A unique highly reducing polyketide synthase (HR-PKS) with a reductase domain was identified in a betaenone biosynthetic gene cluster. Successful heterologous expression and characterization of the HR-PKS and trans-acting enoyl reductase (ER) provide insights into the core structure formation with a decalin scaffold and allow reconstitution of the betaenone biosynthetic machinery.

Oxygenated cyclopentene systems are unique structural motifs found in fungal polyketides such as terrein, cyclohelminthols, and palmaenones. Here we report the identification of the biosynthetic gene clusters for cyclohelminthols and palmaenones and the functional characterization of the polyketide synthases and halogenases involved in the construction of 6‐hydroxymellein derivatives. Heterologous expression in Aspergillus oryzae demonstrated that 6‐hydroxymellein is a common biosynthetic intermediate and that chlorination occurs in the early stages of its products’ biosynthesis. This was further confirmed by in vitro enzymatic reactions conducted in the presence of recombinant proteins. Plausible means of biogenesis of fungal polyketides from 6‐hydroxymellein derivatives, additionally supported by the reported labeling patterns of terrein and structurally related fungal polyketides, are also discussed. This study sets the stage for elucidation of the biosynthetic machinery of fungal polyketides of this type.

Fungal polyketide synthase (PKS)–nonribosomal peptide synthetase (NRPS) hybrids are key enzymes for synthesizing structurally diverse hybrid natural products (NPs) with characteristic biological activities. Predicting their chemical space is of particular importance in the field of natural product chemistry. However, the unexplored programming rule of the PKS module has prevented prediction of its chemical structure based on amino acid sequences. Here, we conducted a phylogenetic analysis of 884 PKS–NRPS hybrids and a modification enzyme analysis of the corresponding biosynthetic gene cluster, revealing a hidden relationship between its genealogy and core structures. This unexpected result allowed us to predict 18 biosynthetic gene cluster (BGC) groups producing known carbon skeletons (number of BGCs; 489) and 11 uncharacterized BGC groups (171). The limited number of carbon skeletons suggests that fungi tend to select PK skeletons for survival during their evolution. The possible involvement of a horizontal gene transfer event leading to the diverse distribution of PKS–NRPS genes among fungal species is also proposed. This study provides insight into the chemical space of fungal PKs and the distribution of their biosynthetic gene clusters.

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