Unprecedented Mushroom Polyketide Synthases Produce the Universal Anthraquinone Precursor

Abstract

(Pre-)anthraquinones are widely distributed natural compounds and occur in plants, fungi, microorganisms, and animals, with atrochrysone (1) as the key biosynthetic precursor. Chemical analyses established mushrooms of the genus Cortinarius-the webcaps-as producers of atrochrysone-derived octaketide pigments. However, more recent genomic data did not provide any evidence for known atrochrysone carboxylic acid (4) synthases nor any other polyketide synthase (PKS) producing oligocyclic metabolites. Here, we describe an unprecedented class of non-reducing (NR-)PKS. In vitro assays with recombinant enzyme in combination with in vivo product formation in the heterologous host Aspergillus niger established CoPKS1 and CoPKS4 of C. odorifer as members of a new class of atrochrysone carboxylic acid synthases. CoPKS4 catalyzed both hepta- and octaketide synthesis and yielded 6-hydroxymusizin (6), along with 4. These first mushroom PKSs for oligocyclic products illustrate how the biosynthesis of bioactive natural metabolites evolved independently in various groups of life.

Keywords: Anthraquinones; Biosynthesis; Fungi; Octaketides; Polyketide Synthase.

Figure 1

Chemical structures of fungal monomeric and dimeric hepta‐ and octaketides.

Figure 2

A) Comparison of domain arrangements of fungal non‐reducing polyketide synthases. Domain acronyms: SAT: starter unit: ACP transacylase; KS: β‐ketoacyl synthase; AT: acyltransferase; PT: product template; ACP: acyl carrier protein; TE: thioesterase. B) Phylogenetic tree reconstructing the relationship of fungal PKSs. Representative products are shown for the respective PKS clades. The bar indicates the uncorrected pairwise distance. The branch lengths are proportional to the number of substitutions per site. Accession numbers of amino acid sequences are provided in the Supporting Information.

Figure 3

In vivo activity assays with Cortinarius PKSs. A) Liquid cultures and chromatograms of ethyl acetate extracts of recombinant A. niger strains expressing C. odorifer PKS genes. The yellow color of some cultures indicates polyketide formation by recombinant strains. Chromatograms were recorded at λ=395 nm. The top trace in panel A represents an overlay of individual chromatograms for standards of doxycycline (Doxy), atrochrysone (1), endocrocin (8), 6‐hydroxymusizin (6), and emodin (9). Trace a: Untransformed A. niger host; b: A. niger tNAL000; traces c–h: A. niger harboring the genes for CoPKS1 (c, A. niger tNAL024), CoPKS2 (d, A. niger tNAL025), CoPKS3 (e, A. niger tNAL026), CoPKS4 (f, A. niger tNAL002), CoPKS5 (g, A. niger tNAL003), and CoPKS6 (h, A. niger tNAL004), respectively. B) Representative HR‐ESIMS spectra (negative mode) of signals, extracted from the CoPKS4 producing A. niger tNAL002. In addition to the major signals (identified as compounds 1, 6, 8, 9, and putative dimers, marked **,***, and ****), the assumed primary PKS product atrochrysone carboxylic acid (4) was detected in traces, as indicated by an asterisk. Calculated masses ([M−H]⁻) are: m/z 317.0667 (4), 273.0769 (1), 313.0354 (8), 597.1039 (**), 597.1039 (***), 231.0663 (6), 553.1140 (****) and 269.0456 (9).

Figure 4

Structure of atrochrysone (1). ¹H–¹H COSY correlations are shown in red, HSQC correlation in blue.

Scheme 1

Sequence of oligocyclic octaketide biosynthesis and modification after heterologous production of C. odorifer PKSs in A. niger. Please see Figure S9 for a model of the CoPKS4‐catalyzed heptaketide biosynthesis.

Figure 5

In vitro product formation by CoPKS4. Extracted ion chromatograms (EICs) of lyophilized reactions are shown for m/z 273 [M−H]⁻ (red traces) and m/z 231 [M−H]⁻ (blue traces) to detect the octaketide atrochrysone (1) and the heptaketide 6‐hydroxymusizin (6), respectively. The top trace represents an overlay of individual EICs of standards. Trace a: negative control with intact CoPKS4 but without any substrates; trace b: negative control with heat‐treated CoPKS4, acetyl‐CoA and malonyl‐CoA; trace c: reaction with CoPKS4, acetyl‐CoA and malonyl‐CoA; trace d: reaction with CoPKS4 and malonyl‐CoA only. The enzymatic reactions in c and d showed a second product at m/z 231 (t _R=2.1 min), which remained unidentified due to low quantity.