Production Of Secondary Metabolites Research Articles

Streptomyces sungeiensis SD3 as a Microbial Chassis for the Heterologous Production of Secondary Metabolites.

We present the newly isolated Streptomyces sungeiensis SD3 strain as a promising microbial chassis for heterologous production of secondary metabolites. S. sungeiensis SD3 exhibits several advantageous traits as a microbial chassis, including genetic tractability, rapid growth, susceptibility to antibiotics, and metabolic capability supporting secondary metabolism. Genomic and transcriptomic sequencing unveiled the primary metabolic capabilities and secondary biosynthetic pathways of S. sungeiensis SD3, including a previously unknown pathway responsible for the biosynthesis of streptazone B1. The unique placement of S. sungeiensis SD3 in the phylogenetic tree designates it as a type strain, setting it apart from other frequently employed Streptomyces chassis. This distinction makes it the preferred chassis for expressing biosynthetic gene clusters (BGCs) derived from strains within the same phylogenetic or neighboring phylogenetic clade. The successful expression of secondary biosynthetic pathways from a closely related yet slow-growing strain underscores the utility of S. sungeiensis SD3 as a heterologous expression chassis. Validation of CRISPR/Cas9-assisted genetic tools for chromosomal deletion and insertion paved the way for further strain improvement and BGC refactoring through rational genome editing. The addition of S. sungeiensis SD3 to the heterologous chassis toolkit will facilitate the discovery and production of secondary metabolites.

Climate-induced changes in essential oil production and terpene composition in alpine aromatic plants

Alpine regions are biodiversity hotspots for endemic species with unique adaptations to harsh climatic conditions. These adaptations often involve specialized behaviours and metabolism including the production of secondary metabolites that protects plants from environmental stresses. This study examines the impacts of elevated CO2 (eCO2) and combined effects of elevated CO2 and temperature (eCO2+T) on two alpine aromatic herbs, namely Angelica glauca and Nardostachys jatamansi. These species were raised in open-top chambers (OTCs) and treated with elevated CO2 (600 ppm) and temperature (+2.0 °C from control) during 2018 to 2020. The primary (total soluble sugar, starch, total soluble protein, amino acid and proline contents), secondary metabolites (Phenolics, essential oil yield, and components using GC-MS) and antioxidant activities (superoxide dismutase and peroxidase) were measured. Total soluble sugar increased under eCO2, while starch content, total amino acid and total soluble protein initially increased but later declined in response to the treatments. Proline content and antioxidant activities were higher under eCO2 and eCO2+T treatments. Total phenolic content showed both increasing (in A. glauca) and decreasing (in N. jatamansi) responses to eCO2 and eCO2+T treatments. GC–MS analysis showed decreased monoterpenes in CO2 and temperature treatments, but some sesquiterpenes showed mixed responses with both increases and decreases. These findings suggest that changes in climate can alter the composition of secondary metabolites in alpine herbs, potentially affecting their therapeutic properties and suitability for use in herbal medicine. This study provides valuable insights into the species-specific physiological responses of plants to changing climate.

Antibacterial activity of endophytic fungi associated with Aspidosperma subincanum Mart. against Gram-positive bacteria

Endophytic fungi are microorganisms that inhabit plant tissues, without causing damage to the host. When in association with medicinal plants, they are able to prompt the production of secondary metabolites with important biological activities. The aim of this study was to evaluate the antibacterial activity of endophytic fungi associated with Aspidosperma subincanum against Gram positive bacteria of clinical relevance and perform the chemical characterization of the extract with the highest antibacterial activity. Endophytic fungi isolated from leaves of A. subincanum were submitted to ethanolic extraction. The antibacterial activity of the extracts was evaluated against S. aureus ATCC 25923, using the microdilution method. From a total of 116 extracts, only 4 (3.44%) were considered positive and then evaluated against B. cereus. The ethanolic endophytic extract AM59.1-98 was able to completely inhibit the growth of both microorganisms and was selected for chemical characterization, which indicated the presence of saponins and tannins. Further studies have to be conducted to confirm if the chemical substances detected are those responsible for its antibacterial activity. The current research provides, for the first time, substantial results concerning the antibacterial activity of endophytes associated with A. subincanum as an alternative path in the search for new drugs.

Enhanced Antioxidant Activity and Secondary Metabolite Production in Tartary Buckwheat under Polyethylene Glycol (PEG)-Induced Drought Stress during Germination

Drought stress is one of the key factors impeding agricultural productivity worldwide. This experiment aimed at investigating the polyethylene glycol (PEG)-induced drought stress effects on seed germination, physiology, and biochemical mechanisms in Tartary buckwheat genotypes. Four PEG-induced stress conditions (0%, 10%, 20%, and 30%) were applied to 14 selected genotypes at the germination stage to evaluate their stress tolerance capacity. Significant differences were obtained in germination percentage, relative water content (RWC), and all growth parameters among the studied 14 genotypes. Based on the stress tolerance index (STI), XiNong 9943, XiNong 9940, and QianKu-5 were found to be tolerant, and QuanKu-4 was susceptible. These cultivars were selected for further physiological and biochemical characterization. The results demonstrated that the activity of enzymes was significantly increased with the increase in PEG dose. SOD (superoxide dismutase), POD (peroxidase), CAT (catalase), and APX (ascorbate peroxidase) levels obtained at 30% PEG in the XiNong 9943 genotype were 2.01, 2.19, 4.92, and 4.46 times higher, respectively, than the normal growth condition (T0). Moreover, the secondary metabolite content also increased with the increase in PEG dose. At 30% PEG, the genotype XiNong 9943 yielded phenols, flavonoids, polyphenol oxidase (PPO), and phenylalanine ammonia lyase (PAL) levels that were higher by 131%, 95%, 154%, and 164%, respectively, than T0 condition. From both the findings of the activity of enzymes and the secondary metabolite content, the genotypic response to drought was ranked in the following order: XiNong 9943 > XiNong 9940 > QianKu-5 > QianKu-4, which supported the STI selection system. Assessing the overall performance, the genotype XiNong 9943 shows drought tolerance, which can be useful material for future buckwheat breeding programs.

Updating the Knowledge on the Secretory Machinery of Hops (Humulus lupulus L., Cannabaceae).

Cannabaceae species garner attention in plant research due to their diverse secretory structures and pharmacological potential associated with the production of secondary metabolites. This study aims to update our understanding of the secretory system in Hops (Humulus lupulus L.), an economically important species especially known for its usage in beer production. For that, stems, leaves, roots, and inflorescences were collected and processed for external morphology, anatomical, histochemical, ultrastructural and cytochemical analyses of the secretory sites. Our findings reveal three types of secretory structures comprising the secretory machinery of Hops: laticifer, phenolic idioblasts and glandular trichomes. The laticifer system is articulated, anastomosing and unbranched, traversing all plant organs, except the roots. Phenolic idioblasts are widely dispersed throughout the leaves, roots and floral parts of the species. Glandular trichomes appear as two distinct morphological types: capitate (spherical head) and peltate (radial head) and are found mainly in foliar and floral parts. The often-mixed chemical composition in the secretory sites serves to shield the plant from excessive UVB radiation, elevated temperatures, and damage inflicted by herbivorous animals or pathogenic microorganisms. Besides the exudate from peltate glandular trichomes (lupulin glands), latex and idioblast content are also likely contributors to the pharmacological properties of different Hop varieties, given their extensive presence in the plant body.

Cultural and Morphological Characterization of Streptomyces and Interaction Study with Sclerotium rolfsii by SEM

Stem rot caused by Sclerotium rolfsii Sacc. is a major constraint in the production and productivity of the groundnut. Management of stem rot disease is very difficult due to soil borne nature, wide host range of S. rolfsii and lack of disease resistance in existing commercial cultivars. Biological control with potential Streptomyces is receiving greater attention all over the world. Studies were conducted on collection and isolation of Streptomyces, their mechanism of action, cultural and morphological characteristics, its efficacy against S. rolfsii with Scanning electron microscope(SEM). Roving survey was conducted in major groundnut growing regions of Andhra Pradesh. During survey 180 soil samples were collected from groundnut rhizosphere and 50 Streptomyces isolates were isolated from different soil samples. Twenty Streptomyces isolates were tested for their growth rate and antifungal activity against groundnut stem rot pathogen, S.rolfsii under in vitro conditions using dual culture method. Among twenty isolates of Streptomyces, five isolates showed maximum inhibition of S. rolfsii. These five isolates were characterized based on cultural and morphological characters. The study involved in differentiating various characters viz., type of spore chain, colony margins, shape of spore chains, colour of spore mass and spore surface. The morphology of the spore chains varied across the isolates showing the filamentous (Ggd), spiral (Kyd and Lrp) and Rectus flexibilis (Kdr and Mkc). SEM studies revealed that inhibition of the growth of S. rolfsii by Streptomyces was mainly due to exhibition of irregular distortions at contact site of the pathogen and collapse of the fungal hyphae and this might be due to the production of secondary metabolites and extracellular enzymes.

SecMet-FISH: labeling, visualization, and enumeration of secondary metabolite producing microorganisms.

Our understanding of the role of secondary metabolites in microbial communities is challenged by intrinsic limitations of culturing bacteria under laboratory conditions and hence cultivation independent approaches are needed. Here, we present a protocol termed Secondary Metabolite FISH (SecMet-FISH), combining advantages of gene-targeted fluorescence in situ hybridization (geneFISH) with in-solution methods (in-solution FISH) to detect and quantify cells based on their genetic capacity to produce secondary metabolites. The approach capitalizes on the conserved nature of biosynthetic gene clusters (BGCs) encoding adenylation (AD) and ketosynthase (KS) domains, and thus selectively targets the genetic basis of non-ribosomal peptide and polyketide biosynthesis. The concept relies on the generation of amplicon pools using degenerate primers broadly targeting AD and KS domains followed by fluorescent labeling, detection, and quantification. Initially, we obtained AD and KS amplicons from Pseuodoalteromonas rubra, which allowed us to successfully label and visualize BGCs within P. rubra cells, demonstrating the feasibility of SecMet-FISH. Next, we adapted the protocol and optimized it for hybridization in both Gram-negative and Gram-positive bacterial cell suspensions, enabling high-throughput single cell analysis by flow cytometry. Ultimately, we used SecMet-FISH to successfully distinguish secondary metabolite producers from non-producers in a five-member synthetic community.

Mechanism of the Synergistic Toxicity of Ampicillin and Cefazoline on Selenastrum capricornutum.

Ampicillin (AMP) and cefazolin (CZO) are commonly used β-lactam antibiotics which are extensively globally produced. Additionally, AMP and CZO are known to have relatively high ecotoxicity. Notably, the mix of AMP and CZO creates a synergistic effect that is more harmful to the environment, and how exposure to AMP-CZO can induce synergism in algae remains virtually unknown. To yield comprehensive mechanistic insights into chemical toxicity, including dose-response relationships and variations in species sensitivity, the integration of multiple endpoints with de novo transcriptomics analyses were used in this study. We employed Selenastrum capricornutum to investigate its toxicological responses to AMP and CZO at various biological levels, with the aim of elucidating the underlying mechanisms. Our assessment of multiple endpoints revealed a significant growth inhibition in response to AMP at the relevant concentrations. This inhibition was associated with increased levels of reactive oxygen species (ROS) and perturbations in nitrogen metabolism, carbohydrate metabolism, and energy metabolism. Growth inhibition in the presence of CZO and the AMP-CZO combination was linked to reduced viability levels, elevated ROS production, decreased total soluble protein content, inhibited photosynthesis, and disruptions in the key signaling pathways related to starch and sucrose metabolism, ribosome function, amino acid biosynthesis, and the production of secondary metabolites. It was concluded from the physiological level that the synergistic effect of Chlorophyll a (Chla) and Superoxide dismutase (SOD) activity strengthened the growth inhibition of S. capricornutum in the AMP-CZO synergistic group. According to the results of transcriptomic analysis, the simultaneous down-regulation of LHCA4, LHCA1, LHCA5, and sodA destroyed the functions of the photosynthetic system and the antioxidant system, respectively. Such information is invaluable for environmental risk assessments. The results provided critical knowledge for a better understanding of the potential ecological impacts of these antibiotics on non-target organisms.

The roles of SARP family regulators involved in secondary metabolism in Streptomyces.

Streptomyces species are best known for their ability to produce abundant secondary metabolites with versatile bioactivities and industrial importance. These metabolites are usually biosynthesized through metabolic pathways encoded by cluster-situated genes. These genes are also known as biosynthetic gene clusters (BGCs) of secondary metabolites. The expression of BGCs is intricately controlled by pyramidal transcriptional regulatory cascades, which include various regulators. Streptomyces antibiotic regulatory proteins (SARPs), a genus-specific family of regulators, are widely distributed and play important roles in regulating the biosynthesis of secondary metabolites in Streptomyces. Over the past decade, the biological functions of SARPs have been extensively investigated. Here, we summarized the recent advances in characterizing the roles of SARPs involved in Streptomyces secondary metabolism from the following three aspects. First, the classification and domain organization of SARPs were summarized according to their size variation. Second, we presented a detailed description of the regulatory mechanisms and modes of action of SARPs involved in secondary metabolism. Finally, the biotechnological application of SARPs was illustrated by improving the production of target secondary metabolites and discovering novel bioactive natural products. This review will help researchers to comprehensively understand the roles of SARPs in secondary metabolite biosynthesis in Streptomyces, which will contribute to building a solid foundation for their future application in synthetic biology.

Induction, Proliferation and Differentiation of Callus in Paris polyphylla Sm. through Leaf Culture

Paris polyphylla Sm. is a vulnerable medicinal plant employed in the treatment of various ailments. This study seeks to establish a protocol for callus induction, proliferation, and differentiation of P. polyphylla. Immature leaf explants were cultured on MS medium with varying concentrations of plant growth regulators (PGRs), including 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (Kn), 6-benzylaminopurine (BAP), Thidiazuron (TDZ), α-Naphthalene acetic acid (NAA), and Gibberellic acid (GA3), along with 10% coconut water. After 12 weeks of primary culture, the optimal callus induction was observed in MS medium supplemented with 0.25 mg/l 2,4-D + 0.5 mg/l Kn. In the secondary culture at 8 weeks, the best callus proliferation, as determined by callus weight or growth index, occurred in MS medium supplemented with 2.0 mg/l BAP alone, 2.0 mg/l Kn alone, 1.0 mg/l TDZ alone, combinations of 2.0 mg/l Kn + 1.0 mg/l BAP + 2.0 mg/l GA3, and combinations of 0.5 mg/l NAA + 2.0 mg/l BAP + 2.0 mg/l GA3, as well as 10% coconut water. Furthermore, callus differentiation into mini rhizomes with root primordia was successfully achieved in MS media containing 2.5 mg/l Kn and 10% coconut water. This study reports, for the first time, the formation and differentiation of callus from leaf explants in P. polyphylla. Large-scale callus generation from leaf explants has the potential to enhance the production of bioactive secondary metabolites for therapeutic purposes and facilitate the development of plantlets through organogenesis.

Postharvest Chemical Treatment of Physiologically Induced Stem End Blockage Improves Vase Life and Water Relation of Cut Flowers

Wound-induced xylem occlusion significantly affects the vase life of cut flowers, as oxidative stress and the polymerization of phenolic compounds lead to the deposition of phenolic compounds/secondary metabolites in the stem ends of cut flowers to heal open tissues of freshly cut stems and prevent microbial invasion. However, this deposition causes blockage of vessels, reduced water uptake, and shortened vase life. The physiological plugging of vessels is linked with various oxidative enzymes’ (PAL, PPOs, LACs, and COs) actions taken to increase the synthesis of different compounds, e.g., lignin, suberin, tyloses, gel, and latex, in wounded areas. The use of chemical preservatives/enzyme inhibitors is one of the safest and most efficient techniques employed to minimize vascular blockage and inhibit phenolic compounds deposition and exudation. This review mainly discusses the types of oxidative enzymes, their pathways and biochemistry along with production of secondary metabolites, their biosynthesis, and their modes of action involved in vascular blockage. It also summarizes the different types of preservatives used in postharvest treatments to improve relative water uptake, flower fresh weight, petal protein content, and hydraulic conductance and prolong the vase life of cut flowers during storage. It is hoped that this elaborate study will help researchers in designing new studies concerning occlusion caused by the accumulation of phenolic compounds in vessels.

Penicillium janthinellum: A Potential Producer of Natural Products

Penicillium is a kind of common filamentous fungi yielding high levels of secondary metabolites with diverse structures and attractive activities. Among these fungi, Penicillium janthinellum is a potential producer of secondary metabolites whose natural products have been noticed due to their various chemical structures and biological activities. This review summarizes the sources, distribution, bioactivities and structural characteristics of compounds isolated from P. janthinellum from 1980 to 2023. A total of 153 natural products have been isolated from P. janthinellum, of which 65 were new compounds. The compounds separated from P. janthinellum exhibit diverse skeletal chemical structures, concentrated in the categories of polyketides (40%), alkaloids (31%) and terpenoids (14%). P. janthinellum-derived compounds display attractive biological activities, such as cytotoxic, antibacterial, antifungal and antiviral activities. These results indicate that P. janthinellum is a potential fungus for producing bioactive secondary metabolites which can be used as precursors for new drugs.

Effect of active layer-by-layer edible coating on quality, biochemicals, and the antioxidant system in ready-to-eat ‘Williams’ pear fruit during cold storage

This study tested an active edible coating on the quality and storage capacity of fresh-cut pear stored at 2 ± 0.5 °C for 10 days by incorporating oxalic (OA) and citric acid (CA) into carboxymethyl cellulose (CMC) and sodium alginate (SA). The physicochemical parameters, secondary metabolite and amino acid contents, and antioxidant activities were determined. Furthermore, enzymes related to oxidative stress and browning, and relative markers were also analysed. The results showed that ready-to-eat pear coated with CMC+SA+OA+CA had a significant inhibitory effect on physiological decay, minimizing the TSS and pH increase and TA decrease. In addition, coating application improved the production of secondary metabolites. The activity of all enzymes involved in ROS detoxification was shown to be promoted by the application of the coating. The degree of browning and oxidative damage that increase progressively with increasing storage time in physiological condition, resulting inhibited by coating. Furthermore, this treatment significantly reduced the protein degradation. Therefore, using the CMC+SA+OA+CA edible coating has the potential to hinder decay and greatly extend the storage period of 'Williams' pear fruit in cold storage.

Identification and genomic characterization of Pseudomonas spp. displaying biocontrol activity against Sclerotinia sclerotiorum in lettuce.

Lettuce is an economically major leafy vegetable that is affected by numerous diseases. One of the most devastating diseases of lettuce is white mold caused by Sclerotinia sclerotiorum. Control methods for this fungus are limited due to the development of genetic resistance to commonly used fungicides, the large number of hosts and the long-term survival of sclerotia in soil. To elaborate a new and more sustainable approach to contain this pathogen, 1,210 Pseudomonas strains previously isolated from agricultural soils in Canada were screened for their antagonistic activity against S. sclerotiorum. Nine Pseudomonas strains showed strong in vitro inhibition in dual-culture confrontational assays. Whole genome sequencing of these strains revealed their affiliation with four phylogenomic subgroups within the Pseudomonas fluorescens group, namely Pseudomonas corrugata, Pseudomonas asplenii, Pseudomonas mandelii, and Pseudomonas protegens. The antagonistic strains harbor several genes and gene clusters involved in the production of secondary metabolites, including mycin-type and peptin-type lipopeptides, and antibiotics such as brabantamide, which may be involved in the inhibitory activity observed against S. sclerotiorum. Three strains also demonstrated significant in planta biocontrol abilities against the pathogen when either inoculated on lettuce leaves or in the growing substrate of lettuce plants grown in pots. They however did not impact S. sclerotiorum populations in the rhizosphere, suggesting that they protect lettuce plants by altering the fitness and the virulence of the pathogen rather than by directly impeding its growth. These results mark a step forward in the development of biocontrol products against S. sclerotiorum.

Optimization of induction and hairy root culture establishment in two mullein species, Verbascum erianthum and Verbascum stachydiforme

The genus Verbascum, belonging to the family Scrophulariaceae, has a significant center of diversity in Iran. Two of its species, V. erianthum and V. stachydiforme, originate in the Iranian-Turanian region, but no studies have been conducted on the induction of their hairy roots. This genus is a valuable source of biologically active compounds such as iridoid glycosides and flavonoids. Hairy root culture is a suitable technique for the production and accumulation of secondary metabolites. Three different studies were conducted to optimize the induction and establishment of hairy roots. In the first experiment, the influence of explant type (leaf and hypocotyl), six infection methods, and co-culture time (48 and 72 h) on the efficiency of hairy root induction was investigated. The results showed that the highest hairy root induction (68.18%) was observed in the leaf explants inoculated by direct infection with three wounds within 72 h co-culture time. In the second experiment, the effect of four Agrobacterium rhizogenes strains (ATCC 15834, A4, A7, and A13) and leaf age (14, 21, and 28 days) on transformation efficiency and some morphological traits examined in both species were studied. The high transformation efficiency of hairy root (80.55%) was detected in the 21-day-old leaf explant of V. erianthum species that was inoculated with the A13 strain. The transformed hairy root colons were confirmed by PCR using rolB gene-specific primers. To optimize hairy root growth and avoid tissue browning, hairy roots were cultured in various media containing different antioxidants and improver agents (including ascorbic acid, citric acid, and NAA). The results showed that the highest fresh growth index (20.42) and the lowest tissue browning (9%) as well as total phenol (8.51 mg GA/g DW), and total flavonoid content (4.42 mg QUE/g DW) were obtained in medium B5 with 1.5 mg/l NAA.

Effect of Some Ecological Factors on Stomata and Phenolic Compounds Production in Aloe Vera L. Gel and Effect Gel to the Adult of Flour Beetle: Tribolium Castaneum (Coleoptera : Tenebriodae)

This search presents the effect of some ecological factors such as (ultraviolet –B radiation , temperature , salinity ) on stomatal index and stomatal frequency and phenolic compounds after exposure to Aloe vera L. UV-B radiation , temperature and salinity . Ultraviolet – B radiation is one of the important abiotic factors that can stimulate the production of secondary metabolites, including phenolic compounds. Aloe vera (Asphodelaceae) is a medicinal plant used in Iraq for treating sunburn, wounds, and heartburn. The flour beetle: Tribolium castaneum is one of the inset causes of loss storage food yield in the world. The study aims the effect of some ecological factors such as (ultraviolet –B radiation , temperature , salinity ) on stomatal index and stomatal frequency and phenolic compounds after exposure to Aloe vera L. UV-B radiation , temperature and salinity and investigate the insecticidal and repellency effect of leaf gel for Aloe vera to estimate the percentage of the insecticidal effect for concentrations 25, 50, 75, and 100 % of alcoholic extract within seven days on adult insects. The gel in leaf exposure to UV-B radiation contained a higher diversity of phenolic compounds and flavonoids. This search showed a high percent kill for an adult with 100% of the alcoholic extract compared with control and an excellent effect of Aloe vera gel at a concentration of 50 % for a repellency effect with a value of 10.842%. Phenolic compounds showed after exposure to UV as : (dioxyflavone ,kampferol, catechuic acid, flavonol glucuronic acid, gabardine isoflavone, isoquercitrin, p-coumaric acid, hispaglabridine ) with a high percentage in the gel after exposure to UV-B compared by control.

Pesticides: Unintended Impact on the Hidden World of Gut Microbiota.

A vast range of pesticides have been routinely employed for plant protection throughout the last few decades. Pesticides can enter non-target organisms in various ways, posing health hazards. Exposure to different environmental pollutants, including pesticides, can affect the human gut flora. Metabolites generated from the gut microbiota play an essential role in the host's health by regulating metabolic homeostasis. A disruption in this equilibrium can lead to the emergence of numerous illnesses and their etiology. Pesticides have been shown in a few recent studies to harm the host's gut microbiome. As a result, there is an urgent need to investigate the impact of pesticides on gut microbiota-mediated immunity. Metabolic alterations in the host may give a better understanding of pesticide-induced harm. This review highlights the potential consequences of pesticide exposure on gut microbiota composition and function, mainly focusing on how it might alter the production of secondary metabolites with potential downstream implications for host health.

Unraveling the functional characteristics of endophytic bacterial diversity for plant growth promotion and enhanced secondary metabolite production in Pelargonium graveolens

The rich diversity of microbial endophytic communities associated with plants, often referred to as the second genome, serves as a compelling illustration of efficient co-evolution. This noteworthy partnership plays a pivotal role in sustaining plant well-being and enhancing plant adaptability across diverse habitats. Therefore, examining the diversity of endophytic microbes associated with their particular host plant is valuable for gaining insights into the vast spectrum of plant-microbe interactions. The present experiments aimed at investigating the bacterial endophytic diversity in both root and shoot tissues of Pelargonium graveolens, employing culture dependent and culture independent high-throughput metagenomics approach. A total of 614 and 620 operational taxonomic units (OTUs), encompassing 291 and 229 genera, were identified in the shoot and root tissues of P. graveolens, respectively. Furthermore, the subsequent classification of OTUs revealed 15 highly abundant phyla, with Proteobacteria dominating both root and shoot tissues. Notably, an exceptionally high abundance of Firmicutes phyla was observed in the shoot compared to the root. Additionally, 30 bacterial endophytes from the root, stem, petiole, and leaves were isolated and molecularly characterized, unveiling a consistent pattern of diversity distribution between the root and shoot of P. graveolens. Upon screening all isolates for plant growth promoting traits, Pseudomonas oryzihabitans was found to be positive for major biochemical test like nitrogen fixation, phosphate solubilization etc. and on inoculation resulted in about two-fold increase in content of essential oil accompanied by a significant rise in the geraniol and citronellol content. Diving deep into the genetic constitution of P. oryzihabitans unveiled a substantial number of genes directly and indirectly contributing to the endophyte’s capability in colonizing host plants effectively. In summary, data obtained from metagenomics and culture dependent approaches including glass house trials suggest potential bacterial endophytes suitable for field applications for yield enhancement and in planta secondary metabolite enhancement investigations.

Growth enhancement, metabolic profile improvement, and DXR and TPS2 gene expression changes in Thymus vulgaris L. by cyanobacterial inoculation.

In recent decades cyanobacterial species have attracted research attention as potential sources of new biostimulants. In this study, the biostimulant effects of five cyanobacterial suspensions on the growth and essential oil composition of Thymus vulgaris L. were evaluated. The expression of key genes involved in the biosynthesis of thymol and carvacrol, such as DXR and TPS2, were investigated. A pot culture experiment revealed that cyanobacterial application significantly improved T. vulgaris L. growth indices, including plant height, dry and fresh weight, leaf and flower number, leaf area, and photosynthetic pigment content. Total phenol and flavonoid content in inoculated plants also showed a significant increase compared with the control. Anabaena torulosa ISB213 inoculation significantly increased root and shoot biomass by about 65.38% and 92.98% compared with the control, respectively. Nostoc calcicola ISB215 inoculation resulted in the highest amount of essential oil accumulation (18.08 ± 0.62) in T. vulgaris leaves, by about 72.19% compared with the control (10.5 ± 0.50%). Interestingly, the amount of limonene in the Nostoc ellipsosporum ISB217 treatment (1.67%) increased significantly compared with the control and other treatments. The highest expression rates of DXR and TPS2 genes were observed in the treatment of N. ellipsosporum ISB217, with 5.92-fold and 5.22-fold increases over the control, respectively. This research revealed the potential of the cyanobacteria that were studied as promising biostimulants to increase the production of biomass and secondary metabolites of T. vulgaris L., which could be a suitable alternative to chemical fertilizers. © 2024 Society of Chemical Industry.

The serine/threonine protein kinase MpSTE1 directly governs hyphal branching in Monascus spp.

Monascus spp. are commercially important fungi due to their ability to produce beneficial secondary metabolites such as the cholesterol-lowering agent lovastatin and natural food colorants azaphilone pigments. Although hyphal branching intensively influenced the production of these secondary metabolites, the pivotal regulators of hyphal development in Monascus spp. remain unclear. To identify these important regulators, we developed an artificial intelligence (AI)–assisted image analysis tool for quantification of hyphae-branching and constructed a random T-DNA insertion library. High-throughput screening revealed that a STE kinase, MpSTE1, was considered as a key regulator of hyphal branching based on the hyphal phenotype. To further validate the role of MpSTE1, we generated an mpSTE1 gene knockout mutant, a complemented mutant, and an overexpression mutant (OE::mpSTE1). Microscopic observations revealed that overexpression of mpSTE1 led to a 63% increase in branch number while deletion of mpSTE1 reduced the hyphal branching by 68% compared to the wild-type strain. In flask cultures, the strain OE::mpSTE1 showed accelerated growth and glucose consumption. More importantly, the strain OE::mpSTE1 produced 9.2 mg/L lovastatin and 17.0 mg/L azaphilone pigments, respectively, 47.0% and 30.1% higher than those of the wild-type strain. Phosphoproteomic analysis revealed that MpSTE1 directly phosphorylated 7 downstream signal proteins involved in cell division, cytoskeletal organization, and signal transduction. To our best knowledge, MpSTE1 is reported as the first characterized regulator for tightly regulating the hyphal branching in Monascus spp. These findings significantly expanded current understanding of the signaling pathway governing the hyphal branching and development in Monascus spp. Furthermore, MpSTE1 and its analogs were demonstrated as promising targets for improving production of valuable secondary metabolites.Key points• MpSTE1 is the first characterized regulator for tightly regulating hyphal branching• Overexpression of mpSTE1 significantly improves secondary metabolite production• A high-throughput image analysis tool was developed for counting hyphal branching