A natural short pathway synthesizes roquefortine C but not meleagrin in three different Penicillium roqueforti strains

Abstract

The production of mycotoxins and other secondary metabolites in Penicillium roqueforti is of great interest because of its long history of use in blue-veined cheese manufacture. In this article, we report the cloning and characterization of the roquefortine gene cluster in three different P. roqueforti strains isolated from blue cheese in the USA (the type strain), France, and the UK (Cheshire cheese). All three strains showed an identical roquefortine gene cluster organization and almost identical (98-99%) gene nucleotide sequences in the entire 16.6-kb cluster region. When compared with the Penicillium chrysogenum roquefortine/meleagrin seven-gene cluster, the P. roqueforti roquefortine cluster contains only four genes (rds, rdh, rpt, and gmt) encoding the roquefortine dipeptide synthetase, roquefortine D dehydrogenase, roquefortine prenyltransferase, and a methyltransferase, respectively. Silencing of the rds or rpt genes by the RNAi strategy reduced roquefortine C production by 50% confirming the involvement of these two key genes in roquefortine biosynthesis. An additional putative gene, orthologous of the MFS transporter roqT, is rearranged in all three strains as a pseudogene. The same four genes and a complete (not rearranged) roqT, encoding a MFS transporter containing 12 TMS domains, occur in the seven-gene cluster in P. chrysogenum although organized differently. Interestingly, the two "late" genes of the P. chrysogenum roquefortine/meleagrin gene cluster that convert roquefortine C to glandicoline B and meleagrin are absent in the P. roqueforti four-gene cluster. No meleagrin production was detected in P. roqueforti cultures grown in YES medium, while P. chrysogenum produces meleagrin in these conditions. No orthologous genes of the two missing meleagrin synthesizing genes were found elsewhere in the recently released P. roqueforti genome. Our data suggest that during evolution, the seven-gene cluster present in P. chrysogenum, and probably also in other glandicoline/meleagrin producing fungi, has been trimmed down to a short cluster in P. roqueforti leading to the synthesis of roquefortine C rather than meleagrin as a final product.

The Biosynthetic Gene Cluster for Andrastin A in Penicillium roqueforti

“…This technology has been widely used to demonstrate the functionality of several biosynthetic gene clusters in P. roqueforti (Kosalková et al, 2015; Del-Cid et al, 2016; Hidalgo et al, 2016). For this purpose, ten suitable plasmids were constructed and used to transform P. roqueforti CECT 2905 (see Material and Methods for details).…”

“…Penicillium roqueforti strain CECT 2905 produces several secondary metabolites and currently, the gene clusters responsible for the biosynthesis of three of them, namely roquefortine C, PR-toxin and mycophenolic acid, have been identified (Kosalková et al, 2015; Del-Cid et al, 2016; Hidalgo et al, 2016). Here we describe the gene cluster responsible for the biosynthesis of another secondary metabolite from this fungus, andrastin A, a promising antitumoral compound.…”

“…The loss of genes in a P. roqueforti gene cluster, compared with the respective P. chrysogenum cluster, has been observed before in the roquefortine C/ meleagrin gene cluster. In P. chrysogenum , this cluster contains seven genes, whereas in P. roqueforti it is shorter and contains four genes (Kosalková et al, 2015). In this case, the evolutionary reorganization of the cluster produced that two genes present in P. chrysogenum were entirely lost in P. roqueforti , whereas a third gene was partially lost and now is found as a residual pseudogene (Kosalková et al, 2015; Martín and Liras, 2016).…”

“…In P. chrysogenum , this cluster contains seven genes, whereas in P. roqueforti it is shorter and contains four genes (Kosalková et al, 2015). In this case, the evolutionary reorganization of the cluster produced that two genes present in P. chrysogenum were entirely lost in P. roqueforti , whereas a third gene was partially lost and now is found as a residual pseudogene (Kosalková et al, 2015; Martín and Liras, 2016). These data and our observation about the pseudogenization of adrB in the adr cluster suggest that the evolutionary reorganization in clusters from P. chrysogenum and P. roqueforti may be a common and extended process.…”

“…Thus, this species produces mycotoxins (such as roquefortine C and PR-toxin), and bioactive compounds such as mycophenolic acid and andrastin A (García-Estrada and Martín, 2016). In P. roqueforti , the gene clusters responsible for the biosynthesis of roquefortine C, PR-toxin and mycophenolic acid have been recently identified (Kosalková et al, 2015; Del-Cid et al, 2016; Hidalgo et al, 2016). However, to date the genes responsible for the biosynthesis of andrastin A in this fungus remain as unknown.…”