A Branched Biosynthetic Pathway Is Involved in Production of Roquefortine and Related Compounds in Penicillium chrysogenum

Abstract

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.

Prototype of an intertwined secondary-metabolite supercluster

“…The data obtained in this study suggest that the putative O-methyltransferase FmaD is responsible for methylation of the C6-hydroxyl group of β-trans-bergamotene during fumagillin biosynthesis. O-methyltransferases in a variety of secondary-metabolite gene clusters in different fungal species have been demonstrated to catalyze similar reactions (35)(36)(37). Identification of a fumagillinaldehyde derivative in the ΔfmaF mutant suggests that the putative phytanoyl-CoA-oxidase is involved in the final oxidation step of the tetraene chain.…”

Novel Key Metabolites Reveal Further Branching of the Roquefortine/Meleagrin Biosynthetic Pathway

“…However, BLAST analysis did not reveal an enzyme in the roquefortine/meleagrin pathway that is able to perform that reaction (6,7). Moreover, a 53 times higher concentration of 8 as compared with 10 in the host strain, but the absence of 8 in the roqO deletion strain with 10 still being present, leads to the conclusion that 8 is not a precursor of 10 (Fig.…”

“…Sample preparation was carried out as described previously (7). Metabolomic profiling was performed on an Agilent 1200 capillary pump (Agilent, Santa Clara, CA) coupled to a Surveyor photodiode array detector (Thermo Scientific) and an LTQ-FT-ICR-Ultra mass spectrometer (Thermo Scientific) equipped with an electrospray interface as described earlier (7).…”

“…Next to penicillins, secondary metabolites such as roquefortines and glandicolines were isolated from liquid cultures of P. chrysogenum, which show pharmaceutically interesting properties, such as neurotoxic (2), antimicrobial (3,4), and antitumor (5) activities. They are structurally closely related and arise from the roquefortine/meleagrin pathway, which contains a dimodular nonribosomal peptide synthetase flanked by six associated genes (6,7). Starting with histidyltryptophanyldiketopiperazine (HTD), 5 synthesized by the core synthetase enzyme RoqA using tryptophan and histidine as substrates, RoqD catalyzes the reversed prenylation of HTD at the C-3 of its indole moiety, utilizing dimethylallyl diphosphate to form roquefortine D. At the same time, RoqR, a cytochrome P450 oxidoreductase, oxidizes HTD at its histidinyl moiety to dehydrohistidyltryptophanyldiketo piperazine (DHTD).…”

“…Although several labeling, silencing, and deletion experiments have been conducted, there is still ambiguity about the subsequent biosynthetic reactions and the genes involved. For instance, roquefortine C is supposed to be converted into glandicoline A and further to glandicoline B with RoqM and RoqO each catalyzing one reaction (6,7). However, their assignment to a particular reaction is still unclear.…”