Characterization of a NRPS-like Protein from Pestalotiopsis fici for Aldehyde Generation

Abstract

Nonribosomal peptide synthetase (NRPS)-like enzymes containing A-T-R domain architecture are also known as carboxylate reductases (CARs) for aldehyde generation. To identify new members of CARs, we established a virtual library containing 84 fungal CARs distributed in seven distinct clades by genome mining and phylogenetic analysis. Nine CARs, including PnlA from Pestalotiopsis fici and eight known CARs, were clustered in clade VI and proposed to catalyze the reduction of nonreducing polyketide synthase (NR-PKS)-derived aryl carboxylic acids. The recombinant protein PnlA was overproduced and purified to apparent homogeneity from Saccharomyces cerevisiae. In vitro enzyme assays of PnlA with 28 different benzoic acid derivatives (1–28) revealed the corresponding aldehyde formation in 14 cases (1–14). Comparison of conversion yields indicated the high preference of PnlA toward 3,5-dimethylorsellinic acid (DMOA, 4) and vanillic acid (10). A specificity-conferring code Q355 in PnlA was postulated by sequence alignment with the known CARs in clade VI. Our study provides an updated virtual library of fungal CAR enzymes and expands the biocatalytic selectivity of CARs.

Identification and partial reconstitution of the biosynthetic pathway of bioactive meroterpenoids fromHericium erinaceus(Lion’s Mane mushroom)

“…S3a). The multiple sequence alignment analysis between the amino acid sequences of known fungal aromatic CARs-retrieved from the UniProt database and described previously [33]-and the sequences of g019530 and g019600 to identify conserved motifs, was performed with MEGA 11 [53] using the MUSCLE algorithm [54] with default settings, and visualized in Jalview [55].…”

“…Orsellinic acid-the core structure shared by hericenones, hericerin, and erinacerins-is one of the simplest aromatic polyketides, and it is biosynthesized by type III PKS in plants and by type I PKS in bacteria and fungi [32]. In several meroterpenoids of H. erinaceus, the carboxylic acid moiety is reduced to an aldehyde, indicating that an aromatic carboxylic acid reductase (CAR) might also be involved in the biosynthesis [33]. Meroterpenoid-producing BGCs that encode these enzymes have been recently identified in the ascomycetes Stachybotrys bisbyi [34] and Acremonium egyptiacum [35].…”