Fig. 7: Replicating norlaudanosoline pathways using homologous enzyme templates.
From: Machine learning discovery of missing links that mediate alternative branches to plant alkaloids
a PsTyDC1 (green) is exchanged with an engineered PpDDC template (blue) via three active site gain-of-function substitutions, Y79F, F80Y, and H181N, to promote DHPAAS activity. In accordance with the resulting increase in AAS activity, these three substitutions result in increased SVM probability scores for AAS prediction, and reduced SVM probability scores for AAAD prediction. Norlaudanosoline (NL) production from L-DOPA was compared using PsTyDC1 in strain T1-10-DE3 (t = 44 h, n = 3) and PpDDC-Y79F-F80Y-H181N (PpDDC-T) in strain DT-02-DE3 (t = 40.5 h, n = 2). Culture conditions for each strain are described in the methods section. b PsPDC1 (green) is exchanged with S. cerevisiae ARO10 (purple) for higher PPDC activity in E. coli. Production of norlaudanosoline (NL) from L-DOPA by PsPDC1 in strain P1-02-AI is shown (t = 44 h, n = 2). Production of norlaudanosoline (NL) from L-DOPA and dopamine by ARO10 in strain A1-01-DE3 is compared (t = 44 h, n = 3). Culture conditions are described in the methods section. For panels a and b, Samples from individual cultures were analyzed two or three times (n = 2 or n = 3) to generate bar graphs in Prism 7, with error bars representing mean values +/− standard deviations. c Strain A1-01-DE3 containing ARO10 (purple) metabolizes tryptophan in TB medium to produce an indole 3-acetaldehyde derived indole alkaloid byproduct (t = 61 h), as indicated by the extracted ion chromatogram (EIC). Strain P1-02-AI containing PsPDC1 (green) did not readily convert indole 3-pyruvate to indole 3-acetaldehyde, as indicated by no detectable indole alkaloid byproduct (t = 61 h). Source data are provided as a Source Data file.
