# Conserved early steps of stemmadenine biosynthesis[image]

## TL;DR

Researchers identified and tested enzymes involved in making a key compound in plant alkaloid production, achieving milligram yields in a model plant system.

## Contribution

The study demonstrates conserved enzymatic steps and identifies shunt products in stemmadenine biosynthesis using heterologous expression.

## Key findings

- Orthologs of key biosynthesis enzymes are catalytically indistinguishable except for geissoschizine synthase.
- Substrate promiscuity of Redox1 leads to shunt products that reduce pathway efficiency.
- Stemmadenine was produced at 6 mg yields in Nicotiana benthamiana using heterologous expression.

## Abstract

Stemmadenine acetate is a pivotal intermediate in the production of pharmacologically active monoterpene indole alkaloids. Here, we identify orthologs of the stemmadenine acetate pathway genes (SGD, GS, GO, Redox1, Redox2, and SAT). We characterize these enzymes in vitro, and in addition, we reconstitute stemmadenine acetate biosynthesis in Nicotiana benthamiana, comparing the formation of intermediates and shunt products that are produced when previously characterized orthologs from the plant Catharanthus roseus are used. Ortholog pairs are catalytically indistinguishable, except in the case of geissoschizine synthase. Surprisingly, the geissoschizine synthase ortholog catalyzes the formation of an alternative stereoisomer, 19Z-geissoschizine, seeding a low-flux Z-series in heterologous reconstitution systems in vitro and in planta. We, in addition, characterize the major shunt products that arose during reconstitution of stemmadenine acetate biosynthesis. We show that the substrate promiscuity of Redox1 results in the formation of the shunt products 16(R/S)-isositsirikines, hampering pathway flux and yields. In addition, we show that stemmadenine can be oxidized by endogenous N. benthamiana enzymes, leading to the shunt product condylocarpine. Nevertheless, we could produce stemmadenine at a 6 mg yield from 19E-geissoschizine by heterologous expression in N. benthamiana. Overall, we highlight the prospects for milligram production of important biosynthetic intermediates in N. benthamiana.