Dataset Information

Biosynthesis of the Pharmaceutically Important Fungal Ergot Alkaloid Dihydrolysergic Acid Requires a Specialized Allele of cloA.

ABSTRACT: Ergot alkaloids are specialized fungal metabolites that are important as the bases of several pharmaceuticals. Many ergot alkaloids are derivatives of lysergic acid (LA) and have vasoconstrictive activity, whereas several dihydrolysergic acid (DHLA) derivatives are vasorelaxant. The pathway to LA is established, with the P450 monooxygenase CloA playing a key role in oxidizing its substrate agroclavine to LA. We analyzed the activities of products of cloA alleles from different fungi relative to DHLA biosynthesis by expressing them in a mutant of the fungus Neosartorya fumigata that accumulates festuclavine, the precursor to DHLA. Transformants expressing CloA from Epichloë typhina × Epichloë festucae, which oxidizes agroclavine to LA, failed to oxidize festuclavine to DHLA. In substrate feeding experiments, these same transformants oxidized exogenously supplied agroclavine to LA, indicating that a functional CloA was produced. A genomic clone of cloA from Claviceps africana, a sorghum ergot fungus that produces a DHLA derivative, was cloned and expressed in the festuclavine-accumulating mutant of N. fumigata, but several introns in this genomic clone were not processed properly. Expression of a synthetic intron-free version of C. africanacloA resulted in the accumulation of DHLA as assessed by fluorescence high-pressure liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). In substrate feeding experiments, the C. africana CloA also accepted agroclavine as the substrate, oxidizing it to LA. The data indicate that a specialized allele of cloA is required for DHLA biosynthesis and that the pharmaceutically important compound DHLA can be produced in engineered N. fumigataIMPORTANCE Ergot alkaloids are fungal metabolites that have impacted humankind historically as poisons and more recently as pharmaceuticals used to treat dementia, migraines, and other disorders. Much is known about the biosynthesis of ergot alkaloids that are derived from lysergic acid (LA), but important questions remain about a parallel pathway to ergot alkaloids derived from dihydrolysergic acid (DHLA). DHLA-derived alkaloids have minor structural differences compared to LA-derived alkaloids but can have very different activities. To understand how DHLA is made, we analyzed activities of a key enzyme in the DHLA pathway and found that it differed from its counterpart in the LA pathway. Our data indicate a critical difference between the two pathways and provide a strategy for producing DHLA by modifying a model fungus. The ability to produce DHLA in a model fungus may facilitate synthesis of DHLA-derived pharmaceuticals.

SUBMITTER: Arnold SL

PROVIDER: S-EPMC5494617 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Biosynthesis of the Pharmaceutically Important Fungal Ergot Alkaloid Dihydrolysergic Acid Requires a Specialized Allele of <i>cloA</i>.

Arnold Stephanie L SL Panaccione Daniel G DG

Applied and environmental microbiology 20170630 14

Ergot alkaloids are specialized fungal metabolites that are important as the bases of several pharmaceuticals. Many ergot alkaloids are derivatives of lysergic acid (LA) and have vasoconstrictive activity, whereas several dihydrolysergic acid (DHLA) derivatives are vasorelaxant. The pathway to LA is established, with the P450 monooxygenase CloA playing a key role in oxidizing its substrate agroclavine to LA. We analyzed the activities of products of <i>cloA</i> alleles from different fungi relat ...[more]

PMID: 28476772

Similar Datasets

Project description:Pregnant Suffolk ewes (n = 19; 81.2kg ± 7.7) estimated to be carrying twins were assigned to endophyte-infected tall fescue seed (E+; 4.14 µg ergovaline + ergovalinine/g seed) or a control diet (CON; 0 µg ergovaline + ergovalinine) and evaluated at different stages of gestation (gd85 [pre-treatment, n = 3], gd110 [n = 4/treatment] or gd133 [n = 4/treatment). Ewes were individually fed a basal diet with (E+) or without (CON) endophyte-infected tall fescue seed from gd86 to gd110 or gd133. A terminal necropsy was performed, and uterine components were evaluated for size, weight, and type. Data were analyzed using a priori contrasts to test effects of stage of gestation (gd85 vs. gd110 or gd110 vs. gd133) and adaptive responses to feeding E+ fescue seed versus CON at gd110 and gd133. Serum prolactin concentrations were greater (P < 0.05) at gd110 than gd85, and then remained constant to gd133. Feeding E+ tall fescue seed reduced (P < 0.05) serum prolactin concentrations compared to CON at both gd110 and gd133. From gd85 to gd110, placentome type and number changed from type A to type B (P < 0.05) but did not change (P > 0.05) after gd110. Total fetal weight per ewe increased 2977 g from gd85 to gd110 (P = 0.011) and then 3151 g from gd110 to 133 (P = 0.012). Brain weight as a percentage of fetal body weight was higher (P = 0.029) for E+ fetuses on gd110 compared to CON, which demonstrates asymmetrical growth and intrauterine growth restriction (IUGR). In total, 15,961 genes were identified in the cotyledon through mapping to the ovine genome across all samples. There were 136 genes differentially expressed (FDR < 0.05; log2foldchange < -1 and > 1) from gd85 to gd110 with ovarian steroidogenesis and steroid biosynthesis pathways enriched and 469 genes differentially expressed from gd110 to gd133 with ribosome and oxidative phosphorylation pathways enriched. Exposure to E+ fescue resulted in differential expression of 22 genes at gd110 but only one gene at gd133 compared to CON. These results show that there are major changes in fetal growth and placental remodeling during the late gestation period that are influenced by exposure to ergot alkaloids from endophyte-infected tall fescue seed; however, major changes in the transcriptome of the cotyledon tissue in response to E+ fescue were not observed and other factors must be involved in the reduction of placental efficiency and fetal growth observed with E+ fescue exposure.

Project description:Ergot alkaloids are specialized fungal metabolites with potent biological activities. They are encoded by well-characterized gene clusters in the genomes of producing fungi. Penicillium camemberti plays a major role in the ripening of Brie and Camembert cheeses. The P. camemberti genome contains a cluster of five genes shown in other fungi to be required for synthesis of the important ergot alkaloid intermediate chanoclavine-I aldehyde and two additional genes (easH and easQ) that may control modification of chanoclavine-I aldehyde into other ergot alkaloids. We analyzed samples of Brie and Camembert cheeses, as well as cultures of P. camemberti, and did not detect chanoclavine-I aldehyde or its derivatives. To create a functioning facsimile of the P. camembertieas cluster, we expressed P. camemberti easH and easQ in a chanoclavine-I aldehyde-accumulating easA knockout mutant of Neosartorya fumigata The easH-easQ-engineered N. fumigata strain accumulated a pair of compounds of m/z 269.1288 in positive-mode liquid chromatography-mass spectrometry (LC-MS). The analytes fragmented in a manner typical of the stereoisomeric ergot alkaloids rugulovasine A and B, and the related rugulovasine producer Penicillium biforme accumulated the same isomeric pair of analytes. The P. camemberti eas genes were transcribed in culture, but comparison of the P. camemberti eas cluster with the functional cluster from P. biforme indicated 11 polymorphisms. Whereas other P. camembertieas genes functioned when expressed in N. fumigata, P. camembertieasC did not restore ergot alkaloids when expressed in an easC mutant. The data indicate that P. camemberti formerly had the capacity to produce the ergot alkaloids rugulovasine A and B.IMPORTANCE The presence of ergot alkaloid synthesis genes in the genome of Penicillium camemberti is significant, because the fungus is widely consumed in Brie and Camembert cheeses. Our results show that, although the fungus has several functional genes from the ergot alkaloid pathway, it produces only an early pathway intermediate in culture and does not produce ergot alkaloids in cheese. Penicillium biforme, a close relative of P. camemberti, contains a similar but fully functional set of ergot alkaloid synthesis genes and produces ergot alkaloids chanoclavine-I, chanoclavine-I aldehyde, and rugulovasine A and B. Our reconstruction of the P. camemberti pathway in the model fungus Neosartorya fumigata indicated that P. camemberti formerly had the capacity to produce these same ergot alkaloids. Neither P. camemberti nor P. biforme produced ergot alkaloids in cheese, indicating that nutritionally driven gene regulation prevents these fungi from producing ergot alkaloids in a dairy environment.

Dataset Information

Biosynthesis of the Pharmaceutically Important Fungal Ergot Alkaloid Dihydrolysergic Acid Requires a Specialized Allele of cloA.

Publications

Biosynthesis of the Pharmaceutically Important Fungal Ergot Alkaloid Dihydrolysergic Acid Requires a Specialized Allele of <i>cloA</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets