Silencing of a second dimethylallyltryptophan synthase of Penicillium roqueforti reveals a novel clavine alkaloid gene cluster

Abstract

Penicillium roqueforti produces several prenylated indole alkaloids, including roquefortine C and clavine alkaloids. The first step in the biosynthesis of roquefortine C is the prenylation of tryptophan-derived dipeptides by a dimethylallyltryptophan synthase, specific for roquefortine biosynthesis (roquefortine prenyltransferase). A second dimethylallyltryptophan synthase, DmaW2, different from the roquefortine prenyltransferase, has been studied in this article. Silencing the gene encoding this second dimethylallyltryptophan synthase, dmaW2, proved that inactivation of this gene does not prevent the production of roquefortine C, but suppresses the formation of other indole alkaloids. Mass spectrometry studies have identified these compounds as isofumigaclavine A, the pathway final product and prenylated intermediates. The silencing does not affect the production of mycophenolic acid and andrastin A. A bioinformatic study of the genome of P. roqueforti revealed that DmaW2 (renamed IfgA) is a prenyltransferase involved in isofumigaclavine A biosynthesis encoded by a gene located in a six genes cluster (cluster A). A second three genes cluster (cluster B) encodes the so-called yellow enzyme and enzymes for the late steps for the conversion of festuclavine to isofumigaclavine A. The yellow enzyme contains a tyrosine-181 at its active center, as occurs in Neosartorya fumigata, but in contrast to the Clavicipitaceae fungi. A complete isofumigaclavines A and B biosynthetic pathway is proposed based on the finding of these studies on the biosynthesis of clavine alkaloids.

Clavine Alkaloids Gene Clusters of Penicillium and Related Fungi: Evolutionary Combination of Prenyltransferases, Monooxygenases and Dioxygenases

“…In addition to the studies with N. fumigata , recently clavine gene clusters have been found in Penicillium commune [ 6 ], in A. japonicus , that forms cycloclavine [ 7 , 8 ], in several Arthrodermataceae family members [ 9 ] and recently in Penicillium roqueforti [ 10 ]. In this article, we focus our attention on a comparative study of the clavine gene clusters and their encoded enzymes in Penicillium , Aspergillus and related Aspergillaceae fungi in comparison with the better known lysergic acid-derived alkaloids produced by Clavicipitaceae fungi.…”

“…During the study of a novel dimethylallyltryptophan synthase gene in P. roqueforti , different from the roquefortine prenyltransferase, Fernández Bodega and coworkers [ 10 ] found that silencing of this second DMATS gene, blocked the biosynthesis of isofumigaclavine A but not that of roquefortine C. These studies indicated that this second DMATS gene of P. roqueforti , named ifgA , is involved in the biosynthesis of isofumigaclavine A. In addition, silencing of the ifgA gene blocked or reduced the biosynthesis of two other compounds [ 10 ] that are likely prenylated intermediates or oxidized products derived of agroclavine [ 1 , 2 ] but does not affect the production of mycophenolic acid or andrastin A. Interestingly, the formation of roquefortine C was increased 30% in the ifgA silenced mutant, suggesting that blocking the clavine pathway diverts the precursors tryptophan and DMA-PP to form roquefortine C. The ifgA gene, encodes a 462 amino acids DMATS enzyme of the direct (normal) type [ 21 ]. Bioinformatic analysis showed that this protein is highly similar to other DMAT synthases in Penicillium camemberti , Penicillium expansum , Penicillium steckii , Penicillium griseofulvum and P. commune (see below).…”

“…The P. roqueforti ifgA encoded protein is similar to the FgaPT2 of N. fumigata (63% identity) and this suggests that IfgA is the first enzyme of the clavine biosynthetic pathway in P. roqueforti . The FgaPT1 enzyme is a reverse prenyltransferase that introduces a dimethylallyl group at the C-2 carbon of fumigaclavine A to form fumigaclavine C; this reverse prenyltransferase is absent in the genome of P. roqueforti and therefore this fungus is able to form isofumigaclavine A but does not convert this product to isofumigaclavine C [ 10 ]. On the other hand, the IfgA protein is more distantly related to DmaW, the Claviceps purpurea homologous protein (58% identity), what suggests interesting implications in the evolution of these genes (see below).…”

“…The organization of these six genes is shown in Figure 2 A. The functionality of ifgA has been proved by RNA silencing [ 10 ]. The role of the proteins encoded by the other genes has been proposed by comparison with the enzymes encoded by the orthologous genes of N. fumigata , characterized by gene disruption, gene complementation and in vitro heterologous expression [ 14 , 25 ] ( Table 1 ).…”

“…Accumulation of prenylated intermediates and lack of fumigaclavine A formation has been reported in mutants of N. fumigata disrupted in the festuclavine hydroxylase [ 5 ]. Similarly, the lack of an authentic orthologous festuclavine hydroxylase in P. roqueforti may explain the observed accumulation of similar high molecular weight compounds [ 10 ] and possibly also of the earlier intermediates agroclavine and festuclavine [ 17 ].…”