Extended Data Fig. 3: Characterization of GsCYP75A109.
From: Discovery and engineering of colchicine alkaloid biosynthesis
a, Addition of GsCYP75A109 to the N. benthamiana transient expression system with co-infiltrated 1 leads to the consumption of 3 (m/z 314.1751) and the production of a new compound that corresponds to a hydroxylation (m/z 330.1700), as shown by the LC–MS chromatograms. These results were confirmed in two independent experiments. b, MS/MS fragmentation spectrum of the generated m/z 330.1700 product (*) at a collision energy of 20 V. Consistent results were obtained in three separate experiments. c, Tabulated list and putative structures for ion fragments generated from MS/MS analysis of the m/z 330.1700 product. See Supplementary Information for a detailed analysis of MS/MS results. d, Untargeted metabolite analysis (XCMS) comparing the presence and absence of GsCYP75A109 in the transient co-expression system (n = 6 independent replicates for each experimental condition). The unique mass signatures (P < 0.1 between samples, as determined by XCMS) are shown in ranked order based on their increasing (top) or decreasing (bottom) fold change in abundance between the two conditions. The presumed product (m/z 330.1700) is shown in red; the mass isotopologues (M0, M1) of the presumed substrate (m/z 314.1751) are shown in blue. e, Proposed reaction catalysed by GsCYP75A109 as supported by MS/MS fragmentation and previously published labelling studies. f, An N-terminal truncation of a predicted mitochondrial localization signal from GsNMT (yielding GsNMTt) increases the yield of putative 4 (m/z 330.1700) in the transient co-expression system, as shown by the representative LC–MS chromatograms. g, Quantification of the heterologous production of 3 (m/z 314) or 4 (m/z 330) with the use of GsNMT or GsNMTt in the co-expression system. Filled-in boxes (grey) indicate the presence of a gene in the co-expression experiment; an empty box (white) indicates its absence. For each reaction, data are mean ± s.d. of 3 distinct biological replicates. Statistical comparisons were made using a one-tailed Student’s t-test, with an assumption of unequal variance. n.d., not detected. Direct comparison between the experimental conditions was performed twice with similar results obtained each time. Activity of GsNMTt in pathway engineering was consistent in more than three experiments.
