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Ergot alkaloids are alkaline natural products that belong to the group of indole alkaloids. They have a broad spectrum of pharmacological, but often toxic effects because of their structural similarity with the neurotransmitters dopamine, serotonin and adrenaline. The main producers are filamentous fungi of the families Clavicipitaceae and Aspergillaceae like Claviceps purpurea, Aspergillus fumigatus or Penicillium commune. Starting with L-tryptophan, the first steps of the biosynthesis of ergot alkaloids are similar in all known producing species. After reaching the branch point of the biosynthetic pathways, different end products are formed.
This study aims to investigate the remaining common step of the biosynthesis of ergot alkaloids: the conversion of 4-DMA-L-abrine to chanoclavine-I. This step includes at least two oxidation and one decarboxylation reactions. Deletion studies showed that the FAD dependent oxidoreductase FgaOx1 and the catalase FgaCat are essential for the formation of chanoclavine-I. To elucidate the reaction mechanism, both enzymes should be overproduced in E. coli or S. cerevisiae and characterized biochemically. In the case of fgaCat, the heterologous expression in E. coli was successful, but no catalytic activity could be detected for this enzyme alone. The sequence of FgaOx1 from the NCBI database was corrected after an analysis of the intron-exon-structure, but the overproduction of the protein in E. coli or S. cerevisiae was not successful.
A bioinformatic comparison of the ergot alkaloid genes from Aspergillus fumigatus, Claviceps purpurea and Arthroderma benhamiae with available genome sequences of Ascomycetes from the NCBI database revealed the presence of ergot alkaloid genes in 15 species that had not been described before. Two of those species are Penicillium roqueforti and Penicillium camemberti which are known for their usage in the industrial production of cheese. In addition to FgaOx1, another 27 protein sequences from the NCBI database were corrected after analysis of their intron-exon-structures in the course of the bioinformatic studies. By identifying the gene clusters and correcting the sequences, this work is a valuable contribution to the future elucidation of the biosynthetic pathways and the analysis of the structural diversity of ergot alkaloids in various producing species.
The production of isofumigaclavine A in Penicillium roqueforti has been reported previously, but there were no publications about its biosynthesis at the beginning of this study. The identification of two ergot alkaloid gene clusters and two additional non-clustered genes in different genomic regions laid the foundation for the elucidation of the biosynthetic pathway. Eight genes are very likely involved in the later steps of the isofumigaclavine A biosynthesis and were chosen for cloning and heterologous expression in E. coli and S. cerevisiae. Three of these genes were successfully expressed in E. coli and the recombinant proteins (FgaDHPr, FgaOx3Pr3 and FgaATPr) were purified via affinity chromatography. The in vitro assays showed no enzymatic activity for FgaATPr. FgaDHPr was characterized as a short-chain dehydrogenase/reductase which catalyzes the conversion of chanoclavine-I to chanoclavine-I aldehyde in the presence of NAD+. The native form of the enzyme is likely a pentamer. The KM value for chanoclavine-I was 573 μM and for NAD+ 82 μM. The mean maximum reaction velocity vmax was 326 nmol mg-1 min-1, corresponding to a turnover number kcat 0.15 s-1. FgaOx3Pr3 belongs to the “Old Yellow Enzymes“ and acts as a FMN containing and NAD(P)H dependent oxidoreductase. FgaOx3Pr3 was characterized as a chanoclavine-I aldehyde reductase which catalyzes the formation of festuclavine in the presence of FgaFS from Aspergillus fumigatus or EasG from Claviceps purpurea in vitro. In addition, the enzyme activities of all investigated chanoclavine-I dehydrogenases (FgaDH, ChaDH, FgaDHPr and FgaDHPca) were enhanced significantly by FgaOx3Pr3. These experiments proved that FgaOx3Pr3 overcomes the product inhibition for the FgaDH reaction, resulting in an enhanced product yield. This feature has not been described for other enzymes of the same class, therefore FgaOx3Pr3 is a unique bifunctional enzyme and an excellent candidate for the chemoenzymatic synthesis of clavine-type ergot alkaloids.
The production of ergot alkaloids in Penicillium camemberti has not been reported in the literature so far. Although a potential gene cluster for the biosynthesis of ergot alkaloids has been identified in this study, no ergot alkaloids were detected in the culture extracts of the investigated strains. Nevertheless, the two genes fgaDHPca and easHPca were cloned and successfully expressed in E. coli and S. cerevisiae. EasHPca showed no enzymatic activities in the in vitro assays, while FgaDHPca was characterized as a chanoclavine-I dehydrogenase. The native Form of FgaDHPca is likely a tetramer and the KM values were 536 μM for chanoclavine-I and 528 μM for NAD+. The mean maximum reaction velocity vmax was 383 nmol mg-1 min-1 and the turnover number kcat 0.18 s-1.Thus, it was shown for the first time in this work, that P. camemberti has the genetic potential for the biosynthesis of ergot alkaloids.