Extended Data Fig. 4: LC/MS analysis of extracts from the heterologous expression of cp1 and cp2 in A. nidulans.

LC/MS analyses include cp1ABCD (i), cp1BCD (ii), cp1ACD (iii), cp1ABC (iv), cp1ABD (v), and cp2ABCD (vi). Selected ion chromatograms correspond to the [M + H]⁺ for 1 ([M + H]⁺ = 358), 4 ([M + H]⁺ = 316), 5 ([M + H]⁺ = 330), 6 ([M + H]⁺ = 358), 7 ([M + H]⁺ = 372), 8 ([M + H]⁺ = 364), 9 ([M + H]⁺ = 380), 10 ([M + H]⁺ = 406), 11 ([M + H]⁺ = 422), 12 ([M + H]⁺ = 318), and 13 ([M + H]⁺ = 300). Y-axis represents ion counts and the chromatograms are presented on the same scale. Heterologous expression of three gene cassette cp1ABD is sufficient for the biosynthesis of 1 and the analogs in A. nidulans. As polyamines are abundant primary metabolites in fungi, the PLP-dependent decarboxylase Cp1C is not essential for the biosynthesis of 1 and the analogs in heterologous host A. nidulans. Interestingly, the heterologous expression of cp1BCD led to the formation of malic acid (12) and fumaric acid (13) derivatives, suggesting the role of Cp1A as an epoxidase. This result further supported the promiscuous substrate specificities of both Cp1B and Cp1D. The structures of all compounds except 11 were determined by NMR.