Identification and Optimization of Kratom Strictosidine Pathway Enabled by Yeast Multiplex Engineering.

Area of Science:

• Plant biochemistry
• Metabolic engineering
• Natural product biosynthesis

Background:

• Monoterpene indole alkaloids (MIAs) are crucial plant natural products with pharmaceutical applications.
• The strictosidine biosynthetic pathway is fully known only in Catharanthus roseus.
• Kratom (Mitragyna speciosa) strictosidine biosynthesis was only partially understood.

Purpose of the Study:

• To elucidate and reconstruct the complete kratom strictosidine biosynthetic pathway.
• To utilize multiplex pathway engineering for rapid discovery and optimization.
• To enhance strictosidine production in a heterologous yeast system.

Main Methods:

• Multiplex pathway engineering in yeast for gene discovery and pathway assembly.
• Iterative integration and screening of functional kratom genes.
• Identification and characterization of a secologanin transporter (MsNPF2.6).
• Pathway optimization by incorporating enzymes from other plant species.

Main Results:

• Successfully reconstructed the complete kratom strictosidine pathway from geranyl pyrophosphate and tryptophan.
• Identified 13 functional kratom genes essential for the pathway.
• Discovered MsNPF2.6, a vacuolar transporter that increased strictosidine production by 62% in yeast.
• Achieved further production enhancement by incorporating nepetalactol-producing enzymes.

Conclusions:

• The strictosidine biosynthetic pathway in kratom has been fully established.
• Multiplex engineering is a powerful strategy for rapid plant pathway discovery and optimization.
• This study provides a foundation for engineering MIA production in heterologous hosts.