Discovery and Reconstitution of the Cycloclavine Biosynthetic Pathway - Enzymatic Formation of a Cyclopropyl Group Dorota Jakubczyk, Lorenzo Caputi, Anaëlle Hatsch, Curt A. F. Nielsen, Melanie Diefenbacher, Jens Klein, Andrea Molt, Hartwig Schröder, Johnathan Z. Cheng, Michael Naesby, and Sarah E. O’Connor ACIE, 2015, 54, 5117-5121 CO2H N NH2 H O O 8 enzymes + P P N O O O H O O L-tryptophan DMAPP NH cycloclavine Steph McCabe Wipf Group- Current Literature 27th June 2015 Steph McCabe@ Wipf Group Slide 1 of 12 6/27/15 N H Ergot Alkaloids NH • Ergot alkaloids are produced by fungi of the Clavicipitaceae and Trichocomaceae families, which infect the seeds of grass or grains • Consumption of ergot contaminated grains leads to ergotism (St. Anthony’s fire); § ergotismus convulsivus characterized by hallucinations and paranoia § ergotismus gangrenous characterized by gangrene of the feet, legs, hands and arms • Ergot alkaloids are characterized by the presence of a tetracyclic Ergoline core and can be further classified into Lysergic acid or Clavine subclasses based on the oxidation state of the substituent at C8 8 NH D H HO2C 8 Me 8 NH C NH D H D H A B 2 C C N H 1 Ergot on wheat stalks A B 2 Ergoline Core A B 2 N N H 1 H 1 Lysergic acid subclass Clavine subclass O O N N NH O Et2N OH H O N O NH N N H H H H H NH NH NH NH ergotamine Lysergic acid diethylamide (LSD) festuclavine cycloclavine Wallwey, C. and Li, S.,Nat. Prod. Rep., 2011, 28, 496 Jakubczyk, D., Cheng, J. Z. and O’Connor, S., Nat. Prod. Rep., 2014, 31, 1328 Steph McCabe@ Wipf Group Slide 2 of 12 6/27/15 N H Biological Activity NH • The broad spectrum of bioactivity exhibited by ergot alkaloids is related to their ability to act as an agonist or antagonist toward neuroreceptors for dopamine, serotonin and adrenaline. 7 8 6 NH H 9 10 5 4 H11 16 3 12 2 13 1 15 NH 14 Ergoline 6 6 NH2 NHMe 6 NH2 5 HO 5 5 10 10 4 11 11 11 HO 16 3 12 16 12 16 12 2 13 13 13 1 15 15 15 N HO 14 HO 14 14 H OH OH serotonin (5-HT) dopamine (DA) epinephrine (adrenalin) Jakubczyk, D., Cheng, J. Z. and O’Connor, S., Nat. Prod. Rep., 2014, 31, 1328 Steph McCabe@ Wipf Group Slide 3 of 12 6/27/15 N H Examples of FDA Approved Drugs with an Ergoline Core NH Br OH N OH NH NH O O O N H O N N H H O N NH R N Ergometrine R = Me R = H Me Nicergoline Use: induces contractions, Methysergide Methylergometrine Use: treatment of senile prevents post partum Use migraine treatment Use: prevents post partum dementia haemorrhage MOA: antagonist of haemorrhage, migraine treatment MOA: partial agonist/antagonist of MOA: alpha-1A adrenergic Mechanism of Action (MOA): serotonin 5-HT2B receptors receptor antagonist acts on alpha-adrenergic serotonergic, dopaminergic and serotonergic receptors and alpha adrenergic receptors N O NH O N N NH O N O S N H OH O N H O N H H H H Br NH NH NH Pergolide Cabergoline Bromocriptine Use: treatment of Parkinson’s Disease Use: treatment of Use: treatment of pituitary tumors, MOA: agonist of dopaminergic receptors hyperprolactinaemia Parkinson's disease, (*withdrawn from the US market in 2007 and Parkinson's disease hyperprolactinaemia, due to increased risk of cardiac MOA: agonist of MOA: agonist valvulopathy) dopamine D2 receptors. of dopamine D2 receptors DrugBank 4.0: Nucleic Acids Res., 2014, 42(1),1091-7. Steph McCabe@ Wipf Group Slide 4 of 12 6/27/15 N H Cycloclavine NH N H NH Cycloclavine • First isolated in 1969 from the seeds of the African morning glory (Ipomea hildebrandtii) by Hoffman and co-workers • Subsequently isolated Aspergillus japonicus, a species of in filamentous fungus • Absolute configuration assigned from X-ray crystallographic analysis of the methobromide salt • No significant biological activities reported thus far Ipomea hildebrandtii • 3 total syntheses, including an earlier synthesis from our group in 14 linear steps and 1.2% overall yield steps Boc O N 1. IMDAF OH HN 2. LAH N N HO H H CO2Me (±)-cycloclavine Hofmann et. al.,Tetrahedron, 1969, 25, 5879 Furuta, T., et al., Agric. Biol. Chem., 1982, 46, 1921 Incze, M., et al., Tetrahedron, 2008, 64, 2924 Petronijevic, F. R. and P. Wipf JACS, 2011, 133, 7704 Jabre, N. D., et al. ,Tetradhedron Lett,, 2014, 56, 197 Steph McCabe@ Wipf Group Slide 5 of 12 6/27/15 N Biosynthetic Pathway Elucidation: H Reconstitution of the Ergot Alkaloid Synthesis (EAS) Genes NH • All ergot alkaloids are derived from common a biosynthetic intermediate - chanoclavine-I. The mechanisms of most downstream biosynthetic transformations are unknown • Recently a 16.8 kbp biosynthetic cluster, containing 8 genes in the genome of cycloclavine producer Aspergillus japonicus was identified (O'Connor et al) • 7 genes are homologous to genes previously implicated in the biosynthesis of festuclavine or agroclavine. • Role of easH unknown equipped with a 1.7mm cryogenic TCI probe. The structure of cycloclavine 6 was solved by H 1 13 1 1 1 13 lysergic acid N means H-NMR, C-NMR, H, H-ROESY, and H, C-HMBC.subclass H OH O HN SI-2 Supporting Figures agroclavine H H EasD/ FgaDH H H N N H Figure S1 Organization of the AspergillusH japonicus cycloclavine 6 gene cluster NAD NADH HN HN H N ?? N chanoclavine-I chanoclavine-I aldehyde clavine subclass H H NAD NADH HN HN festuclavine cycloclavine Steph McCabe@ Wipf Group Slide 6 of 12 6/27/15 13 N Biosynthetic Pathway Elucidation: H Reconstitution of the Ergot Alkaloid Synthesis (EAS) Genes NH • This paper aimed to validate whether this cluster was responsible for cycloclavine biosynthesis by reconstitution of the eight genes in S. cerevisiae • Chanoclavine-I was synthesized by heterologous expression of dmaW, easF, easE and easC in a S. cerevisiae strain CO2H CO2H DmaW/ EasF/ NH2 prenyl transferase NH2 methyl transferase N DMAPP PPi N SAM S-homo H H cysteine L-tryptophan DMAT OH HN H H EasE/ oxidase N EasC/ catalase H CO2H N HN H Chanoclavine-I N-Me-DMAT Microbial Cell Factories, 2014, 13, 95 Nat. Prod. Rep., 2011, 28, 496 Steph McCabe@ Wipf Group Slide 7 of 12 6/27/15 Angewandte. Communications N Biosynthetic Pathway Elucidation: H Reconstitution of the Ergot Alkaloid Synthesis (EAS) Genes NH • In Vivo transformation of the chanoclavine-I producing strain with easD, easA, and easG produced festuclavine • Addition of easH produced cycloclavine as the major product along with festuclavine • In vitro incubation of chanoclavine-I aldehyde with easA, eas G easH produced cycloclavine • Cycloclavine was reconstituted in S. cerevisiae with a final concentration of 529 mg. L-1 OH O easD/ eas A/ H H H H Reductase N oxidase N H H NAD NADH NADH NAD HN HN chanoclavine-I chanoclavine-I aldehyde H eas G/ N Reductase H O NADH NAD HN festuclavine H H N H N H N H HN H easH/ eas G/ incubation of chanoclavine-I aldehyde dioxygenase Reductase with easA, eas G and easH HN HN cycloclavine festuclavineFigure 1. Ergot-alkaloid biosynthetic pathway. a) Biosynthesis of festuclavine (4), agroclavine (5), and cycloclavine (6) from l-tryptophan and NADH NAD optimised ratio > 7:1 dimethylallyl pyrophosphate (DMAPP). b) LC–MS chromatograms showing that EasA, EasG, and EasH are required to generate cycloclavine (6) from chanoclavine-I aldehyde (3). i–iv) Authentic standards of chanoclavine-I aldehyde (3; i), agroclavine (5; ii), festuclavine (4; iii), and cycloclavine (6, iv). v,vi) Reaction products from the incubation of Aj_EasA, Aj_EasG, and Aj_EasH with chanoclavine-I aldehyde (3) and cofactors: Steph McCabe@ Wipf Group Slide 8 of 12 v) by-product, festuclavine (4); vi) predominant enzymatic6/27/15 product, cycloclavine (6). both cycloclavine (6) and festuclavine (4) have been isolated derivatized ergot alkaloids have not been subject to such from A. japonicus,[3a] we hypothesize that this gene cluster efforts. To examine whether we could produce the complex produces a mixture of these two compounds in the native ergot alkaloid cycloclavine (6) in high yields in S. cerevisiae, host, though how this ratio is impacted by environmental we integrated multiple copies of cycloclavine-pathway genes conditions is unknown. To assess whether increased amounts into the genome of the commonly used yeast strain S288C. of EasH would impact the festuclavine/cycloclavine ratio, we The best strain had an additional copy of easG, two additional constructed a strain carrying the entire eight-gene cluster copies of dmaW and easD, and three additional copies of easC supplemented with additional copies of easH from plasmid and easH. Moreover, additional copies of the host genes pdi1 vectors. We observed a clear gene-dose-dependent increase in (protein disulfide isomerase) and fad1 (FAD synthetase) the ratio of cycloclavine (6) to festuclavine (4) as the copy were also included to assist in the production of the disulfide- number of easH increased (see Figure S4). and flavin-containing enzyme EasE (see Table S1 in the Complex ergot alkaloids constitute a rich source of Supporting Information). This strain resulted in the produc- biologically active compounds, and a robust production tion of cycloclavine (6) with a final concentration of 1 platform for these molecules will improve accessibility and 529 mgLÀ in the growth medium when fermentation was prospects for commercial application. Whereas chanoclavine- carried out for 160 h in a 1 L fermenter and a fed-batch I(2) has been successfully reconstituted in S.