Discovery and engineering of colchicine alkaloid biosynthesis

Abstract

SUMMARY PARAGRAPH: Few complete pathways are established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture. 1 A lead example is colchicine, an FDA-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species. 2 - 5 Here we use a combination of transcriptomics, metabolic logic, and pathway reconstitution to elucidate a near complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome, or genetic tools in the native host. We have uncovered eight genes from Gloriosa superba for the biosynthesis of N -formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine. 6 Notably, in doing so we have identified a non-canonical cytochrome P450 that catalyzes the remarkable ring expansion reaction required to produce the distinct carbon scaffold of colchicine. We further utilize the newly identified genes to engineer a biosynthetic pathway (16 enzymes total) to N -formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This work establishes a metabolic route to tropolone-containing colchicine alkaloids and provides new insights into the unique chemistry plants use to generate complex, bioactive metabolites from simple amino acids.

Engineered Microbes for Producing Anticholinergics

“…As recently observed in other models, the adopted strategy to split the whole TA biosynthetic pathway into distinct modules truly helps in identifying and disrupting yeast endogenous processes negatively influencing the different biosynthetic branches. [12,13] In sum, this outstanding reconstruction of a heterologous metabolism, involving 26 gene integrations and eight gene disruptions constitutes one of the most elaborated whole-cell system producing NPs to date.…”

Metabolic Engineering for Glycyrrhetinic Acid Production in Saccharomyces cerevisiae

“…With the development of bioinformatics and computational biology, it has been possible to identify all potential biosynthetic pathways of the natural products in one species by establishing a multi-layered omics technology platform (Mochida and Shinozaki, 2011). The platform includes omics data analyses of transcripts, genomes, proteomes, metabolomes, the prediction of gene function, and the verification of key genes in the biosynthetic pathways of natural products (Nett et al, 2020).…”

“…First, plants harboring GA or other plant bioactive components are identified. Secondly, transcriptomics, genomics, proteomics and metabolomics analyses are used to recover pathways and key enzymes related to the synthesis of GA or other natural products (Chen et al, 2020;Gao et al, 2020;Nett et al, 2020;Srinivasan and Smolke, 2020). After that, appropriate chassis cells which provide precursors for natural product synthesis are selected for construction and optimization of associated microbial cell factories.…”

Nature's Chemists: The Discovery and Engineering of Phytochemical Biosynthesis

“…For example, eight genes from Gloriosa superba involved in the biosynthesis of the alkaloid colchicine were discovered using N. benthamiana as a screening tool. Subsequently, the newly discovered genes plus eight previously described genes were used to engineer N. benthamiana to synthesise N-formyldemecolcine, a colchicine precursor, from phenylalanine and tyrosine (Nett et al, 2020). The use of N. benthamiana for the heterologous production of whole pathways is discussed further in section Plants as Chasses for Production of High-Value Phytochemicals.…”