Fig 1.
Identification of polyketide synthase for the biosynthesis of cristazarin.
PKS gene expression profiles of the Cladonia metacorallifera mycobiont grown on culture media containing either fructose, glucose, ribitol or sorbitol as a single carbon source and on malt extract agar (MYA). Expression values (Log2-transformed (RPKM+1)) are shown as heat maps for 39 PKS genes in C. metacorallifera.
Fig 2.
Demarcation of the cristazarin BGC boundaries by mapped reads of RNA-seq.
(A) Mapped reads of five RNA-seq samples of the C. metacorallifera mycobiont growing on different carbon sources were shown for a genomic locus harboring the PKS22 (crz7; gene ID: Cmt_01711). RNA-seq reads mapped on the C. metacorallifera reference genome were subsampled to 60 million reads for visual comparison of expression levels between samples. Arrows on the x-axis indicate genes (Cmt_01704–Cmt_01717). The numbers on the y-axis are per-base coverage of mapped reads. (B) Synteny of the cristazarin BGCs in Cladonia species: C. metacorallifera (top), C. macilenta (middle), and C. borealis (bottom). Arrows indicate open reading frames (ORFs) found in the BGCs, and homologous genes were represented with different colors. Numbers above the arrows indicate crz1–crz9.
Table 1.
Annotation of the cristazarin biosynthetic gene cluster in Cladonia metacorallifera.
Fig 3.
Phylogenetic dereplication of polyketide synthases related to melanin production.
A maximum likelihood phylogenetic tree of non-reducing polyketide synthases (NR-PKSs). Colored strips on the right side of leaves indicate polyketide backbones produced by the functionally characterized NR-PKSs. Clades of the previously described NR-PKS groups (Kim et al., 2021; Mosunova et al., 2022) were shaded with different colors (see inset). Arrows indicate clades of the Cladonia PKS families, PKS13, PKS14, PKS15, and PKS22, that belong to group II NR-PKS (Kim et al., 2021). Leaves highlighted in red are lichen NR-PKSs. Crz7 in C. metacorallifera was denoted by a red asterisk. An NR-PKS in Aspergillus fumigatus (EDP55264) that produce melanin pigment using YWA as a precursor was set as an outgroup. Bootstrap values of greater than 75% were shown. Branch lengths are proportional to the inferred amount of evolutionary change, and the scale represents 0.1 amino acid sequence substitutions per site. T4HN, 1,3,6,8-tetrahydroxynaphthalene; AT4HN, 2-acetyl-1,3,6,8-tetrahydroxynaphthalene.
Fig 4.
Divergent biosynthetic routes of cristazarin.
A proposed biosynthetic pathway of cristazarin from a polyketide precursor, 2-acetyl-1,3,6,8-tetrahydoxynaphthalene (AT4HN), can be deduced from putative function of tailoring enzymes in the cristazarin gene cluster (red). Note that a biosynthetic pathway for 6-O-methylasparvenone (a naphthalenone) is analogous to that for cristazarin, involving several biosynthetic genes in the naphthalenone BGC (blue) in Aspergillus parvulus (Mosunova et al. 2022). ACP, acyl carrier protein domain.