Fig. 4: Synthesis and modification of substituted tetrahydroisoquinolines from supplemented amino acids.

a NCS-catalyzed formation of substituted THIQs through catabolism of externally supplied amino acids via the Ehrlich pathway. b Ion-extracted LC–MS chromatograms of strain LP501 grown on l-methionine (25), l-DOPA (7), l-2-aminobutyrate (29), l-norvaline (33), l-norleucine (37), or l-2-aminoheptanoic acid (41) as the chief source of nitrogen (green). Aldehydes derived from amino acids were incorporated into the corresponding substituted THIQs and methylated by BIA tailoring enzymes (Ps6OMT and PsCNMT) produced by strain LP501. Substituted THIQs and their methylated derivatives shifted in retention time relative to the canonical BIA products from l-tyrosine, namely (S)-norcoclaurine (3) and (S)-N-methylcoclaurine (9), which were formed de novo on all amino acid substrates. Growth of strain LP501 on urea as the major nitrogen source (orange) failed to generate peaks corresponding to substituted THIQs with the exception of 31 and 40, which co-elute with singly methylated salsolinol (by Ps6OMT or PsCNMT) and lophocerine, respectively. Product 36 elutes closely with both singly methylated derivatives of 22 (by Ps6OMT or PsCNMT), which are synthesized de novo from l-leucine. Products 35 and 39 are isomers of N-methylisosalsoline and 22, respectively, but do not co-elute with these de novo products. Methylation of norlaudanosoline (24) by Ps6OMT and PsCNMT yields 3-hydroxy-N-methylcoclaurine (10), which is produced de novo by strain LP501 irrespective of the nitrogen source. All m/z values were calculated based on the expected structures of the respective compounds of interest (Supplementary Fig. 16 and Supplementary Table 1). DMN dopamine.