Production Of Metabolites Research Articles

In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Fritillaria cirrhosa

The bulb of Fritillaria cirrhosa D. Don is widely used for the anti-asthmatic, anti-tussive, and anti-cancer agents, etc., while the yield is limited by an endangered status, a long juvenile phase, and restricted growth habitat. Ancillary approaches to improve the bulb yield by micropropagation and bioactive metabolites production by bioreactor have not been established. Here is reported the plant regeneration, suspension cell culture, and bioactive metabolite production at different treatments. The embryogenic calli were successfully induced via the histomorphological identification. The highest proliferation times (4.11-fold) were observed with a select combination of hormones [NAA (0.2 mg/L) + 6-BA (1.0 mg/L) + GA3 (1.0 mg/L)] and culture conditions (red light and 20 °C), the highest content of imperialine (0.13 mg/g) was observed under blue light, total phenolic (0.52 mg/g) under red light, polysaccharides (36.57 mg/g) and total flavonoids (2.67 mg/g) as well as antioxidant capacity under white light. The plantlets were regenerated within 125 d from the induced embryogenic calli to acclimation and transplantation of seedlings. For the suspension cell culture, a 6.30-, 1.78-, 1.37-, and 1.51-fold increase of proliferation times, imperialine, polysaccharides, and total phenolic contents was observed at 40 d, respectively. Based on the above observations, an effective and complete in vitro approach has been proposed to regenerate plants and produce bioactive metabolites in F. cirrhosa.

Rethinking Aspergillosis in the Era of Microbiota and Mycobiota.

Aspergillosis encompasses a wide range of clinical conditions based on the interaction between Aspergillus and the host. It ranges from colonization to invasive aspergillosis. The human lung provides an entry door for Aspergillus. Aspergillus has virulence characteristics such as conidia, rapid growth at body temperature, and the production of specific proteins, carbohydrates, and secondary metabolites that allow A. fumigatus to infiltrate the lung's alveoli and cause invasive aspergillosis. Alveolar epithelial cells play an important role in both fungus clearance and immune cell recruitment via cytokine release. Although the innate immune system quickly clears conidia in immunocompetent hosts, A. fumigatus has evolved multiple virulence factors in order to escape immune response such as ROS detoxifying enzymes, the rodlet layer, DHN-melanin and toxins. Bacterial co-infections or interactions can alter the immune response, impact Aspergillus growth and virulence, enhance biofilm formation, confound diagnosis, and reduce treatment efficacy. The gut microbiome's makeup influences pulmonary immune responses generated by A. fumigatus infection and vice versa. The real-time PCR for Aspergillus DNA detection might be a particularly useful tool to diagnose pulmonary aspergillosis. Metagenomics analyses allow quick and easy detection and identification of a great variety of fungi in different clinical samples, although optimization is still required particularly for the use of NGS techniques. This review will analyze the current state of aspergillosis in light of recent discoveries in the microbiota and mycobiota.

Oligomycin-producing Streptomyces sp. newly isolated from Swiss soils efficiently protect Arabidopsis thaliana against Botrytis cinerea.

This study reports the isolation of new Streptomyces strains with strong plant-protective potential mediated by their production of specialized metabolites. Using the broad host range pathogenic fungus Botrytis cinerea, we demonstrate that the cell-free filtrate of selected Streptomyces isolates efficiently inhibits different developmental stages of the fungus, including mycelial growth and the epidemiologically relevant spore germination. Beyond in vitro experiments, the strains and their metabolites also efficiently protected plants against the disease caused by this pathogen. This work further identifies oligomycins as active compounds involved in the observed antifungal activity of the strains. This work shows that we can harness the natural ability of soil-borne microbes and of their metabolites to efficiently fight other microbes responsible for significant crop losses. This opens the way to the development of environmentally friendly health protection measures for crops of agronomical relevance, based on these newly isolated strains or their metabolic extracts containing oligomycins.

Commercial wine yeast nitrogen requirement influences the production of secondary metabolites (aroma, hydroxytyrosol, melatonin and other bioactives) during alcoholic fermentation

During alcoholic fermentation, Saccharomyces cerevisiae synthesizes different compounds, which are crucial for product quality: volatile compounds with sensory impact, and bioactive compounds such as melatonin (MEL) and hydroxytyrosol (HT), linked to health benefits. As many of these compounds are related with yeast's nitrogen metabolism, their production have been studied in four different commercial strains with different nitrogen requirement (Red Fruit, Uvaferm VRB, Lalvin Rhone 2323 and Lalvin QA23) being, Uvaferm UVR the higher nitrogen demander strain. All strains produced the secondary metabolites, notably Uvaferm UVR produced the highest HT concentration, despite its low growth. Uvaferm UVR emerged also as a significant producer of MEL, indicating a potential role in fermentation related stress. Moreover, Uvaferm UVR shows the highest total concentrations of volatile compounds. Multivariate analysis revealed distinct clustering based on nitrogen requirements of the strains, highlighting the strain-dependent metabolic responses.

Isolation and Characterization of Phenylalanine Ammonia Lyase (PAL) Genes in Ferula pseudalliacea: Insights into the Phenylpropanoid Pathway.

Phenylalanine ammonia lyase (PAL) is a key enzyme regulating the biosynthesis of the compounds of the phenylpropanoid pathway. This study aimed to isolate and characterize PAL genes from Ferula pseudalliacea Rech.f. (Apiales: Apiaceae) to better understand the regulation of metabolite production. Three PAL gene isoforms (FpPAL1-3) were identified and cloned using the 3'-RACE technique and confirmed by sequencing. Bioinformatics analysis revealed important structural features, such as phosphorylation sites, physicochemical properties, and evolutionary relationships. Expression analysis by qPCR demonstrated the differential transcription profiles of each FpPAL isoform across roots, stems, leaves, flowers, and seeds. FpPAL1 showed the highest expression in stems, FpPAL2 in roots and flowers, and FpPAL3 in flowers. The presence of three isoforms of PAL in F. pseudalliacea, along with the diversity of PAL genes and their tissue-specific expression profiles, suggests that complex modes of regulation exist for phenylpropanoid biosynthesis in this important medicinal plant. The predicted interaction network revealed associations with key metabolic pathways, emphasizing the multifaceted roles of these PAL genes. In silico biochemical analyses revealed the hydrophilicity of the FpPAL isozyme; however, further analysis of substrate specificity and enzyme kinetics can clarify the specific role of each FpPAL isozyme. These comprehensive results increase the understanding of PAL genes in F. pseudalliacea, helping to characterize their contributions to secondary metabolite biosynthesis.

CECAL SLURRY AS AN ALTERNATIVE MODEL TO CECAL LIGATION AND PUNCTURE FOR THE STUDY OF SEPSIS-INDUCED CARDIOVASCULAR DYSFUNCTION.

Sepsis is a life-threatening condition widely studied by animal models. Cecal ligation and puncture (CLP) is still regarded as the gold standard model for sepsis. However, CLP has limitations due to its invasiveness and variability. Cecal slurry (CS) model is a nonsurgical and thus less invasive alternative. However, the lack of standardization of the CS model in the literature limits its practical application. Additionally, it is not well studied whether CS model reproduces septic cardiovascular dysfunction in rats, which is a crucial issue in septic patients. Thus, this study aimed to standardize the CS model in Wistar rats and evaluate sepsis-induced cardiovascular dysfunction compared to CLP. Our results showed that CS model induced important features of sepsis cardiovascular dysfunction 24 h after its onset, such as hypotension, tachycardia, and decreased contractile response to vasoconstrictors both in vivo and ex vivo as well changes in renal blood flow. Increases in blood lactate, AST, ALT, creatinine, and urea indicated organ dysfunction. CS model also induced increased production of nitric oxide metabolites and bacterial spread to tissues. CS model causes less animal suffering, it is a nonsurgical model, and, more importantly, it replicates the cardiovascular dysfunction induced by sepsis with better homogeneity than CLP. Therefore, CS model serves as an alternative and possibly as a better model for sepsis research.

Aureobasidium pullulans formulations: evaluation of the effectiveness against grey mould of table grape

Antagonism against Botrytis cinerea is often carried out using yeast as direct antagonists. Aureobasidium pullulans strain AP1 was tested in two different formulations: wettable powder (WP) and oil dispersion (OD). By in vitro assays, the viability of the strain cells was constantly evaluated for seven months and the OD formulation ensured the highest cells viability. The efficacy of the formulations was assayed by evaluating the production of volatile and non-volatile metabolites. Results showed that the formulation affected the non-volatile less than the volatile metabolites. Both AP1 WP and AP1 OD non-volatile metabolites displayed almost 50% of mycelial pathogen inhibition. Comparing the two products, the lowest EC50 value (518.15 mg L− 1) was detected for the AP1 OD formulation that was thus chosen for postharvest in vivo assays. The preventative treatments (200, 400, 800 mg L− 1) were active in reducing the pathogen incidence on table grape on average by 52%. Instead, in the curative application assay, the highest concentration (800 mg L− 1) reduced grey mold incidence by 86%. The present study reported the potential of two new formulations to use against the postharvest grey mold of table grape for a possible further commercial product development.

Eficiencia biológica y evaluación de los compuestos bioactivos de cepas silvestres mexicanas de Hericium erinaceus

The basidiomycete Hericium erinaceus is one of the most consumed edibles and medicinal mushrooms in the world and appreciated in traditional Chinese medicine. In Mexico it is known as lion's mane. The biological efficiency of wild strains of H. erinaceus cultivated in different substrates in the Durango region, mainly agave bagasse, a waste from mezcal production, was evaluated. The CCH003 strain from Espinazo del diablo showed the highest biological efficiency of 42.33 % and a productivity rate of 0.47 %, with a total cultivation period of 90 days and three harvests. Regarding the evaluation of bioactive compounds, the same strain obtained significant differences compared to the others, it presented the highest values for all determinations; 60 ± 0.018 mg EAG.g ES-1 in phenolic content, 4.21 ± 0.013 mg EQ.g ES-1 for flavonoids, 71.16 ± 0.002 mg EAA.g ES-1 in CAT, 0.0012 ± 0.001 mg AA.g ES-1 for by ABTS and 121 ± 0.107 µg EAG.mL-1 by DPPH. The variability of the results in the tests carried out provides information on how the type of substrate, climatological and geographical conditions and stage of maturity influence the development of the fungus, including the production of secondary metabolites, even if it is the same species. It is expected that this information will be useful to promote the use of agave bagasse as a substrate in the cultivation of H. erinaceus and thereby diversify rural activities in the region, and in the future generate new studies on the effect of conditions on the production of bioactive compounds. Keywords: agave bagasse, antioxidant, medicinal mushrooms.

Exogenous methyl jasmonate (MeJA) altered phytochemical composition and enhanced the expression of PatAACT gene of in vitro culture-derived patchouli var. Sidikalang (Pogostemon cablin Benth.)

The demand for economically valuable terpenoid-derived compounds, especially those extracted from patchouli plants, is experiencing exponential growth. These compounds are potentially applied in a wide range of industrial sectors including food manufacturing, perfumeries, and pharmaceuticals. Nevertheless, there is a need for further development in patchouli plant cultivation to align with market demands. Therefore, employing robust in vitro-based propagation methods could offer significant advantages. Furthermore, this technique is also used for plant metabolites enhancement. This strategy applies the methyl jasmonate (MeJA) elicitor to boost the production of secondary metabolites. In Indonesia, patchouli var. Sidikalang is extensively cultivated in many areas. This study aims to evaluate the effect of naphtaleneacetic acid (NAA) and 6-benzylamino purine (BAP) to increase plant growth via in vitro culture. Furthermore, we also investigate the effect of MeJA on the metabolite profiles and on the AACT gene expression. Our findings revealed that the combination of 2.5 µM of BAP and 0.25 µM of NAA resulted in optimal growth and high rate of organogenesis across all growth parameters. Our data also successfully identified 50 dominant compounds, each with varying percentage areas. Notably, patchouli alcohol emerged as the primary compound in the tricyclic sesquiterpene group, consistently present in all treatment groups. Furthermore, the expression of the PatAACT gene significantly increased by 7.42-fold after treatment with MeJA at 100 µM and by 2,9-fold after treatment with MeJA at 300 µM, compared to the control. Altogether, our findings might offer new insight in P. cablin propagation and new strategy for patchouli-derived metabolites using exogenous MeJA.

An in silico investigation of the toxicological effects and biological activities of 3-phenoxybenzoic acid and its metabolite products

We aimed to elucidate the toxic effects and biological activities of 3-phenoxybenzoic acid (3PBA) and its metabolite products. Numerous in silico methods were used to identify the toxic effects and biological activities of 3PBA, including PASS online, molecular docking, ADMETlab 2.0, ADMESWISS, MetaTox, and molecular dynamic simulation. Ten metabolite products were identified via Phase II reactions (O-glucuronidation, O-sulfation, and methylation). All of the investigated compounds were followed by Lipinski’s rule, indicating that they were stimulants or inducers of hazardous processes. Because of their high gastrointestinal absorption and ability to reach the blood-brain barrier, the studied compounds’ physicochemical and pharmacokinetic properties matched existing evidence of harmful effects, including haematemesis, reproductive dysfunction, allergic dermatitis, toxic respiration, and neurotoxicity. The studied compounds have been linked to the apoptotic pathway, the reproductivity system, neuroendocrine disruptors, phospholipid-translocating ATPase inhibitors, and JAK2 expression. An O-glucuronidation metabolite product demonstrated higher binding affinity and interaction with CYP2C9, CYP3A4, caspase 3, and caspase 8 than 3PBA and other metabolite products, whereas metabolite products from methylation were predominant and more toxic. Our in silico findings partly meet the 3Rs principle by minimizing animal testing before more study is needed to identify the detrimental effects of 3PBA on other organs (liver, kidneys). Future research directions may involve experimental validation of in silico predictions, elucidation of molecular mechanisms, and exploration of therapeutic interventions. These findings contribute to our understanding of the toxicological profile of 3PBA and its metabolites, which has implications for risk assessment and regulatory decisions.

Production of high levels of 2,3-butanediol from renewable crude glycerol by Klebsiella pneumoniae GEM167 using metabolic engineering and control of oxygen transfer efficiency

Numerous industries use 2,3-butanediol as a bulk chemical. This study aimed to develop a technique for the commercial production of 2,3-butanediol from glycerol using K. pneumoniae GEM167 strain with enhanced oxidative pathways. We developed K. pneumoniae GEM167ΔadhEΔldhA, a strain that has decreased production of competitive metabolites (ethanol and lactate) and increased production of 2,3-butanediol. We investigated the correlation between the oxygen transfer coefficient and 2,3-butanediol production by modifying the agitation speed and aeration rate. K. pneumoniae GEM167ΔadhEΔldhA provided a high titer (102.9 ± 3.1 g/L) and productivity (2.14 ± 0.06 g/L/h) of 2,3-butanediol with no detectable 1,3-propaendiol production under specific culture conditions (37 °C, 700 rpm, 2.0 vvm, and pH 6.0). To the best of our knowledge, the productivity of 2,3-butanediol from glycerol in this study is the highest yet reported. And the substitution with crude glycerol enhanced the titer (105.3 ± 2.8 g/L) and productivity (2.19 ± 0.06 g/L/h) of 2,3-butanediol.

Unraveling radiation resistance strategies in two bacterial strains from the high background radiation area of Chavara-Neendakara: A comprehensive whole genome analysis.

This paper reports the results of gamma irradiation experiments and whole genome sequencing (WGS) performed on vegetative cells of two radiation resistant bacterial strains, Metabacillus halosaccharovorans (VITHBRA001) and Bacillus paralicheniformis (VITHBRA024) (D10 values 2.32 kGy and 1.42 kGy, respectively), inhabiting the top-ranking high background radiation area (HBRA) of Chavara-Neendakara placer deposit (Kerala, India). The present investigation has been carried out in the context that information on strategies of bacteria having mid-range resistance for gamma radiation is inadequate. WGS, annotation, COG and KEGG analyses and manual curation of genes helped us address the possible pathways involved in the major domains of radiation resistance, involving recombination repair, base excision repair, nucleotide excision repair and mismatch repair, and the antioxidant genes, which the candidate could activate to survive under ionizing radiation. Additionally, with the help of these data, we could compare the candidate strains with that of the extremely radiation resistant model bacterium Deinococccus radiodurans, so as to find the commonalities existing in their strategies of resistance on the one hand, and also the rationale behind the difference in D10, on the other. Genomic analysis of VITHBRA001 and VITHBRA024 has further helped us ascertain the difference in capability of radiation resistance between the two strains. Significantly, the genes such as uvsE (NER), frnE (protein protection), ppk1 and ppx (non-enzymatic metabolite production) and those for carotenoid biosynthesis, are endogenous to VITHBRA001, but absent in VITHBRA024, which could explain the former's better radiation resistance. Further, this is the first-time study performed on any bacterial population inhabiting an HBRA. This study also brings forward the two species whose radiation resistance has not been reported thus far, and add to the knowledge on radiation resistant capabilities of the phylum Firmicutes which are abundantly observed in extreme environment.

Sirtuin E deacetylase is required for full virulence of Aspergillus fumigatus

Aspergillus fumigatus represents a public health problem due to the high mortality rate in immunosuppressed patients and the emergence of antifungal-resistant isolates. Protein acetylation is a crucial post-translational modification that controls gene expression and biological processes. The strategic manipulation of enzymes involved in protein acetylation has emerged as a promising therapeutic approach for addressing fungal infections. Sirtuins, NAD+-dependent lysine deacetylases, regulate protein acetylation and gene expression in eukaryotes. However, their role in the human pathogenic fungus A. fumigatus remains unclear. This study constructs six single knockout strains of A. fumigatus and a strain lacking all predicted sirtuins (SIRTKO). The mutant strains are viable under laboratory conditions, indicating that sirtuins are not essential genes. Phenotypic assays suggest sirtuins’ involvement in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. Deletion of sirE attenuates virulence in murine and Galleria mellonella infection models. The absence of SirE alters the acetylation status of proteins, including histones and non-histones, and triggers significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

Complete genome sequence of Bacillus velezensis strain Ag109, a biocontrol agent against plant-parasitic nematodes and Sclerotinia sclerotiorum

Soybean is the main oilseed cultivated worldwide. Even though Brazil is the world’s largest producer and exporter of soybean, its production is severely limited by biotic factors. Soil borne diseases are the most damaging biotic stressors since they significantly reduce yield and are challenging to manage. In this context, the present study aimed to evaluate the potential of a bacterial strain (Ag109) as a biocontrol agent for different soil pathogens (nematodes and fungi) of soybean. In addition, the genome of Ag109 was wholly sequenced and genes related to secondary metabolite production and plant growth promotion were mined. Ag109 showed nematode control in soybean and controlled 69 and 45% of the populations of Meloidogyne javanica and Pratylenchus brachyurus, respectively. Regarding antifungal activity, these strains showed activity against Macrophomia phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum. For S. sclerotiorum, this strain increased the number of healthy plants and root dry mass compared to the control (with inoculation). Based on the average nucleotide identity and digital DNA-DNA hybridization, this strain was identified as Bacillus velezensis. Diverse clusters of specific genes related to secondary metabolite biosynthesis and root growth promotion were identified, highlighting the potential of this strain to be used as a multifunctional microbial inoculant that acts as a biological control agent while promoting plant growth in soybean.

BBS Gene Expression and Its Diversity in the Genus Dendrobium

Dendrobium officinale Kimura & Migo in the genus Dendrobium of Orchidaceae is an important medicinal plant that produces various bibenzyl and phenanthrene derivatives. In some orchids, these derivatives have been reported to increase with fungal infection. Bibenzyl biosynthesis is regulated by bibenzyl synthase (BBS). Although six genes of the BBS family have been registered from D. officinale, their gene regulation mechanisms are unclear. The infection of Dendrobium with mycorrhizal fungi also reportedly increases the expression of genes involved in biosynthesis; however, the effect of mycorrhizal fungi on bibenzyl production is unknown. The present study examined the effects of three mycorrhizal fungi isolated from D. officinale on BBS gene expression and bibenzyl production over time. One of the Tulasnellaceae operational taxonomic units induced BBS gene expression and increased two representative bibenzyls, gigantol and dendrophenol, at specific time points. Furthermore, 19 BBS sequences were cloned from 12 Dendrobium species, and a phylogenetic analysis was performed. The results indicated that repeated BBS gene duplication occurred during the evolution of the genus, and further duplication occurred after speciation. These results suggest that it is possible to optimize metabolite production by selecting suitable symbiotic fungi.

The repertoire and levels of secondary metabolites in microbial cocultures depend on the inoculation ratio: a case study involving Aspergillus terreus and Streptomyces rimosus

ObjectiveThe aim of this study was to determine the influence of the inoculation volume ratio on the production of secondary metabolites in submerged cocultures of Aspergillus terreus and Streptomyces rimosus.ResultsThe shake flask cocultures were initiated by using 23 inoculum variants that included different volumes of A. terreus and S. rimosus precultures. In addition, the axenic controls were propagated in parallel with the cocultures. UPLC‒MS analysis revealed the presence of 15 secondary metabolites, 12 of which were found both in the “A. terreus vs. S. rimosus” cocultures and axenic cultures of either A. terreus or S. rimosus. The production of the remaining 3 molecules was recorded solely in the cocultures. The repertoire and quantity of secondary metabolites were evidently dependent on the inoculation ratio. It was also noted that detecting filamentous structures resembling typical morphological forms of a given species was insufficient to predict the presence of a given metabolite.ConclusionsThe modification of the inoculation ratio is an effective strategy for awakening and enhancing the production of secondary metabolites that are not biosynthesized under axenic conditions.

Two-year investigation of spore-formers through the production chain at two cheese plants in Norway

Spore-forming bacteria are the most complex group of microbes to eliminate from the dairy production line due to their ability to withstand heat treatment usually used in dairy processing. These ubiquitous microorganisms have ample opportunity for multiple points of entry into the milk chain, creating issues for food quality and safety. Certain spore-formers, namely bacilli and clostridia, are more problematic to the dairy industry due to their possible pathogenicity, growth, and production of metabolites and spoilage enzymes. This research investigated the spore-forming population from raw milk reception at two Norwegian dairy plants through the cheesemaking stages until ripening. Samples were collected over two years and examined by amplicon sequencing in a culture independent manner and after an anaerobic spore-former enrichment step. In addition, a total of 608 isolates from the enriched samples were identified at the genus or species level using MALDI-TOF analysis. Most spore-forming isolates belong to the genera Bacillus or Clostridium, with the latter dominating the enriched MPN tubes of raw milk and bactofugate. Results showed a great variation among the clostridia and bacilli detected in the enriched MPN tubes. However, B. licheniformis and C. tyrobutyricum were identified in all sample types from both plants throughout the 2-year study. In conclusion, our results shed light on the fate of different spore-formers at different processing stages in the cheese production chain, which could facilitate targeted actions to reduce quality problems.

Genome mining of Lactiplantibacillus plantarum PA21: insights into its antimicrobial potential

BackgroundThe dramatic increase of antimicrobial resistance in the healthcare realm has become inexorably linked to the abuse of antibiotics over the years. Therefore, this study seeks to identify potential postbiotic metabolites derived from lactic acid bacteria such as Lactiplantibacillus plantarum that could exhibit antimicrobial properties against multi-drug resistant pathogens.ResultsIn the present work, the genome sequence of Lactiplantibacillus plantarum PA21 consisting of three contigs was assembled to a size of 3,218,706 bp. Phylogenomic analysis and average nucleotide identity (ANI) revealed L. plantarum PA21 is closely related to genomes isolated from diverse niches such as dairy products, food, and animals. Genome mining through the BAGEL4 and antiSMASH database revealed four bacteriocins in a single cluster and four regions of biosynthetic gene clusters responsible for the production of bioactive compounds. The potential probiotic genes indirectly responsible for postbiotic metabolites production were also identified. Additionally, in vitro studies showed that the L. plantarum PA21 cell-free supernatant exhibited antimicrobial activity against all nine methicillin-resistant Staphylococcus aureus (MRSA) and three out of 13 Klebsiella pneumoniae clinical isolates tested.ConclusionResults in this study demonstrates that L. plantarum PA21 postbiotic metabolites is a prolific source of antimicrobials against multi-drug resistant pathogens with potential antimicrobial properties.

Comprehensive analyses of the ARF gene family in cannabis reveals their potential roles in regulating cannabidiol biosynthesis and male flower development.

Cannabidiol (CBD), as an important therapeutic property of the cannabis plants, is mainly produced in the flower organs. Auxin response factors (ARFs) are play a crucial role in flower development and secondary metabolite production. However, the specific roles of ARF gene family in cannabis remain unknown. In this study, various bioinformatics analysis of CsARF genes were conducted using online website and bioinformatics, quantitative real time PCR technology was used to investigate the expression patterns of the CsARF gene family in different tissues of different cannabis varieties, and subcellular localization analysis was performed in tobacco leaf. In this study, 22 CsARF genes were identified and found to be unevenly distributed across 9 chromosomes of the cannabis genome. Phylogenetic analysis revealed that the ARF proteins were divided into 4 subgroups. Duplication analysis identified one pair of segmental/whole-genome duplicated CsARF, and three pairs of tandemly duplicated CsARF. Collinearity analysis revealed that two CsARF genes, CsARF4 and CsARF19, were orthologous in both rice and soybean. Furthermore, subcellular localization analysis showed that CsARF2 was localized in the nucleus. Tissue-specific expression analysis revealed that six genes were highly expressed in cannabis male flowers, and among these genes, 3 genes were further found to be highly expressed at different developmental stages of male flowers. Meanwhile, correlation analysis between the expression level of CsARF genes and CBD content in two cultivars 'H8' and 'Y7' showed that the expression level of CsARF13 was negatively correlated with CBD content, while the expression levels of six genes were positively correlated with CBD content. In addition, most of CsARF genes were responsive to IAA treatment. Our study laid a foundation for the further studies of CsARFs function in cannabis, and provides candidate genes for breeding varieties with high CBD yield in cannabis production.

Screening and evaluation of antibacterial active strains of Actinomycetes isolated from Northern Indian soil for biofilm inhibition against selected ESKAPE pathogens

AbstractThe growing problem of antimicrobial resistance (AMR) globally has warranted the search for new and alternate anti-infective strategies. This study aimed to isolate and screen soil Actinomycetes from three different locations in Northern India for their broad-spectrum antibacterial and antibiofilm activity against certain drug-resistant bacteria from ESKAPE group. A total of 110 isolates of Actinomycetes were screened for antibacterial activity using overlay and agar-well diffusion assay against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Ninety-five isolates showed antibacterial activity against one or more test bacteria. These isolates were tentatively identified as members of genus Streptomyces. Five isolates (IMA13, IMA25, IMA43, IMA46, and IMA47) demonstrated broad-spectrum activity against test pathogens and were subjected to production of secondary metabolites in liquid medium. Ethyl acetate fraction of culture filtrate showed MIC in the range of 64–512 µg/ml against test bacteria. The most active isolate, IMA-46, was identified by 16srRNA gene sequence analysis as Streptomyces rameus. Further, ethyl acetate fraction from S. rameus-IMA46 was used to inhibit biofilm formation at sub-MIC values. A significant level (50% to 92%) of biofilm inhibition was recorded against test pathogens. Light microscopy and SEM analysis revealed the biofilm structural changes, reduction in cell aggregation and EPS production. LC/MS analysis of the active extract of S. rameus IMA46 showed the presence of many biologically active compounds, including tetracycline and doxycycline. The present study highlights the potential of soil Actinomycetes in production of broad-spectrum anti-infective compounds in combating growing problem of AMR.