Correction: Heterologous Reconstitution of the Intact Geodin Gene Cluster in Aspergillus nidulans through a Simple and Versatile PCR Based Approach

Abstract

Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing. However, the lack of solid methodology for genetic manipulation of most species severely hampers pathway characterization. Here we present a simple PCR based approach for heterologous reconstitution of intact gene clusters. Specifically, the putative gene cluster responsible for geodin production from Aspergillus terreus was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to transformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were successfully transferred between the two species enabling geodin synthesis in A. nidulans. Subsequently, functions of three genes in the cluster were validated by genetic and chemical analyses. Specifically, ATEG_08451 (gedC) encodes a polyketide synthase, ATEG_08453 (gedR) encodes a transcription factor responsible for activation of the geodin gene cluster and ATEG_08460 (gedL) encodes a halogenase that catalyzes conversion of sulochrin to dihydrogeodin. We expect that our approach for transferring intact biosynthetic pathways to a fungus with a well developed genetic toolbox will be instrumental in characterizing the many exciting pathways for secondary metabolite production that are currently being uncovered by the fungal genome sequencing projects.

Halogenase-Targeted Genome Mining Leads to the Discovery of (±) Pestalachlorides A1a, A2a, and Their Atropisomers

“…Flavin-dependent halogenases (FDHs), the most characterized halogenases based on their substrates, can be categorized into the following five main classes: free-standing phenol, free-standing indole, carrier protein-dependent phenol, carrier protein-dependent pyrrole, and aliphatic FDHs [ 9 ]. GedL is a free-standing phenol FDH from Aspergillus terreus NIH2624 [ 26 ]. It is involved in the biosynthesis of geodin and halogenates the substrate at the late stage of biosynthesis [ 26 ].…”

“…GedL is a free-standing phenol FDH from Aspergillus terreus NIH2624 [ 26 ]. It is involved in the biosynthesis of geodin and halogenates the substrate at the late stage of biosynthesis [ 26 ]. Here, we used GedL as the probe to conduct tBlastp analysis on our in-house fungal genome sequences.…”

“…Here, we used GedL as the probe to conduct tBlastp analysis on our in-house fungal genome sequences. An antiSMASH analysis was subsequently performed, and a gene ptaK , encoding a putative flavin-dependent halogenase with 51% amino acid sequence identity to GedL [ 26 ], was found to be contained in a cryptic BGC of endolichenic Pestalotiopsis rhododendri LF-19-12. Succeeding phylogenetic analysis showed that PtlK grouped with free-standing phenol FDHs ( Figure 2 ), suggesting that its substrate might hold a phenol moiety.…”

“…As a part of our efforts to investigate new natural products [ 21 , 22 , 23 , 24 , 25 ], we used the fungal FDH GedL [ 26 ] as a probe to explore the halogenase-containing BGCs from our in-house fungal genome database. A putative halogenase gene, ptlK , was mined from an endolichenic fungus, Pestalotiopsis rhododendri LF-19-12, and further bioinformatics analysis disclosed that ptlK was located in a cryptic BGC ptl .…”

Halogenation in Fungi: What Do We Know and What Remains to Be Discovered?

“…Contrary to PtaM on isosulochrin, the halogenation catalyzed by GedL on sulochrin occurs on the other phenolic moiety, showing the interesting regioselectivity of this type of enzyme. The BCG description has been realized after heterologous reconstitution of the geodin gene cluster from A. terreus in the expression platform Aspergillus nidulans [ 36 ]. Geodin has been described to enhance fibrinolytic activity of vascular endothelial bovine cells [ 125 ].…”