Partial Reconstruction of the Ergot Alkaloid Pathway by Heterologous Gene Expression in Aspergillus nidulans

Ryan, Kathleen J.; Moore, Chris; Panaccione, Daniel G.

doi:10.3390/toxins5020445

Cited by 50 publications

(61 citation statements)

References 19 publications

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“…Standard prepared in this manner was analyzed for fluorescence properties characteristic of DHLA (excitation at 272 nm with emission at 372 nm) and by high-resolution mass spectrometry (calculated m/z, 271.1441; measured m/z, 271.1440; deviation, 0.37 ppm). Electrospray ionization liquid chromatography-mass spectrometry (LC-MS) was performed in positive mode on a Thermo LCQ Deca XP Plus equipped with a Surveyor HPLC as described by Ryan et al (32). DHLA standard was analyzed on a Thermo Q Exactive mass spectrometer as described by Robinson and Panaccione (21).…”

Section: Modification Of Fungimentioning

confidence: 99%

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

Arnold

Panaccione

2017

Appl Environ Microbiol

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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 ϫ 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. africana cloA resulted in the accumulation of DHLA as assessed by fluorescence high-pressure liquid chromatography (HPLC) and liquid chromatographymass 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. fumigata. IMPORTANCE 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.KEYWORDS ergot alkaloids, lysergic acid, dihydrolysergic acid, P450 monooxygenase E rgot alkaloids are specialized fungal metabolites that have impacted humankind historically as toxins and more recently as pharmaceuticals used to treat dementia, migraines, hyperprolactinemia, and Parkinson's disease (1-4). Many natural ergot alka-

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Section: Modification Of Fungimentioning

confidence: 99%

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

Arnold

Panaccione

2017

Appl Environ Microbiol

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“…Very recently, heterologous partial reconstitution of the ergot alkaloid biosynthetic pathway was accomplished in Aspergillus nidulans by introducing a segment of the A. fumigatus genome covering a portion of the ergot alkaloid biosynthetic gene cluster into the A. nidulans chromosome [65]. The integrated A. fumigatus genomic DNA segment contained four genes, dimethylallyltryptophan synthase-coding dmaW, dimethylallyltryptophan N-methyltransferase-coding easF, oxidoreductaselike protein-coding easE and catalase-like protein-coding easC.…”

Section: Ergotaminementioning

confidence: 99%

“…The transformed A. nidulans was able to produce 16 and 17, early intermediates in the D-lysergic acid biosynthetic pathway (Figure 10). Using this heterologous production system, gene deletion experiments were performed to determine for the first time that the four genes were sufficient for the biosynthesis of 17 [65]. This successful production of ergot alkaloid biosynthetic intermediates in A. nidulans would facilitate the effort toward achieving engineered biosynthesis of this class of natural products.…”

Section: Opiatesmentioning

confidence: 99%

Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

et al. 2016

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Abstract:Varieties of alkaloids are known to be produced by various organisms, including bacteria, fungi and plants, as secondary metabolites that exhibit useful bioactivities. However, understanding of how those metabolites are biosynthesized still remains limited, because most of these compounds are isolated from plants and at a trace level of production. In this review, we focus on recent efforts in identifying the genes responsible for the biosynthesis of those nitrogen-containing natural products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid), saframycin (tetrahydroisoquinoline alkaloid), strictosidine (monoterpene indole alkaloid), ergotamine (ergot alkaloid) and opiates (benzylisoquinoline and morphinan alkaloid). This review also discusses the engineered biosynthesis of these compounds, primarily through heterologous reconstitution of target biosynthetic pathways in suitable hosts, such as Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. Those heterologous biosynthetic systems can be used to confirm the functions of the isolated genes, economically scale up the production of the alkaloids for commercial distributions and engineer the biosynthetic pathways to produce valuable analogs of the alkaloids. In particular, extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details.

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“…Although the production of the intermediate chanoclavine‐I has been reported previously,5, 11 cycloclavine ( 6 ) is the first downstream ergot alkaloid to be successfully reconstituted in excellent yields. EasH was identified as the enzyme responsible for switching the pathway from the biosynthesis of festuclavine ( 4 ) or agroclavine ( 5 ) to that of cycloclavine ( 6 ).…”

mentioning

confidence: 96%

“…Whereas chanoclavine‐I ( 2 ) has been successfully reconstituted in S. cerevisiae as well as another heterologous host, Aspergillus nidulans ,5, 11 more derivatized ergot alkaloids have not been subject to such efforts. To examine whether we could produce the complex ergot alkaloid cycloclavine ( 6 ) in high yields in S. cerevisiae , we integrated multiple copies of cycloclavine‐pathway genes into the genome of the commonly used yeast strain S288C.…”

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confidence: 99%

Discovery and Reconstitution of the Cycloclavine Biosynthetic Pathway—Enzymatic Formation of a Cyclopropyl Group

et al. 2015

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The ergot alkaloids, a class of fungal‐derived natural products with important biological activities, are derived from a common intermediate, chanoclavine‐I, which is elaborated into a set of diverse structures. Herein we report the discovery of the biosynthetic pathway of cycloclavine, a complex ergot alkaloid containing a cyclopropyl moiety. We used a yeast‐based expression platform along with in vitro biochemical experiments to identify the enzyme that catalyzes a rearrangement of the chanoclavine‐I intermediate to form a cyclopropyl moiety. The resulting compound, cycloclavine, was produced in yeast at titers of >500 mg L−1, thus demonstrating the feasibility of the heterologous expression of these complex alkaloids.

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Partial Reconstruction of the Ergot Alkaloid Pathway by Heterologous Gene Expression in Aspergillus nidulans

Cited by 50 publications

References 19 publications

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

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

Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

Discovery and Reconstitution of the Cycloclavine Biosynthetic Pathway—Enzymatic Formation of a Cyclopropyl Group

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