Total Biosynthesis of the Tubulin-Binding Alkaloid Colchicine

Abstract

Colchicine (1) is a bioactive plant alkaloid from Colchicum and Gloriosa species that is used as a pharmaceutical treatment for inflammatory diseases, including gouty arthritis and familial Mediterranean fever. The activity of this alkaloid is attributed to its ability to bind tubulin dimers and inhibit microtubule assembly, which not only promotes anti-inflammatory effects, but also makes colchicine a potent mitotic poison. The biochemical origins of colchicine biosynthesis have been investigated for over 50 years, but only recently has the underlying enzymatic machinery become clear. Here, we report the discovery of multiple pathway enzymes from Gloriosa superba that allows for the reconstitution of a complete metabolic route to 1. This includes three enzymes that process a previously established tropolone-containing intermediate into 1 via tailoring of the nitrogen atom. We further demonstrate the total biosynthesis of enantiopure (-)-1 from primary metabolites via heterologous production in a model plant, thus enabling future efforts for the metabolic engineering of this medicinal alkaloid. Additionally, our results provide insight into the timing and tissue specificity for the late stage modifications required in colchicine biosynthesis, which are likely connected to the biological functions for this class of medicinal alkaloids in native producing plants.

Figure 1.. Proposed biosynthetic pathway for colchicine.

a) Previously established biosynthetic pathway for the engineered production of 3. Native Gloriosa superba genes are shown in blue, while those from other plant sources are in red. b) Proposed remaining biosynthetic transformations to produce 1 from 3.

Figure 2.. Discovery of three enzymes that transform N-formyldemecolcine (3) into colchicine (1).

Transiently co-expressed combinations of G. superba genes in N. benthamiana with co-infiltration of 3 as substrate leads to stepwise transformation into 1. Shown here are the quantified ion abundances (m/z, [M+H]⁺) of the verified pathway intermediates/products observed upon the addition of each biosynthetic gene, as measured by LC-MS analysis. Shown above the quantified ion abundances is the corresponding biosynthetic proposal for the stepwise formation of 1 from 3.

Figure 3.. Complete engineered biosynthesis of colchicine in N. benthamiana.

Addition of the three identified enzymes to the previously engineered biosynthetic pathway to 3 allows for total biosynthesis of 1 in N. benthamiana, as shown here via LC-MS chromatograms. EIC = extracted ion chromatogram (EIC = m/z 400.1755 [M+H]⁺ and m/z 422.1574 [M+Na]⁺ for both compounds). Native Gloriosa superba genes are shown in blue, while those from other plant sources are brown.

Figure 4.. Expression variation of colchicine biosynthetic pathway genes.

Transcript abundances in four sequenced tissues are shown for each biosynthetic pathway gene that acts downstream of 1-phenethylisoquinoline scaffold (2) formation. Shown for each tissue is the trimmed mean of M (TMM)-normalized, log₂-transformed transcript counts per million (CPM) with ± standard deviation. n = 5 transcript libraries for rhizome, n = 2 for the three other tissue types. Shown above the bar graphs for each gene is the Pearson correlation coefficient (Pearson’s r) when comparing its transcript expression profile to that of GsOMT1.