Fig. 8: Optimization of norcocluarine, reticuline, and N-methylcoclaurine production for analysis of flux through hybrid pathways.
From: Machine learning discovery of missing links that mediate alternative branches to plant alkaloids
a PpDDC-Y79F-F80Y-H181N (PpDDC-T) and PsPDC1 containing strain P1-07-AI (olive green) prefers the norlaudanosoline containing pathway. Combination of PpDDC-T, ARO10 and PsTyDC1 in strain A1-06-AI (dark grey) promotes both norcoclaurine and norlaudanosoline containing pathways. ARO10 expressing strain A1-01-DE3 (light purple) converts tyrosine and dopamine to norcoclaurine and N-methylcoclaurine. N-Methylcoclaurine and reticuline were extracted with ethyl acetate from cultures 40 h after addition of tyrosine together with L-DOPA or dopamine. Tested strains P1-06-DE3 (blue), P1-07-AI (olive green), A1-06-AI (dark grey) and A1-01-DE3 (light purple) each contain Cj6OMT, CjCNMT, Cj4OMT, NCS, plus the indicated genes of the bottom 4 rows. P1-06-DE3 and P1-07-AI contain the same genes, but P1-06-DE3 was induced with only IPTG, without including arabinose for PsPDC1 expression. Cultures containing PpDDC-T and L-DOPA were supplemented with additional sodium ascorbate. The BL21(AI) derived strain P1-07-AI was induced with IPTG and arabinose. For improved N-methylcoclaurine production, A1-01-DE3 was supplemented with the aldehyde reductase/dehydrogenase inhibitor gossypol. Additional culture conditions are described in the methods section. Extracted N-methylcoclaurine and reticuline were TMS-derivatized and analyzed with GC-MS (t = 40 h, n = 3). After extraction, cultures were stored at 4 °C and stable norcoclaurine titers from culture medium were analyzed with LC-MS (n = 2). b Isotope profiling of strains P1-02-AI (expressing PsPDC1) and P1-04-AI (expressing PsPDC1 and PsTyDC1), which produce N-methylcoclaurine-d6 (P1-02-AI - 62 nM, P1-04-AI 160 nM) and reticuline-d5 from tyrosine-d4 and L-DOPA-d3 (t = 61 h, n = 3). There is a synergistic improvement in BIA production when combining PsPDC1 and PsTyDC1. Here, NCS catalyzes the loss of a deuterium from in vivo generated dopamine-d3. c For tracing aromatic isotope flux from tyrosine to norcoclaurine and N-methylcoclaurine, alkaloids were extracted with ethyl acetate from the A1-01-DE3 culture 40 h after addition of tyrosine-d3 and dopamine, according to the methods section. Extracted alkaloids were TMS-derivatized and analyzed with GC-MS (n = 3). After extraction, cultures were stored at 4 °C and stable BIA titers from culture medium were analyzed with CE-MS (n = 3); the fraction of labeled BIA-d4 and unlabeled BIA from natural tyrosine in the rich TB broth can be quantified. With exception to unlabeled norcoclaurine (n = 2), all other individual samples were analyzed 3 times (n = 3) to generate bar graphs in Prism 7, with error bars representing mean values +/− standard deviations. Source data are provided as a Source Data file.
