Conserved early steps of stemmadenine biosynthesis

Area of Science:

• Plant biotechnology
• Metabolic engineering
• Natural product biosynthesis

Background:

• Stemmadenine acetate is a crucial intermediate for synthesizing pharmacologically active monoterpene indole alkaloids.
• Understanding its biosynthesis is key for efficient production of valuable compounds.

Purpose of the Study:

• To identify and characterize stemmadenine acetate pathway genes.
• To reconstitute stemmadenine acetate biosynthesis in Nicotiana benthamiana.
• To compare plant-derived pathway enzymes and identify factors limiting production.

Main Methods:

• Identification and in vitro characterization of stemmadenine acetate pathway gene orthologs (SGD, GS, GO, Redox1, Redox2, SAT).
• Heterologous reconstitution of the pathway in Nicotiana benthamiana.
• Analysis of intermediates and shunt products using plant orthologs from Catharanthus roseus.

Main Results:

• Most ortholog pairs were catalytically similar, except for GS, which produced an alternative stereoisomer (19Z-geissoschizine).
• Substrate promiscuity of Redox1 led to shunt products (16(R/S)-isositsirikines), reducing pathway flux.
• Endogenous N. benthamiana enzymes oxidized stemmadenine to condylocarpine.
• Milligram-scale production (6 mg) of stemmadenine was achieved from 19E-geissoschizine via heterologous expression.

Conclusions:

• Enzyme characterization and pathway reconstitution in N. benthamiana reveal insights into stemmadenine acetate biosynthesis.
• Identified bottlenecks include GS stereoisomer formation and Redox1 promiscuity.
• Demonstrated potential for milligram-scale production of stemmadenine acetate in N. benthamiana.