Molecular Cloning and Functional Analysis of Gene Clusters for the Biosynthesis of Indole-Diterpenes in Penicillium crustosum and P. janthinellum

Abstract

The penitremane and janthitremane families of indole-diterpenes are abundant natural products synthesized by Penicillium crustosum and P. janthinellum. Using a combination of PCR, cosmid library screening, and Illumina sequencing we have identified gene clusters encoding enzymes for the synthesis of these compounds. Targeted deletion of penP in P. crustosum abolished the synthesis of penitrems A, B, D, E, and F, and led to accumulation of paspaline, a key intermediate for paxilline biosynthesis in P. paxilli. Similarly, deletion of janP and janD in P. janthinellum abolished the synthesis of prenyl-elaborated indole-diterpenes, and led to accumulation in the latter of 13-desoxypaxilline, a key intermediate for the synthesis of the structurally related aflatremanes synthesized by Aspergillus flavus. This study helps resolve the genetic basis for the complexity of indole-diterpene natural products found within the Penicillium and Aspergillus species. All indole-diterpene gene clusters identified to date have a core set of genes for the synthesis of paspaline and a suite of genes encoding multi-functional cytochrome P450 monooxygenases, FAD dependent monooxygenases, and prenyl transferases that catalyse various regio- and stereo- specific oxidations that give rise to the diversity of indole-diterpene products synthesized by this group of fungi.

Keywords: gene clusters; indole-diterpene; janthitremanes; penitrems; shearinines.

Figure 1

Chemical structures of paspaline (a), penitrem A (b), and shearinine A (c).

Figure 2

Physical maps of the PAX (paxilline) locus in P. paxilli (a), PEN (penitremane) locus in P. crustosum (b) and the JAN (janthitremane) locus in P. janthinellum (c). For each locus the top line represents the entire sequence and subsequent lines represent unique cosmid clones that were identified for each genome. The positions and transcriptional orientations of identified genes are indicated by arrows on the sequences with genes homologous to paxilline and post-paxilline biosynthesis in P. paxilli shown in red.

Figure 3

Indole-diterpene biosynthesis gene replacements for (a) janD and (b) janP at the JAN locus in P. janthinellum (mutants #38, and #1-9 and #2-5, respectively), and (c) penP at the PEN locus in P. crustosum (mutant #2-9). Physical maps of the respective genomic regions with linear replacement constructs and mutant alleles with autoradiographs of Southern blots of 1 μg genomic digest probed with [³²P]-dCTP-labelled fragments from within the deleted region of each gene. Expected bands in the Southern analyses were 5.7-kb for janD (a) 6.7-kb for janP (b) 5.5-kb for penP (c). (d) PCR amplification analysis of targeted and flanking genes from wild-type and mutant strains using primers described in Table A2.

Figure 3

Indole-diterpene biosynthesis gene replacements for (a) janD and (b) janP at the JAN locus in P. janthinellum (mutants #38, and #1-9 and #2-5, respectively), and (c) penP at the PEN locus in P. crustosum (mutant #2-9). Physical maps of the respective genomic regions with linear replacement constructs and mutant alleles with autoradiographs of Southern blots of 1 μg genomic digest probed with [³²P]-dCTP-labelled fragments from within the deleted region of each gene. Expected bands in the Southern analyses were 5.7-kb for janD (a) 6.7-kb for janP (b) 5.5-kb for penP (c). (d) PCR amplification analysis of targeted and flanking genes from wild-type and mutant strains using primers described in Table A2.

Figure 4

Extracted ion chromatograms for P. crustosum wild-type (WT) and ∆penP deletion mutant showing LC-MS peaks for 13-desoxypaxilline (17.5 min, m/z + 420), paspaline (26.4 min, m/z + 422) and penitrems E, A, D, B, and F (respectively 11.3 min, m/z + 568; 14.9 min, m/z + 584; 19.4 min, m/z + 600; 20.8 min, m/z + 618; and 25.0 min, m/z + 634).

Figure 5

Extracted ion chromatograms for P. janthinellum wild-type (WT) and ∆janP and ∆janD deletion mutants showing LC-MS peaks for 13-desoxypaxilline (17.6 min, m/z + 420), and prenyl-elaborated indole-diterpenes (25.5 min, m/z + 584 attributable to shearinine A or F; 32.0 min and 33.2 min, both m/z + 570 attributable to shearinine K or isomers).

Figure 6

Proposed biosynthetic scheme for shearinine K and A biosynthesis in P. janthinellum.