Extended Data Figure 12. Dropout analysis of module 3 biosynthetic genes.

Metabolic engineering of the full pathway to N-formyldemecolcine (10) in N. benthamiana was compared to transient co-expression systems in which individual module 3 enzymes were removed. a) Accumulation of proposed pathway intermediates within dropout experiments. Gray boxes to the left of the graph indicate biosynthetic genes/modules included within a co-expression experiment, while white boxes indicate their absence. Shown for each intermediate is the mean extracted ion abundance (n=3, ± standard deviation) for the exact ion mass [M+H]⁺ (for 10, both [M+H]⁺ and [M+Na]⁺), as well as the retention time (r.t.) that corresponds to each compound. b) Dropout of GsOMT1 from the full engineered pathway leads to accumulation of a new compound with a mass equivalent to 2 (m/z 300.1594), as shown via LC-MS chromatograms. The newly identified peak is indicated via the arrow. EIC = extracted ions chromatogram. c) Transient co-expression of GsOMT1 or GsNMTt with module 1, module 2, and Δ24-CjNCS (for production of 1). Shown are the LC-MS chromatograms for the substrate (1, m/z 286.1438), singly-methylated products (2a and 2b, m/z 300.1574) and doubly-methylated product (3, m/z 314.1751). d) MS/MS fragmentation spectrum of 2a/2 (collision energy of 20V), as well as a tabulated list and putative structures for the ion fragments. e) MS/MS fragmentation spectrum of 2b (collision energy of 20V), as well as a tabulated list and putative structures for the ion fragments. Note that fragment B (m/z 269) supports the placement of the methyl group on the nitrogen. For reference, compare to fragment B of 2a in panel “d”. f) Comparative consumption of 1 by GsOMT1 and GsNMTt. Gray boxes indicate the presence of a gene/module within the co-expression experiment, while a white box indicates its absence. n=3 for each reaction; statistical comparisons made using Dunnett’s test with comparison to the module 1/module 2/Δ24-CjNCS control. g) Proposed scheme for the initial methylations of 1. All experiments shown in this figure were performed once.