Extended Data Fig. 2: Comparison analysis of the biosynthetic gene clusters of PIAs and in vivo characterization of NmrA-like proteins CtdO, CtdR, and CtdP.

a, Annotation of the biosynthetic gene clusters of PIAs. 21R-citrinadin A (ctd in P. citrinum ATCC 9849), citrinadin A (cnd in P. citrinum DSM1997)²⁴, paraherquamides (phq in P. fellutanum ATCC20841)⁶⁵, (−)-notoamide A (not in A. protuberus MF297-2)⁶⁶, (+)-notoamide A (not’ in A. versicolor NRRL35600)⁶⁶ and malbrancheamide (mal in Malbranchea aurantiaca RRC1813)⁸. NRPS: non-ribosomal peptide synthetase; FPMO: flavoprotein monooxygenase; P450: cytochrome P450; SDR: short-chain dehydrogenase/reductase. SDRs MalC and PhqE, and NmrA-like oxidoreductase CtdP are characterized as Diels–Alderases. b, LC–MS analysis (extracted ion chromatogram, EIC) of P. citrinum wild-type (WT) and mutant ΔctdN. Four ΔctdN mutants all retained the production of compound 1. c, LC–MS analysis (EIC) of P. citrinum WT and mutants ΔctdO, ΔctdR, ΔctdN, and ΔctdP. The ΔctdR mutant major produces R1, which is identified as an α-anti product by NMR (Supplementary Table 10) and ECD (Supplementary Fig. 6b) analysis, indicating that CtdR might work on the biosynthetic steps after Diels–Alder cycloaddition. The ΔctdO mutant showed reduced production of citrinadin compounds, which possibly serves as a regulator. Both ΔctdR and ΔctdO produce trace amounts of 5 and 6, indicating the deletions of these genes have some influence and result in accumulations of the shunt products. Only the ΔctdP mutant could abolish the production of 1 and 2, as well as have a remarkable accumulation of 5 and 6.