 μg mL−1], mainly to Staphylococcus sp. Caatinga extracts showed higher production of flavonoids and antioxidants in the summer [7.36 ± 0.19 μg QE mg−1 extract; IC50ABTS = 4.86 ± 0.05 μg mL−1], spring [5.96 ± 0.10 μg QE mg−1 extract; IC50ABTS = 5.96 ± 0.08 μg mL−1 ], winter [4.89 ± 0.25 μg QE mg−1 extract; IC50ABTS = 6.72 ± 0.08 μg mL−1 ]. Regarding habitat, two discriminating compound patterns in the studied biomes were revealed by NMR. The results indicated that the Caatinga biome offers better conditions for activating the production of phenolics [336.34 ± 18.1 μgGAE mg−1 extract], tannins [328.38 ± 30.19 μgTAE mg−1 extract] in the summer and flavonoids in winter, spring, and summer. The extracts that showed the best antioxidant activities were also those from the Caatinga. In turn, extracts from the Atlantic Forest are more promising for discovering antibacterial compounds against Staphylococcus sp and cytotoxic for C6 glioma cells. These findings corroborated the traditional use of L. ferrea bark powder for treating skin wounds and suggest the cytotoxic potential of these extracts for glioblastoma cell lines.

Environmental Factors Modulate Saxitoxins (STXs) Production in Toxic Dinoflagellate Alexandrium: An Updated Review of STXs and Synthesis Gene Aspects

The marine dinoflagellate Alexandrium is known to form harmful algal blooms (HABs) and produces saxitoxin (STX) and its derivatives (STXs) that cause paralytic shellfish poisoning (PSP) in humans. Cell growth and cellular metabolism are affected by environmental conditions, including nutrients, temperature, light, and the salinity of aquatic systems. Abiotic factors not only engage in photosynthesis, but also modulate the production of toxic secondary metabolites, such as STXs, in dinoflagellates. STXs production is influenced by a variety of abiotic factors; however, the relationship between the regulation of these abiotic variables and STXs accumulation seems not to be consistent, and sometimes it is controversial. Few studies have suggested that abiotic factors may influence toxicity and STXs-biosynthesis gene (sxt) regulation in toxic Alexandrium, particularly in A. catenella, A. minutum, and A. pacificum. Hence, in this review, we focused on STXs production in toxic Alexandrium with respect to the major abiotic factors, such as temperature, salinity, nutrients, and light intensity. This review informs future research on more sxt genes involved in STXs production in relation to the abiotic factors in toxic dinoflagellates.

First report on in-depth genome and comparative genome analysis of a metal-resistant bacterium Acinetobacter pittii S-30, isolated from environmental sample.

A newly isolated bacterium Acinetobacter pittii S-30 was recovered from waste-contaminated soil in Ranchi, India. The isolated bacterium belongs to the ESKAPE organisms which represent the major nosocomial pathogens that exhibit high antibiotic resistance. Furthermore, average nucleotide identity (ANI) analysis also showed its closest match (>95%) to other A. pittii genomes. The isolate showed metal-resistant behavior and was able to survive up to 5 mM of ZnSO4. Whole genome sequencing and annotations revealed the occurrence of various genes involved in stress protection, motility, and metabolism of aromatic compounds. Moreover, genome annotation identified the gene clusters involved in secondary metabolite production (biosynthetic gene clusters) such as arylpolyene, acinetobactin like NRP-metallophore, betalactone, and hserlactone-NRPS cluster. The metabolic potential of A. pittii S-30 based on cluster of orthologous, and Kyoto Encyclopedia of Genes and Genomes indicated a high number of genes related to stress protection, metal resistance, and multiple drug-efflux systems etc., which is relatively rare in A. pittii strains. Additionally, the presence of various carbohydrate-active enzymes such as glycoside hydrolases (GHs), glycosyltransferases (GTs), and other genes associated with lignocellulose breakdown suggests that strain S-30 has strong biomass degradation potential. Furthermore, an analysis of genetic diversity and recombination in A. pittii strains was performed to understand the population expansion hypothesis of A. pittii strains. To our knowledge, this is the first report demonstrating the detailed genomic characterization of a heavy metal-resistant bacterium belonging to A. pittii. Therefore, the A. pittii S-30 could be a good candidate for the promotion of plant growth and other biotechnological applications.

Species-level distribution of microbiota with secondary metabolic potential in Chinese Baijiu fermentation

Chinese Baijiu fermentation is a typical spontaneous fermentation with complex microbial community. However, the species-level distribution of microbiota with secondary metabolic potential in Baijiu fermentation has not been well studied so far. In this study, taking soy sauce aroma type Baijiu and light aroma type Baijiu fermentations as examples, we identified 1575 biosynthetic gene clusters (BGCs) belonging to 874 gene cluster families (GCFs) through metagenomic analysis. Bioactivity prediction analysis revealed 210 BGCs producing potential bioactive secondary metabolites (probabilities of bioactivities ≥70%). We recovered 66 bacterial metagenome-assembled genomes (MAGs) to explore distribution of bioactive secondary metabolites at a higher resolution taxonomic level. A total of 16 MAGs were identified as BGC-rich MAGs (>3 BGCs), and they belonged to 11 genera. Four MAGs, annotated to be Bacillus velezensis, Bacillus licheniformis, Kroppenstedtia eburnea and Kroppenstedtia spp., contained more than 3 BGC-producing potential bioactive secondary metabolites and had more than 1 counts per million (CPM) abundance in fermentations. They were identified as the potential producers of bioactive secondary metabolites. This study contributed to characterising the diversity and distribution of microbiota with secondary metabolic potential in Baijiu fermentation, and mining novel secondary metabolites and their producers.

Development of a group II intron-based genetic manipulation tool for Streptomyces.

The availability of an alternative and efficient genetic editing technology is critical for fundamental research and strain improvement engineering of Streptomyces species, which are prolific producers of complex secondary metabolites with significant pharmaceutical activities. The mobile group II introns are retrotransposons that employ activities of catalytic intron RNAs and intron-encoded reverse transcriptase to precisely insert into DNA target sites through a mechanism known as retrohoming. We here developed a group II intron-based gene editing tool to achieve precise chromosomal gene insertion in Streptomyces. Moreover, by repressing the potential competition of RecA-dependent homologous recombination, we enhanced site-specific insertion efficiency of this tool to 2.38%. Subsequently, we demonstrated the application of this tool by screening and characterizing the secondary metabolite biosynthetic gene cluster (BGC) responsible for synthesizing the red pigment in Streptomyces roseosporus. Accompanied with identifying and inactivating this BGC, we observed that the impair of this cluster promoted cell growth and daptomycin production. Additionally, we applied this tool to activate silent jadomycin BGC in Streptomyces venezuelae. Overall, this work demonstrates the potential of this method as an alternative tool for genetic engineering and cryptic natural product mining in Streptomyces species.

Paraburkholderia translucens sp. nov. and Paraburkholderia sejongensis sp. nov., isolated from grassland soil and showing plant growth promoting potential.

Two Gram-negative bacterial strains designated MMS20-SJTN17T and MMS20-SJTR3T were isolated from a grassland soil sample, and taxonomically characterized using a polyphasic approach. The 16S rRNA gene sequence analysis indicates that both strains belong to the genus Paraburkholderia of the class Betaproteobacteria, with strain MMS20-SJTN17T being mostly related to Paraburkholderia sprentiae WSM5005T (96.45 % sequence similarity) and strain MMS20-SJTR3T to Paraburkholderia tuberum STM678T (98.59 % sequence similarity). MMS20-SJTN17T could grow at 15-40 °C (optimum, 25-30 °C) and at pH 6.0-8.0 (optimum, pH 6.0-7.0), whereas MMS20-SJTR3T could grow at 10-40 °C (optimum, 30-37 °C) and at pH 6.0-8.0 (optimum, pH 6.0). Both strains tolerated up to 1 % (w/v) NaCl (optimum, 0 %). The major fatty acids of MMS20-SJTN17T were C16 : 0 and C19 : 0 cyclo ω8c, and those of MMS20-SJTR3T were C17 : 0 cyclo and a summed feature comprising C18 : 1 ω7c and/or C18 : 1 ω6c. The major isoprenoid quinone of both strains was ubiquinone-8 and the diagnostic polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Regarding plant growth promoting potential, both strains were capable of producing indole acetic acid, siderophore and 1-aminocyclopropane-1-carboxylic acid deaminase, and also showed phosphate-solubilizing activity. A genome-based comparison using orthologous average nucleotide identity and digital DNA-DNA hybridization values indicates that strain MMS20-SJTN17T shares highest relatedness with Paraburkholderia monticola JC2948T and MMS20-SJTR3T with Paraburkholderia antibiotica G-4-1-8T, with values clearly below the cutoffs for species distinction. Examination of biosynthetic gene clusters responsible for secondary metabolite production reveals unique characteristics distinguishing each strain from closely related Paraburkholderia species. On the basis of genotypic, phenotypic, chemotaxonomic and phylogenomic data, each strain should be classified as a novel species of the genus Paraburkholderia, for which the names Paraburkholderia translucens sp. nov. (=MMS20-SJTN17T=LMG 32366T=KCTC 82783T) and Paraburkholderia sejongensis sp. nov. (=MMS20-SJTR3T=LMG 32367T=KCTC 82784T) are proposed.

Exploring low-dose gamma radiation effects on monoterpene biosynthesis in Thymus vulgaris: insights into plant defense mechanisms.

Thymus vulgaris, commonly known as thyme, is a plant renowned for producing monoterpenes. This study aimed to understand the effects of low-dose gamma radiation, specifically in the range of 1-5Gy, on various traits of Thymus vulgaris, providing context on its importance in agricultural and medicinal applications. The research explored morpho-physiological, biochemical, and gene-expression responses in thyme plants under no gamma- and gamma-ray exposure conditions. The study revealed complex relationships between gamma-ray doses and plant characteristics. In particular, shoot and root lengths initially increased with low doses (1-3Gy) but decreased at higher doses (5Gy), suggesting a dose-dependent threshold effect. Similarly, shoot and root fresh weights displayed an initial increase followed by a decline with increasing doses. Biochemical parameters showed dose-dependent responses, with low to moderate doses (1-3Gy) stimulating enzyme activities and high doses (5Gy) inhibiting them. Gene expression analysis was focused on the following specific genes: thymol synthase, γ-terpinene synthase, and carvacrol synthase. Low to moderate doses increased the expression of these genes, resulting in increased production of bioactive compounds. However, higher doses had diminished effects or suppressed gene expression. Metabolite analysis demonstrated dose-dependent responses, with moderate doses enhancing secondary metabolite production, while higher doses provided limited benefits. These findings underscore the implications of using gamma radiation to enhance secondary metabolite production in plants and its potential applications in agriculture, medicine, and environmental science. The study emphasizes the potential of gamma radiation as an external stressor to influence plant responses and highlights the importance of understanding such effects in various fields.

Strategies for the Enhancement of Secondary Metabolite Production via Biosynthesis Gene Cluster Regulation in Aspergillus oryzae

The filamentous fungus Aspergillus oryzae (A. oryzae) has been extensively used for the biosynthesis of numerous secondary metabolites with significant applications in agriculture and food and medical industries, among others. However, the identification and functional prediction of metabolites through genome mining in A. oryzae are hindered by the complex regulatory mechanisms of secondary metabolite biosynthesis and the inactivity of most of the biosynthetic gene clusters involved. The global regulatory factors, pathway-specific regulatory factors, epigenetics, and environmental signals significantly impact the production of secondary metabolites, indicating that appropriate gene-level modulations are expected to promote the biosynthesis of secondary metabolites in A. oryzae. This review mainly focuses on illuminating the molecular regulatory mechanisms for the activation of potentially unexpressed pathways, possibly revealing the effects of transcriptional, epigenetic, and environmental signal regulation. By gaining a comprehensive understanding of the regulatory mechanisms of secondary metabolite biosynthesis, strategies can be developed to enhance the production and utilization of these metabolites, and potential functions can be fully exploited.

Different wavelengths of LED irradiation promote secondary metabolite production in Pycnoporus sanguineus for antioxidant and immunomodulatory applications.

Pycnoporus sanguineus is a fungus of the phylum Basidiomycota that has many applications in traditional medicine, modern pharmaceuticals, and agricultural industries. Light plays an essential role in the metabolism, growth, and development of fungi. This study evaluated the mycelial growth and antioxidant and anti-inflammatory activities in P. sanguineus fermentation broth (PFB) cultured under different wavelengths of LED irradiation or in the dark. Compared to the dark cultures, the dry weight of mycelia in red- and yellow-light cultures decreased by 37 and 35% and the yields of pigments increased by 30.92 ± 2.18mg and 31.75 ± 3.06mg, respectively. Compared with the dark culture, the DPPH free radical scavenging ability, ABTS+ free radical scavenging capacity, and reducing power of yellow-light cultures increased significantly, and their total phenolic content peaked at 180.0 ± 8.34μg/mL. However, the reducing power in blue-light cultures was significantly reduced, though the total phenol content did not vary with that of dark cultures. In LPS- and IFN-γ-stimulated RAW 264.7 cells, nitrite release was significantly reduced in the red and yellow light-irradiated PFB compared with the dark culture. In the dark, yellow-, and green-light cultures, TNF-α production in the inflamed RAW 264.7 cells was inhibited by 62, 46, and 14%, respectively. With red-, blue-, and white-light irradiation, TNF-α production was significantly enhanced. Based on these results, we propose that by adjusting the wavelength of the light source during culture, one can effectively modulate the growth, development, and metabolism of P. sanguineus.

The gut microbiome of farmed Arctic char (Salvelinus alpinus) is shaped by feeding stage and nutrient presence.

The gut microbiome plays an important role in maintaining health and productivity of farmed fish. However, the functional role of most gut microorganisms remains unknown. Identifying the stable members of the gut microbiota and understanding their functional roles could aid in the selection of positive traits or act as a proxy for fish health in aquaculture. Here, we analyse the gut microbial community of farmed juvenile Arctic char (Salvelinus alpinus) and reconstruct the metabolic potential of its main symbionts. The gut microbiota of Arctic char undergoes a succession in community composition during the first weeks post-hatch, with a decrease in Shannon diversity and the establishment of three dominant bacterial taxa. The genome of the most abundant bacterium, a Mycoplasma sp., shows adaptation to rapid growth in the nutrient-rich gut environment. The second most abundant taxon, a Brevinema sp., has versatile metabolic potential, including genes involved in host mucin degradation and utilization. However, during periods of absent gut content, a Ruminococcaceae bacterium becomes dominant, possibly outgrowing all other bacteria through the production of secondary metabolites involved in quorum sensing and cross-inhibition while benefiting the host through short-chain fatty acid production. Whereas Mycoplasma is often present as a symbiont in farmed salmonids, we show that the Ruminococcaceae species is also detected in wild Arctic char, suggesting a close evolutionary relationship between the host and this symbiotic bacterium.

Dynamic microbial and metabolic changes during Apulian Caciocavallo cheese-making and ripening produced according to a standardized protocol

The cheese microbiota plays a critical role in influencing its sensory and physicochemical properties. In this study, traditional Apulian Caciocavallo cheese coming from 4 different dairies in the same area and produced following standardized procedures have been examined, as well as the different bulk milks and natural whey starter cultures used. Moreover, considering the cheese wheels as the blocks of Caciocavallo cheeses as whole, these were characterized at different layers (i.e., core, under-rind, and rind) of the block using a multi-omics approach. In addition to physical-chemical characterization, culturomics, quantitative PCR, metagenomics, and metabolomics analysis, have been carried out post-salting and throughout ripening time (2 mo) to investigate the major shifts in the succession of the microbiota and flavor development. Culture-dependent and 16S rRNA metataxonomics results clearly clustered samples based on the microbiota biodiversity related to the production dairy plant as the result of the use of different NWS or intrinsic conditions of each production site. At the beginning of the ripening, cheeses were dominated by the Lactobacillus and, in 2 dairies (Art and SdC), Streptococcus genera associated with the NWS. The analysis allowed us to show that, although the diversity of identified genera did not change significantly between the rind, under-rind and core fractions of the same samples, there was an evolution in the relative abundance and absolute quantification, modifying and differentiating profiles during ripening. The qPCR mainly differentiated the temporal adaptation of those species originating from bulk milks and those provided by NWSs. The primary starter detected in NWS and cheese reassured the high relative concentration of 1-butanol, 2-butanol, 2-heptanol, 2-butanone, acetoin, delta-dodecalactone, hexanoic acid ethyl ester, octanoic acid ethyl ester, and VFFA during ripening, while cheeses displaying low abundances of Streptococcus and Lactococcus (dairy Del) have a lower total concentration of acetoin compared with Art and SdC. However, the sub-dominant strains and NSLAB present in cheeses are responsible for the production of secondary metabolites belonging to the chemical classes of ketones, alcohols, and organic acids, reaffirming the importance and relevance of autochthonous strains of each dairy plant although considering a delimited production area.

Characterization of anthranilic acid produced by Virgibacillus salarius MML1918 and its bio-imaging application.

Anthranilic acid (AA) holds significant importance in the chemical industry. It serves as a crucial building block for the amino acid tryptophan by manipulating the tryptophan biosynthesis pathway, it is possible to increase the production of anthranilic acid. In this study, we utilized metabolic engineering approaches to produce anthranilic acid from the halophilic bacterium Virgibacillus salarius MML1918. The halophilic bacteria were grown in an optimized production medium, and mass production of secondary metabolites was made in ATCC medium 1097 Proteose peptone-for halophilic bacteria and subjected to column chromatography followed by sub-column chromatography the single band for the purified compound was confirmed. Further, various spectral analyses were made for the partially purified compounds, and fluorescence microscopy for fungal cell observation was performed. The purified compound was confirmed by single crystal X-ray diffraction (XRD) analysis, and it was identified as 2-amino benzoic acid. The Fourier transform infrared Spectroscopy (FT-IR) spectrum and nuclear magnetic resonance (NMR) spectrum also confirm the structural characteristic of 2-amino benzoic acid. The UV-Vis absorption spectrum of AA shows the maximum absorption at 337.86nm. The emission spectrum of 2-amino benzoic acid showed the maximum emission at 453nm. The bio-imaging application of 2-amino benzoic acid was examined with fungal mycelium of Rhizoctonia solani. It was effectively bound and emitted the blue color at the concentration of 200 and 300µg/mL. The halophilic bacterium (V. salarius), may have unique metabolic pathways and requirements compared to non-halophilic organisms, to produce AA effectively. This could have implications for industrial biotechnology, particularly in manufacturing environments where high salt concentrations are present and also it can be used as bio-imaging agent.

Tools to make Stachybotrys chartarum genetically amendable: Key to unlocking cryptic biosynthetic gene clusters

The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of the toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, a number of secondary metabolites remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 gene cluster, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as generating deletions and overexpressing single cluster genes. This toolbox can now be readily employed to unravel not only this particular biosynthetic gene cluster but also other candidate biosynthetic gene clusters present in S. chartarum, making this fungus accessible for genetic engineering.