scholarly journals Several Metarhizium Species Produce Ergot Alkaloids in a Condition-Specific Manner

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Caroline E. Leadmon ◽  
Jessi K. Sampson ◽  
Matthew D. Maust ◽  
Angie M. Macias ◽  
Stephen A. Rehner ◽  
...  
Keyword(s):  
Yeast Extract ◽  
Ergot Alkaloid ◽  
Genomic Sequence ◽  
Close Association ◽  
Acid Amide ◽  
Ergot Alkaloids ◽  
Lysergic Acid ◽  
Content Type ◽  
Extract Agar ◽  
Yeast Extract Agar

ABSTRACT Genomic sequence data indicate that certain fungi in the genus Metarhizium have the capacity to produce lysergic acid-derived ergot alkaloids, but accumulation of ergot alkaloids in these fungi has not been demonstrated previously. We assayed several Metarhizium species grown under different conditions for accumulation of ergot alkaloids. Isolates of M. brunneum and M. anisopliae accumulated the lysergic acid amides lysergic acid α-hydroxyethyl amide, ergine, and ergonovine on sucrose-yeast extract agar but not on two other tested media. Isolates of six other Metarhizium species did not accumulate ergot alkaloids on sucrose-yeast extract agar. Conidia of M. brunneum lacked detectable ergot alkaloids, and mycelia of this fungus secreted over 80% of their ergot alkaloid yield into the culture medium. Isolates of M. brunneum, M. flavoviride, M. robertsii, M. acridum, and M. anisopliae produced high concentrations of ergot alkaloids in infected larvae of the model insect Galleria mellonella, but larvae infected with M. pingshaense, M. album, M. majus, and M. guizhouense lacked detectable ergot alkaloids. Alkaloid concentrations were significantly higher when insects were alive (as opposed to killed by freezing or gas) at the time of inoculation with M. brunneum. Roots of corn and beans were inoculated with M. brunneum or M. flavoviride and global metabolomic analyses indicated that the inoculated roots were colonized, though no ergot alkaloids were detected. The data demonstrate that several Metarhizium species produce ergot alkaloids of the lysergic acid amide class and that production of ergot alkaloids is tightly regulated and associated with insect colonization. IMPORTANCE Our discovery of ergot alkaloids in fungi of the genus Metarhizium has agricultural and pharmaceutical implications. Ergot alkaloids produced by other fungi in the family Clavicipitaceae accumulate in forage grasses or grain crops; in this context they are considered toxins, though their presence also may deter or kill insect pests. Our data report ergot alkaloids in Metarhizium species and indicate a close association of ergot alkaloid accumulation with insect colonization. The lack of accumulation of alkaloids in spores of the fungi and in plants colonized by the fungi affirms the safety of using Metarhizium species as biocontrol agents. Ergot alkaloids produced by other fungi have been exploited to produce powerful pharmaceuticals. The class of ergot alkaloids discovered in Metarhizium species (lysergic acid amides) and their secretion into the growth medium make Metarhizium species a potential platform for future studies on ergot alkaloid synthesis and modification.

scholarly journals Genetic Reprogramming of the Ergot Alkaloid Pathway of Metarhizium brunneum

2020 ◽  
Vol 86 (19) ◽  
Author(s):  
Kyle A. Davis ◽  
Jessi K. Sampson ◽  
Daniel G. Panaccione
Keyword(s):  
Ergot Alkaloid ◽  
High Performance ◽  
Neurological Diseases ◽  
Ergot Alkaloids ◽  
Lysergic Acid ◽  
Efficient Production ◽  
Natural Sources ◽  
Content Type ◽  
Metarhizium Brunneum ◽  
Alkaloid Synthesis

ABSTRACT Ergot alkaloids are important specialized fungal metabolites that are used to make potent pharmaceuticals for neurological diseases and disorders. Lysergic acid (LA) and dihydrolysergic acid (DHLA) are desirable lead compounds for pharmaceutical semisynthesis but are typically transient intermediates in the ergot alkaloid and dihydroergot alkaloid pathways. Previous work with Neosartorya fumigata demonstrated strategies to produce these compounds as pathway end products, but their percent yield (percentage of molecules in product state as opposed to precursor state) was low. Moreover, ergot alkaloids in N. fumigata are typically retained in the fungus as opposed to being secreted. We used clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) and heterologous expression approaches to engineer these compounds in Metarhizium brunneum, representing an alternate expression host from a different lineage of fungi. The relative percent yields of LA (86.9%) and DHLA (72.8%) were much higher than those calculated here for previously engineered strains of N. fumigata (2.6% and 2.0%, respectively). Secretion of these alkaloids also was measured, with averages of 98.4% of LA and 87.5% of DHLA being secreted into the growth medium; both values were significantly higher than those measured for the N. fumigata derivatives (both of which were less than 5.6% secreted). We used a similar approach to engineer a novel dihydroergot alkaloid in M. brunneum and, through high-performance liquid chromatography-mass spectrometry (LC-MS) analyses, provisionally identified it as the dihydrogenated form of lysergic acid α-hydroxyethylamide (dihydro-LAH). The engineering of these strains provides a strategy for producing novel and pharmaceutically important chemicals in a fungus more suitable for their production. IMPORTANCE Ergot alkaloids derived from LA or DHLA are the bases for numerous pharmaceuticals with applications in the treatment of dementia, migraines, hyperprolactinemia, and other conditions. However, extraction of ergot alkaloids from natural sources is inefficient, and their chemical synthesis is expensive. The ability to control and redirect ergot alkaloid synthesis in fungi may allow more efficient production of these important chemicals and facilitate research on novel derivatives. Our results show that Metarhizium brunneum can be engineered to efficiently produce and secrete LA and DHLA and, also, to produce a novel derivative of DHLA not previously found in nature. The engineering of dihydroergot alkaloids, including a novel species, is important because very few natural sources of these compounds are known. Our approach establishes a platform with which to use M. brunneum to study the production of other ergot alkaloids, specifically those classified as lysergic acid amides and dihydroergot alkaloids.

Independent evolution of a lysergic acid amide in Aspergillus species

2021 ◽  
Author(s):  
Abigail M. Jones ◽  
Chey R. Steen ◽  
Daniel G. Panaccione
Keyword(s):  
Ergot Alkaloid ◽  
Phylogenetic Analyses ◽  
Acid Amide ◽  
Ergot Alkaloids ◽  
Gene Clusters ◽  
Lysergic Acid ◽  
Alkaloid Synthesis ◽  
Genes Encoding ◽  
Selection For ◽  
High Concentrations

Ergot alkaloids derived from lysergic acid have impacted humanity as contaminants of crops and as the bases of pharmaceuticals prescribed to treat dementia, migraines, and other disorders. Several plant-associated fungi in the Clavicipitaceae produce lysergic acid derivatives, but many of these fungi are difficult to culture and manipulate. Some Aspergillus species, which may be more ideal experimental and industrial organisms, contain an alternate branch of the ergot alkaloid pathway but none were known to produce lysergic acid derivatives. We mined genomes of Aspergillus species for ergot alkaloid synthesis ( eas ) gene clusters and discovered three species–– A. leporis, A. homomorphus, and A. hancockii ––had eas clusters indicative of the capacity to produce a lysergic acid amide. In culture, A. leporis, A. homomorphus, and A. hancockii produced lysergic acid amides, predominantly lysergic acid α-hydroxyethylamide (LAH). Aspergillus leporis and A. homomorphus produced high concentrations of LAH and secreted most of their ergot alkaloid yield into the culture medium. Phylogenetic analyses indicated genes encoding enzymes leading to the synthesis of lysergic acid were orthologous to those of the lysergic acid amide-producing Clavicipitaceae; however, genes to incorporate lysergic acid into an amide derivative evolved from different ancestral genes in the Aspergillus species. Our data demonstrate fungi outside the Clavicipitaceae produce lysergic acid amides and indicate the capacity to produce lysergic acid evolved once, but the ability to insert it into LAH evolved independently in Aspergillus species and the Clavicipitaceae. The LAH-producing Aspergillus species may be useful for study and production of these pharmaceutically important compounds. IMPORTANCE Lysergic acid derivatives are specialized metabolites with historical, agricultural, and medical significance and were known heretofore only from fungi in one family, the Clavicipitaceae. Our data show that several Aspergillus species, representing a different family of fungi, also produce lysergic acid derivatives and that the ability to put lysergic acid into its amide forms evolved independently in the two lineages of fungi. From microbiological and pharmaceutical perspectives, the Aspergillus species may represent better experimental and industrial organisms than the currently employed, lysergic acid producers of the plant-associated Clavicipitaceae. The observation that both lineages independently evolved the derivative lysergic acid α-hydroxyethylamide (LAH), among many possible lysergic acid amides, suggests a selection for this metabolite.

scholarly journals A Baeyer-Villiger monooxygenase gene involved in the synthesis of lysergic acid amides affects the interaction of the fungus Metarhizium brunneum with insects

2021 ◽  
Author(s):  
Chey R. Steen ◽  
Jessi K. Sampson ◽  
Daniel G. Panaccione
Keyword(s):  
Ergot Alkaloid ◽  
Galleria Mellonella ◽  
Insect Pests ◽  
Biological Activities ◽  
Plant Root ◽  
Total Concentration ◽  
Acid Amide ◽  
Ergot Alkaloids ◽  
Structural Similarity ◽  
Lysergic Acid

Several fungi, including the plant root symbiont and insect pathogen Metarhizium brunneum , produce lysergic acid amides via a branch of the ergot alkaloid pathway. Lysergic acid amides include important pharmaceuticals and pharmaceutical lead compounds and have potential ecological significance, making knowledge of their biosynthesis relevant. Many steps in the biosynthesis of lysergic acid amides have been determined, but terminal steps in the synthesis of lysergic acid α-hydroxyethylamide (LAH)––by far the most abundant lysergic acid amide in M. brunneum ––are unknown. Ergot alkaloid synthesis ( eas ) genes are clustered in the genomes of fungi that produce these compounds, and the eas clusters of LAH producers contain two uncharacterized genes ( easO and easP ) not found in fungi that do not produce LAH. Knockout of easO via a CRISPR-Cas9 approach eliminated LAH and resulted in accumulation of alternate lysergic acid amides lysergyl-alanine and ergonovine. Despite the elimination of LAH, the total concentration of lysergic acid derivatives was not affected significantly by the mutation. Complementation with a wild-type allele of easO restored the ability to synthesize LAH. Substrate feeding studies indicated that neither lysergyl-alanine nor ergonovine were substrates for the product of easO (EasO). EasO had structural similarity to Baeyer-Villiger monooxygenases (BVMOs), and labeling studies with deuterated alanine supported a role for a BVMO in LAH biosynthesis. The easO knockout had reduced virulence to larvae of the insect Galleria mellonella , indicating that LAH contributes to virulence of M. brunneum on insects and that LAH has biological activities different from ergonovine and lysergyl-alanine. Importance Fungi in the genus Metarhizium are important plant root symbionts and insect pathogens. They are formulated commercially to protect plants from insect pests. Several Metarhizium species including M. brunneum were recently shown to produce ergot alkaloids, a class of specialized metabolites studied extensively in other fungi because of their importance in agriculture and medicine. A biological role for ergot alkaloids in Metarhizium species had not been demonstrated previously. Moreover, the types of ergot alkaloids produced by Metarhizium species are lysergic acid amides, which have served directly or indirectly as important pharmaceutical compounds. The terminal steps in the synthesis of the most abundant lysergic acid amide in Metarhizium species and several other fungi (LAH) have not been determined. The results of this study demonstrate the role of a previously unstudied gene in LAH synthesis and indicate that LAH contributes to virulence of M. brunneum on insects.

scholarly journals Heterologous Expression of Lysergic Acid and Novel Ergot Alkaloids in Aspergillus fumigatus

2014 ◽  
Vol 80 (20) ◽  
pp. 6465-6472 ◽  
Author(s):  
Sarah L. Robinson ◽  
Daniel G. Panaccione
Keyword(s):  
Aspergillus Fumigatus ◽  
Candidate Genes ◽  
Mass Spectra ◽  
Ergot Alkaloids ◽  
Lysergic Acid ◽  
Precursor Feeding ◽  
Gene Encoding ◽  
Content Type ◽  
Multiple Reactions ◽  
Important Pathway

ABSTRACTDifferent lineages of fungi produce distinct classes of ergot alkaloids. Lysergic acid-derived ergot alkaloids produced by fungi in the Clavicipitaceae are particularly important in agriculture and medicine. The pathway to lysergic acid is partly elucidated, but the gene encoding the enzyme that oxidizes the intermediate agroclavine is unknown. We investigated two candidate agroclavine oxidase genes from the fungusEpichloë festucaevar.lolii×Epichloë typhinaisolate Lp1 (henceforth referred to asEpichloësp. Lp1), which produces lysergic acid-derived ergot alkaloids. Candidate geneseasHandcloAwere expressed in a mutant strain of the moldAspergillus fumigatus, which typically produces a subclass of ergot alkaloids not derived from agroclavine or lysergic acid. Candidate genes were coexpressed with theEpichloësp. Lp1 allele ofeasA, which encodes an enzyme that catalyzed the synthesis of agroclavine from anA. fumigatusintermediate; the agroclavine then served as the substrate for the candidate agroclavine oxidases. Strains expressingeasAandcloAfromEpichloësp. Lp1 produced lysergic acid from agroclavine, a process requiring a cumulative six-electron oxidation and a double-bond isomerization. Strains that accumulated excess agroclavine (as a result ofEpichloësp. Lp1easAexpression in the absence ofcloA) metabolized it into two novel ergot alkaloids for which provisional structures were proposed on the basis of mass spectra and precursor feeding studies. Our data indicate that CloA catalyzes multiple reactions to produce lysergic acid from agroclavine and that combining genes from different ergot alkaloid pathways provides an effective strategy to engineer important pathway molecules and novel ergot alkaloids.

Comparison of agar-based methods for the isolation and enumeration of heterotrophic bacteria with the new Multidose IDEXX(tm) SimPlate method

2004 ◽  
Vol 50 (1) ◽  
pp. 277-280
Author(s):  
M. Vulindlu ◽  
A. Charlett ◽  
S. Surman ◽  
J.V. Lee
Keyword(s):  
Yeast Extract ◽  
Heterotrophic Bacteria ◽  
Treatment Plant ◽  
Plant Performance ◽  
Extract Agar ◽  
Quality Of Water ◽  
Heterotrophic Microorganisms ◽  
External Sources ◽  
Conventional Methods ◽  
Yeast Extract Agar

Pour and spread plates are the conventional methods of choice for the isolation and enumeration of heterotrophic microorganisms in treated water supplies. The tests are performed at 22°C and 37°C for 72 h and 48 h respectively. Counts at 22°C are associated with pollution of water systems from external sources, while counts at 37°C are used as an indication of treatment plant performance and the deterioration of the general quality of water. Conventional methods using Yeast Extract Agar for a pour plate and R2A agar for a spread plate were compared with the multidose IDEXXTM SimPlate method for the isolation and enumeration of heterotrophic bacteria in water. SimPlate gave a significantly higher count on average than the conventional methods. The R2A method showed the next highest count, being significantly higher than Yeast Extract Agar. In addition, unlike the pour and spread plate methods, SimPlate was easier to use, reduced labour, and the test results were far easier to read.

Nannocystis exedens: A Potential Biocompetitive Agent against Aspergillus flavus and Aspergillus parasiticus

2001 ◽  
Vol 64 (7) ◽  
pp. 1030-1034 ◽  
Author(s):  
WILLIE J. TAYLOR ◽  
FRANCES A. DRAUGHON
Keyword(s):  
Aspergillus Flavus ◽  
Yeast Extract ◽  
Biocontrol Agent ◽  
Aspergillus Parasiticus ◽  
Extract Agar ◽  
Potential Biocontrol Agent ◽  
Aflatoxigenic Fungi ◽  
Close Proximity ◽  
Zones Of Inhibition ◽  
Yeast Extract Agar

This study examined the potential for controlling toxigenic Aspergillus flavus and Aspergillus parasiticus by biological means using a myxobacterium commonly found in soil. The ability of Nannocystis exedens to antagonize A. flavus ATCC 16875, A. flavus ATCC 26946, and A. parasiticus NRRL 3145 was discovered. Cultures of aflatoxigenic fungi were grown on 0.3% Trypticase peptone yeast extract agar for 14 days at 28°C. When N. exedens was grown in close proximity with an aflatoxigenic mold, zones of inhibition (10 to 20 mm) developed between the bacterium and mold colony. A flattening of the mold colony on the sides nearest N. exedens and general stunting of growth of the mold colony were also observed. When N. exedens was added to the center of the cross-streak of a mold colony, lysis of the colony by the bacterium was observed after 24 h. Microscopic observations revealed that N. exedens grew on spores, germinating spores, hyphae, and sclerotia of the molds. These results indicate that N. exedens may be a potential biocontrol agent against A. flavus and A. parasiticus.

scholarly journals ISOLASI DAN IDENTIFIKASI KAPANG PADA PINDANG BANDENG (Chanos chanos) PRESTO

2019 ◽  
Vol 2 (1) ◽  
pp. 15
Author(s):  
Resmi Rumenta Siregar
Keyword(s):  
Yeast Extract ◽  
Rose Bengal ◽  
Fusarium Solani ◽  
Penicillium Citrinum ◽  
Malt Extract Agar ◽  
Malt Extract ◽  
Chanos Chanos ◽  
Extract Agar ◽  
Fusarium Sp ◽  
Yeast Extract Agar

Ikan pindang adalah salah satu olahan yang sangat disukai oleh masyarakat Indonesia. Hal ini dapat dilihat dari produksi ikan pindang yang setiap tahunnya mengalami peningkatan. Sebagai contoh di Kabupaten Bogor, produksi ikan pindang pada tahun 2013 sebesar 3.643,56 ton, meningkat menjadi 10.334,44ton pada tahun 2015. Ikan pindang disisi lain, sangat mudah mengalami kemunduran mutu disebabkan masih tingginya kadar air, pengemasan yang tidak memenuhi standar serta proses pengolahan yang pada umumnya kurang menerapkan prinsip sanitasi yang baik. Penelitian ini bertujuan untuk mengisolasi dan mengidentifikasi kapang yang tumbuh pada bandeng presto. Sampel Bandeng presto diambil dari CV. Cindy Group. Kapang diisolasi dengan metode pengenceran bertingkat menggunakan media DRBC (Dichloran Rose Bengal Chloramphenicol Agar), kemudian diidentifikasi secara morfologi menggunakan media Malt Extract Agar dan Czapek’s Yeast Extract Agar. Nilai Aktivitas air (aw) bandeng presto memiliki kisaran rata-rata 0,96 – 0,97. Secara makroskopis terlihat adanya pertubuhan kapang pada permukaan ikan bandeng presto setelah penyimpanan selama 3 hari pada suhu ruang (20-250C). Sebanyak 5 isolat kapang diisolasi dari ikan pindang sampel bandeng presto. Hasil identifikasi secara mikroskopis diketahui bahwa kapang yang tumbuh ada ikan pindang tersebut adalah spesies Penicillium citrinum, Eurotium chevalieri, Fusarium solani, Fusarium sp, dan Cladosporium sp. Kadar aw ikan pindang resto yang masih tinggi (0,96-0,97) menyebabkan ikan pindang mengalami pembusukan yang diakibatkan oleh bakteri.  

Evaluation of Recovery Media for Heated Clostridium sporogenes Spores1

1979 ◽  
Vol 42 (12) ◽  
pp. 946-947 ◽  
Author(s):  
I. J. PFLUG ◽  
M. SCHEYER ◽  
G. M. SMITH ◽  
M. KOPELMAN
Keyword(s):  
Yeast Extract ◽  
Culture Media ◽  
Clostridium Sporogenes ◽  
Extract Agar ◽  
Significant Difference ◽  
Yeast Extract Agar

The efficiency of four culture media for recovery of heat-activated and heated Clostridium sporogenes spores was studied. Yeast extract agar gave the highest spore recovery. The effect of the method of preparing the yeast extract agar on the recovery of heated spores was also evaluated. The results indicate that (a) a significantly lower spore recovery was obtained when the dextrose was omitted completely or when added to the medium before autoclaving, and (b) no significant difference in spore recovery was found between yeast extract agar freshly made or prepared and stored at 4 C up to 11 days before use.

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