Alkaloid Production Research Articles

Repurposing endogenous type I-E CRISPR-Cas systems for natural product discovery in Streptomyces.

The multifunctional proteins of class 2 CRISPR systems such as Cas9, have been employed to activate cryptic biosynthetic gene clusters (BGCs) in Streptomyces, which represent a large and hidden reservoir of natural products. However, such approaches are not applicable to most Streptomyces strains with reasons to be comprehended. Inspired by the prevalence of the class 1 subtype especially the type I-E CRISPR system in Streptomyces, here we report the development of the type I-E CRISPR system into a series of transcriptional regulation tools. We further demonstrate the effectiveness of such activators in nine phylogenetically distant Streptomyces strains. Using these tools, we successfully activate 13 out of 21 BGCs and lead to the identification and characterization of one polyketide, one Ripp and three alkaloid products. Our work is expected to have a profound impact and to facilitate the discovery of numerous structurally diverse compounds from Streptomyces.

De novo production of protoberberine and benzophenanthridine alkaloids through metabolic engineering of yeast

Protoberberine alkaloids and benzophenanthridine alkaloids (BZDAs) are subgroups of benzylisoquinoline alkaloids (BIAs), which represent a diverse class of plant-specialized natural metabolites with many pharmacological properties. Microbial biosynthesis has been allowed for accessibility and scalable production of high-value BIAs. Here, we engineer Saccharomyces cerevisiae to de novo produce a series of protoberberines and BZDAs, including palmatine, berberine, chelerythrine, sanguinarine and chelirubine. An ER compartmentalization strategy is developed to improve vacuole protein berberine bridge enzyme (BBE) activity, resulting in >200% increase on the production of the key intermediate (S)-scoulerine. Another promiscuous vacuole protein dihydrobenzophenanthridine oxidase (DBOX) has been identified to catalyze two-electron oxidation on various tetrahydroprotoberberines at N7-C8 position and dihydrobenzophenanthridine alkaloids. Furthermore, cytosolically expressed DBOX can alleviate the limitation on BBE. This study highlights the potential of microbial cell factories for the biosynthesis of a diverse group of BIAs through engineering of heterologous plant enzymes.

Multi-Omics Analysis Reveals Molecular Responses of Alkaloid Content Variations in Lycoris aurea Across Different Locations.

Lycoris aurea, celebrated for its visually striking flowers and significant medicinal value due to the presence of alkaloids such as lycorine and galanthamine, has intricate yet poorly understood regulatory mechanisms. This study provides a detailed examination of the transcriptomic, metabolomic and ecological dynamics of L. aurea, aiming to elucidate the underlying molecular mechanisms of alkaloid biosynthesis. Our comparative analysis across different ecological settings highlighted key genes involved in alkaloid biosynthesis, such as genes encoding aldehyde dehydrogenase and norbelladine 4'-O-methyltransferase, which were distinctively increased in the high alkaloids-producing group. We identified a total of 6871 differentially expressed genes and 915 metabolites involved in pathways like terpenoid backbone biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis. Protein interaction network analysis revealed significant upregulation of photosynthesis, photosystem and photosynthetic membrane pathways in the alkaloids-producing region. Furthermore, our research delineated the interactions among soil microbial communities, genes and plant and soil biochemical properties, noting that bacterial populations correlate with soil properties that favour the activation of metabolic pathways essential for alkaloid production. Collectively, this study advances our understanding of the genetic and metabolic alkaloid biosynthesis pathways in L. aurea, shedding light on the complex interactions that govern alkaloid production.

Unveiling the impact of nitrogen deficiency on alkaloid synthesis in konjac corms (Amorphophallus muelleri Blume)

BackgroundKonjac corms are known for their alkaloid content, which possesses pharmacological properties. In the primary cultivation areas of konjac, nitrogen deficiency is a common problem that significantly influences alkaloid synthesis. The impact of nitrogen deficiency on the alkaloids in konjac corms remains unclear, further complicated by the transition from mother to daughter corms during their growth cycle.ResultsThis study examined 21 alkaloids, including eight indole alkaloids, five isoquinoline alkaloids, and eight other types of alkaloids, along with the associated gene expressions throughout the development of Amorphophallus muelleri Blume under varying nitrogen levels. Nitrogen deficiency significantly reduced corm diameter and fresh weight and delayed the transformation process. Under low nitrogen conditions, the content of indole alkaloids and the expression of genes involved in their biosynthesis, such as tryptophan synthase (TRP) and tryptophan decarboxylase (TDC), exhibited a substantial increase in daughter corms, with fold changes of 61.99 and 19.31, respectively. Conversely, in the mother corm, TDC expression was markedly reduced, showing only 0.04 times the expression level observed under 10 N treatment. The patterns of isoquinoline alkaloid accumulation in corms subjected to nitrogen deficiency were notably distinct from those observed for indole alkaloids. The accumulation of isoquinoline alkaloids was significantly higher in mother corms, with expression levels of aspartate aminotransferase (GOT), chorismate mutase (CM), tyrosine aminotransferase (TAT), and pyruvate decarboxylase (PD) being 4.30, 2.89, 921.18, and 191.40 times greater, respectively. Conversely, in daughter corms, the expression levels of GOT and CM in the 0 N treatment were markedly lower (0.01 and 0.83, respectively) compared to the 10 N treatment.ConclusionsThe study suggests that under nitrogen deficiency, daughter corms preferentially convert chorismate into tryptophan to synthesize indole alkaloids, while mother corms convert it into tyrosine, boosting the production of isoquinoline alkaloids. This research provides valuable insights into the mechanisms of alkaloid biosynthesis in A. muelleri and can aid in developing nitrogen fertilization strategies and in the extraction and utilization of alkaloids.

Functional characterization of CYP96T1-like cytochrome P450 from Lycoris aurea catalyzing para-para' and para-ortho' oxidative coupling in Amaryllidaceae alkaloids biosynthesis.

Amaryllidaceae alkaloids (AAs) are complex plant secondary metabolites possessing a wide range of biological activities. 4'-O-methylnorbelladine (4OMN) is the branchpoint intermediate for the entire AAs, and was the last common intermediate before AA pathway branches diverge. The cyclization of 4OMN by C-C oxidative coupling, which can afford para-para', ortho-para', and para-ortho' scaffold, was catalyzed by cytochrome P450 96T (CYP96T) family enzymes. To clarify the mechanisms involved in this controversial step, four CYP96T homologs (LauCYP96T1, LauCYP96T1-like-1, LauCYP96T1-like-2 and LauCYP96T1-like-3) were cloned from the full-length transcriptome of Lycoris aurea. All the four LauCYP96T are localized to endoplasmic reticulum. Functional analysis reveals that LauCYP96T1 and LauCYP96T1-like proteins display inverted regioselectivity for oxidative coupling of 4OMN, in which LauCYP96T1 and LauCYP96T1-like-2 dominantly afford para-para' scaffold, and LauCYP96T1-like-1 and LauCYP96T1-like-3 are responsible for para-ortho' scaffold formation. Using molecular homology modeling and docking studies, we predicted models for the binding of 4OMN to LauCYP96T, and identified two amino acid residues that might be responsible for the dominant changes in generated products of para-ortho' and para-para' oxidative coupling. Our results highlight the functional diversity and promiscuity of LauCYP96T enzymes and might provide valuable information for Amaryllidaceae alkaloid production.

Enhancement of the Production of Tropane Alkaloids in the Hyoscyamus Niger L. Callus Using Different Biotic Elicitors

This study looked at the amount of tropane alkaloids in both the leaves of the mother plant and the callus cultures of Hyoscyamus niger L. It also looked at how biotic elicitation affects the growth of callus cultures and increases the production of alkaloids. The investigation identified and quantified three primary tropane alkaloids (Hyoscyamine, Scopolamine, and Atropine) from both sources. Remarkably, the callus cultures exhibited obviously higher levels of alkaloids in comparison to the leaves of the mother plant, implying their potential as a valuable reservoir for alkaloid production. Concerning the effects of biotic elicitation, the study showed that the application of chitosan (CHT), yeast extract (YE), and fungal extract of Trichoderma asperellum yielded a substantial decrease in callus weight when contrasted with the control group. This decrease became more pronounced with escalating concentrations of the elicitors. In terms of alkaloid synthesis, a clear correlation between the concentration of bio stimulants and tropane alkaloid levels was established. Particularly, CHT elicitation displayed the most pronounced enhancement in levels of tropane alkaloids among the three elicitors. Notably, the highest CHT concentration of 40 mg/L yielded the most elevated levels of alkaloids, measuring at 25.3 µg/g for Hyoscyamine, 31.2 µg/g for Scopolamine, and 21.5 µg/g for Atropine. This represents approximate percentage increases in concentration of 208%, 183%, and 198%, respectively, when compared to the control treatment. Generally, these findings carry significant implications for advancing tropane alkaloid biosynthesis, with potential applications spanning the pharmaceutical and biotechnology sectors.

L-phenylalanine and trans-cinnamic acid combined with Fe3O4-NPs treatment induce oxidative stress and enhances alkaloid production in Narcissus tazetta L. by increasing PAL and N4OMT gene expression

L-phenylalanine and trans-cinnamic acid combined with Fe3O4-NPs treatment induce oxidative stress and enhances alkaloid production in Narcissus tazetta L. by increasing PAL and N4OMT gene expression

Mycorrhizal fungi compromise production of endophytic alkaloids, increasing plant susceptibility to an aphid herbivore

Abstract Symbiosis plays a critical role in plant biology. Temperate grasses often associate with several symbiotic fungi simultaneously, including Epichloë endophytes and arbuscular mycorrhizal (AM) fungi, in shoots and roots, respectively. These symbionts often modulate plant–herbivore interactions by influencing nutritional traits (i.e. AM fungi‐mediated nutrient uptake) and/or the secondary chemistry (i.e. endophytic alkaloids) of their host plant. Moreover, such grasses also accumulate large amounts of silicon (Si) from the soil, which can be deposited in tissues to act as a physical anti‐herbivore defence. Recent evidence suggests that both endophytes and AM fungi independently facilitate Si uptake. However, the consequences of their interactions with piercing‐sucking insects (i.e. aphids), or whether Si supply, endophytes, and AM fungi interact in this regard, are currently unknown. While Si deposition may be less effective against aphids than other herbivores (i.e. chewing caterpillars), Si supply can also alter plant secondary metabolite defences, which could affect sucking insects. In a factorial greenhouse experiment, we evaluated whether these components, acting alone or in combination, altered (1) foliar primary chemistry, (2) Si and symbiont‐chemical (endophytic alkaloids) defences, as well as (3) performance of the bird cherry‐oat aphid (Rhopalosiphum padi) feeding on tall fescue (Festuca arundinacea). Endophytes decreased all aphid performance parameters, including population growth and reproduction by 40%, but their impact was reversed by the presence of AM fungi, leading to a 52% increase in aphid performance compared with plants solely hosting endophytes. This improvement in performance was associated with reduced loline alkaloid levels and higher shoot nitrogen in AM‐endophytic plants. Endophytes and AM fungi exhibited antagonism, with endophytes reducing AM colonization by 34% and AM presence decreasing endophyte loline alkaloids by 44%. While both fungi jointly increased Si accumulation by 39% under Si‐supplied conditions, Si had no noticeable effects on aphids. Moreover, although Si supply had no identifiable effects on AM colonization, it reduced endophyte peramine alkaloids by 24%. Synthesis. Our findings indicate that symbiotic fungal partnerships and silicon provision may benefit plants but could weaken anti‐herbivore defences when combined. Revealing the complex interactions among diverse fungal symbionts and showcasing their effects on different anti‐herbivore defences (chemical and physical) and herbivore performance for the first time.

Enhanced production of scopolamine and atropine alkaloids in in vitro cell suspension cultures of Atropa belladonna L. under 2-iP, 2,4-D, and ornithine

Enhanced production of scopolamine and atropine alkaloids in in vitro cell suspension cultures of Atropa belladonna L. under 2-iP, 2,4-D, and ornithine

Impact of External Sources of Indole Acetic Acid and 2,3,5-Triiodobenzoic Acid on Alkaloid Production and Their Relationships with Primary Metabolism and Antioxidant Activity in Annona emarginata (Schltdl.) H. Rainer.

Annona emarginata is a native Brazilian species capable of producing at least ten alkaloids of ecological, agronomic, and pharmacological importance. Some studies have explored the effect of external phytoregulators on the production of alkaloids, including the effect of auxins, which, like alkaloids, derive from the shikimic acid pathway. Thus, this study aimed to evaluate how indole acetic acid (IAA) and its inhibitor 2,3,5-triiodobenzoic acid (TIBA) impact the production of alkaloids and the primary metabolism of A. emarginata, which brings advances in the understanding of the mechanisms of alkaloid synthesis and can aid in the bioprospection of molecules of interest in Annonaceae. The design was completely randomized, with three treatments (control, IAA [10-6 M] and TIBA [10-6 M]) and five collection times (12, 36, 84, 156, and 324 h). The following variables were analyzed: total alkaloids, alkaloid profile, nitrate reductase activity, gas exchange in photosynthesis, chlorophyll a fluorescence, sugars, starch, and antioxidant activity. Of the twelve alkaloids analyzed, discretine and xylopine were not detected in the control plants; however, both were detected when IAA was applied (in roots and leaves) and xylopine (in roots) when the inhibitor was applied. The alkaloid asimilobine was not detected with the use of TIBA. Variations in alkaloid concentrations occurred in a punctual manner, without significant variations in photosynthesis and nitrate reductase activity, but with variations in the antioxidant system and sugar concentrations, mainly at 156 h, when the highest alkaloid concentrations were observed with the use of TIBA. It could be concluded that IAA is capable of selectively modulating the production of alkaloids in A. emarginata, either due to an external source or by the application of its inhibitor (TIBA).

A developmental gradient reveals biosynthetic pathways to eukaryotic toxins in monocot geophytes

Numerous eukaryotic toxins that accumulate in geophytic plants are valuable in the clinic, yet their biosynthetic pathways have remained elusive. A notable example is the >150 Amaryllidaceae alkaloids (AmAs), including galantamine, an FDA-approved treatment for Alzheimer's disease. We show that while AmAs accumulate to high levels in many daffodil tissues, biosynthesis is localized to nascent, growing tissue at the leaf base. A similar trend is found in the production of steroidal alkaloids (e.g., cyclopamine) in corn lily. This model of active biosynthesis enabled the elucidation of a complete set of biosynthetic genes that can be used to produce AmAs. Taken together, our work sheds light on the developmental and enzymatic logic of diverse alkaloid biosynthesis in daffodils. More broadly, it suggests a paradigm for biosynthesis regulation in monocot geophytes, where plants are protected from herbivory through active charging of newly formed cells with eukaryotic toxins that persist as above-ground tissue develops.

Lamiide and Ipolamiide: A Comprehensive Review of Their Bioactive Properties and Therapeutic Potential.

There is an increasing interest in using iridoids and secoiridoids as major targets for chemical synthesis and biosynthesis. Iridoids can be found in numerous species of Lamiaceae, Verbenaceae, Scrophulariaceae, and other families. Iridoids possess a chemical structure characterized by a cyclopentane ring with oxidative substituents, forming a six-membered ring. Various research groups have used these structures as valuable starting materials for regioselective and stereoselective synthesis. This approach has enormous potential for the production of bioactive alkaloids, prostaglandin analogues, and other bioactive natural compounds. Because there is currently no review on lamiide and ipolamiide, this review intends to pique researchers' interest in this vital topic of natural science for drug discovery from naturally occurring iridoids. Lamiide and ipolamiide have the potential to be useful tools in the pharmaceutical sector, enabling the use of these plant metabolites in a variety of medicinal compositions. Given that these molecules appear to be potential natural substances for treating human ailments, we get light on them as alternative therapeutic approaches using these compounds alone or in combination with other substances which will potentially lead to future (pre)-clinical investigations.

Relationship between sprouting, glucoside alkaloids and chlorophyll production in potato tubers (Solanum tuberosum)

Consumers are troubled by whether the sprouting and greening of potato tubers accompanied with glucoside alkaloids production. To controlling the production of glucoside alkaloids and sprouting, controlled illumination and gaseous sprouting inhibitors, included citronella essential oil, methyl salicylate and ethylene, were employed, the changes in sprouting rate, chlorophyll, glucoside alkaloids and related key genes expression in biosynthesis pathways of potato tubers during storage were measured. Results showed that potatoes contained more glucoside alkaloids with high sprouting rate, whereas the greening was only affected by light, which was not a sign of glucoside alkaloid production. The sprouting rates of potato (Solanum tuberosum L. cv. Favorita) in the fumigated groups was 31.11 %-37.01 %, the glucoside alkaloids content was 83.35–90.33 mg kg−1, there was no significant difference. However, the potato sprouting rates in the CK were 100 %, and the contents of glucoside alkaloids were 153.34–161.36 mg kg−1, but the chlorophyll contents of potatoes in illumination groups were 19.27–20.51 mg kg−1, which was significantly higher (P > 0.05) than that in dark (1.46–1.85 mg kg−1). These results indicated that the synthesis of glucoside alkaloids was associated with sprouting, and it was feasible to inhibit glucoside alkaloids synthesis by gaseous sprouting suppressants.

Influence of the Culture Conditions on Camellia sinensis Cell Cultures.

Since the last century, it has been shown that dedifferentiated cells of Camellia sinensis can produce catechins and other secondary metabolites under in vitro conditions, with potential applications in the cosmetic, pharmaceutical and food industries. In this work, cell suspension cultures of a C. sinensis cell line (LSC-5Y) were established in a liquid medium in order to optimize the biomass productivity, catechin monomer (GC, EGC, C, EC, CG, and ECG) and alkaloid (TB and CAF) productivity. The following factors were evaluated: concentration of growth regulators (BA and IBA), inoculum size, age of the cell line, light exposure, and effect of biotic elicitors (MeJA and extracts of Ciborinia camelliae). GC, EGC, and ECG increased approximately 1.80-fold when the auxin IBA concentration was increased from 0.1 to 2.0 mg/L. In addition, better productivity of EGC, C, EC, and CAF was achieved by using inoculum densities between 50 and 100 g/L. Although lower inoculum densities (25 g/L) showed a higher growth rate (0.20 d-1), the use of inoculum densities higher than 25 g/L favors a 2-4-fold increase in total catechin (TC) productivity, with maximum productivity being reached after 21 days of culture. However, the cell line showed instability in TC productivity: in the short term (in three successive subcultures), the coefficient of variation was 32.80%, and catechin production capacity was 2.5 years with maximum productivity at 0.5 years. Finally, it was observed that ethanol, used as an elicitor solvent, has a strong elicitor effect capable of increasing the accumulation of catechins up to 5.24 times compared to the treatment without an elicitor.

Verazine biosynthesis from simple sugars in engineered Saccharomyces cerevisiae

Steroidal alkaloids are FDA-approved drugs (e.g., Zytiga) and promising drug candidates/leads (e.g., cyclopamine); yet many of the ≥697 known steroidal alkaloid natural products remain underutilized as drugs because it can be challenging to scale their biosynthesis in their producing organisms. Cyclopamine is a steroidal alkaloid produced by corn lily (Veratrum spp.) plants, and it is an inhibitor of the Hedgehog (Hh) signaling pathway. Therefore, cyclopamine is an important drug candidate/lead to treat human diseases that are associated with dysregulated Hh signaling, such as basal cell carcinoma and acute myeloid leukemia. Cyclopamine and its semi-synthetic derivatives have been studied in (pre)clinical trials as Hh inhibitor-based drugs. However, challenges in scaling the production of cyclopamine have slowed efforts to improve its efficacy and safety profile through (bio)synthetic derivatization, often limiting drug development to synthetic analogs of cyclopamine such as the FDA-approved drugs Odomzo, Daurismo, and Erivedge. If a platform for the scalable and sustainable production of cyclopamine were established, then its (bio)synthetic derivatization, clinical development, and, ultimately, widespread distribution could be accelerated. Ongoing efforts to achieve this goal include the biosynthesis of cyclopamine in Veratrum plant cell culture and the semi-/total chemical synthesis of cyclopamine. Herein, this work advances efforts towards a promising future approach: the biosynthesis of cyclopamine in engineered microorganisms. We completed the heterologous microbial production of verazine (biosynthetic precursor to cyclopamine) from simple sugars (i.e., glucose and galactose) in engineered Saccharomyces cerevisiae (S. cerevisiae) through the inducible upregulation of the native yeast mevalonate and lanosterol biosynthetic pathways, diversion of biosynthetic flux from ergosterol (i.e., native sterol in S. cerevisiae) to cholesterol (i.e., biosynthetic precursor to verazine), and expression of a refactored five-step verazine biosynthetic pathway. The engineered S. cerevisiae strain that produced verazine contains eight heterologous enzymes sourced from seven different species. Importantly, S. cerevisiae-produced verazine was indistinguishable via liquid chromatography-mass spectrometry from both a commercial standard (Veratrum spp. plant-produced) and Nicotiana benthamiana-produced verazine. To the best of our knowledge, this is the first report describing the heterologous production of a steroidal alkaloid in an engineered yeast. Verazine production was ultimately increased through design-build-test-learn cycles to a final titer of 83 ± 3 μg/L (4.1 ± 0.1 μg/g DCW). Together, this research lays the groundwork for future microbial biosynthesis of cyclopamine, (bio)synthetic derivatives of cyclopamine, and other steroidal alkaloid natural products.

A Countermeasure Strategy against Peramine Developed by Chilesia rudis in the Endophyte-Ryegrass-Herbivore Model.

Exploitation of the symbiotic relationship between endophytic fungi and ryegrass is a crucial technique for reducing the incidence of insect pests. This is primarily due to the production of alkaloids, such as peramine, by the fungi. This alkaloid has been reported as both a deterrent and toxic to a variety of insects. However, insects have developed various strategies to counteract plant defenses. One of the most studied methods is their ability to sequester toxic compounds from plants. In this study, we examined the feeding preferences and adaptation to peramine in Chilesia rudis, a native Chilean larva. Using a no-choice assay, we assessed larval feeding preferences and mass gain on seven experimental lines and two commercial cultivars of endophyte-infected and non-infected ryegrass. Pupal development time and adult performance were evaluated post-assay. Additionally, we measured peramine content in larval carcasses, feces, and ryegrass leaves. Jumbo was the most preferred cultivar with 32 mm2 of leaf tissues consumed. The longest pupal development time was observed in L161 and ALTO AR1, both at 28 days. Wing length in adults was greatest in the Jumbo and L163 cultivars, measuring 1.25 cm and 1.32 cm, respectively. Peramine concentrations were detected in the bodies of C. rudis. In conclusion, this larva can adapt to endophyte-infected ryegrass and develop counter-adaptation mechanisms to mitigate the effects of peramine.

Foliar phosphorus concentration modulates the defensive mutualism of an endophytic fungus in a perennial host grass

Abstract Grasses hosting Epichloë endophytes are protected against herbivores due to the production of various fungal alkaloids. Previous research has found that high foliar phosphorus concentrations reduce the level of the alkaloid ergovaline, thereby reducing the endophyte‐mediated herbivore resistance. Yet, the impact of phosphorus on ergovaline biosynthesis versus its influence on endophyte growth and synthesis of other fungal alkaloids remains unresolved. Our objective was to elucidate these relationships. We grew endophyte‐symbiotic and non‐symbiotic Festuca arundinacea plants and fertilised them with different doses of phosphorus. Later, half of the plants from each treatment were challenged with larvae of the generalist chewing insect Spodoptera frugiperda. We assessed the relationships between foliar phosphorus levels, fungal mycelium and alkaloid concentrations, as well as their impacts on larvae performance, herbivore‐caused damage and plant biomass. Endophyte mycelial biomass in plant tissue was found to be independent of foliar phosphorus concentration. The alkaloids lolines and peramine showed a linear relationship with mycelial biomass but no correlation with foliar phosphorus. Surprisingly, high ergovaline concentrations were positively associated with an interaction between endophyte mycelial biomass and foliar phosphorus concentration. Although herbivory increased loline concentration, only high concentrations of ergovaline and peramine were related to reduced S. frugiperda larvae weight gain. However, endophyte presence did not reduce herbivory damage on plants. Contrary to expectation, we did not find a negative but a positive association between concentrations of foliar phosphorus and ergovaline alkaloid, through its interaction with endophyte mycelial biomass. Alternatively, our findings suggest that phosphorus plays a crucial role in modulating the Epichloë‐mediated defensive mutualism, primarily through its effects on ergovaline rather than on endophyte concentration or production of other alkaloids. Read the free Plain Language Summary for this article on the Journal blog.

Metabolite profiles of Paragliomastix luzulae (formerly named as Acremonium striatisporum) KMM 4401 and its co-cultures with Penicillium hispanicum KMM 4689

The marine holothurian-derived fungal strain KMM 4401 has been identified as Paragliomastix luzulae using 28S rDNA, ITS regions and the partial TEF1 gene sequences. The metabolite profile of the fungal culture was studied by UPLC-MS technique. The strain KMM 4401 is a source of various virescenoside-type isopimarane glycosides suggested as chemotaxonomic feature for this fungal species. Also Px. luzulae KMM 4401 was proposed as possible source of new bioactive secondary metabolites especially antimicrobials. Moreover, the co-cultures of Px. luzulae KMM 4401 with another marine fungus Penicillium hispanicum KMM 4689 inoculated simultaneously or after two weeks were investigated by same way. It was shown, that P. hispanicum KMM 4689 suppressed the production of most of Px. luzulae KMM 4401 metabolites. On the other hand, the co-cultivation of P. hispanicum KMM 4689 and Px. luzulae KMM 4401 resulted in increasing of production of main deoxyisoaustamide alkaloids of P. hispanicum KMM 4689 on 50–190%.Graphical

Effect of Menthol Treatment on the Sprouting and Quality of Potato Tuber

This study used Dutch potatoes at the end of dormancy as a material to explore the impact of menthol (0.2 and 0.5 g/kg based on potato mass) treatment on sprouting inhibition and potato quality. The findings revealed that a menthol concentration of 0.5 g/kg effectively inhibited potato tuber sprouting and significantly reduced glucoside alkaloid production. After a storage period of 15 days, the sprouting percentage and glucoside alkaloid content of potatoes treated with 0.5 g/kg menthol were observed to be significantly lower at 4.17% and 68.63 mg/kg, respectively, compared to the control group which exhibited values of 100% and 282.01 mg/kg, respectively (p < 0.05). Throughout the storage period, 0.5 g/kg of menthol promoted respiration, reduced malondialdehyde production in potatoes, inhibited polyphenol oxidase activity, and slowed down tissue browning. Additionally, it mitigated the decline in starch and soluble protein content, inhibiting the accumulation of reducing sugars.

Maramycin, a Cytotoxic Isoquinolinequinone Terpenoid Produced through Heterologous Expression of a Bifunctional Indole Prenyltransferase/Tryptophan Indole-Lyase in S. albidoflavus.

Isoquinolinequinones represent an important family of natural alkaloids with profound biological activities. Heterologous expression of a rare bifunctional indole prenyltransferase/tryptophan indole-lyase enzyme from Streptomyces mirabilis P8-A2 in S. albidoflavus J1074 led to the activation of a putative isoquinolinequinone biosynthetic gene cluster and production of a novel isoquinolinequinone alkaloid, named maramycin (1). The structure of maramycin was determined by analysis of spectroscopic (1D/2D NMR) and MS spectrometric data. The prevalence of this bifunctional biosynthetic enzyme was explored and found to be a recent evolutionary event with only a few representatives in nature. Maramycin exhibited moderate cytotoxicity against human prostate cancer cell lines, LNCaP and C4-2B. The discovery of maramycin (1) enriched the chemical diversity of natural isoquinolinequinones and also provided new insights into crosstalk between the host biosynthetic genes and the heterologous biosynthetic genes in generating new chemical scaffolds.