Streamlined synthesis of β-elemene using a spatially organized multi-enzyme machinery

Area of Science:

• Biocatalysis
• Synthetic Biology
• Metabolic Engineering

Background:

• β-Elemene is a pharmaceutically significant sesquiterpene.
• Current plant-based and chemical synthesis methods for β-elemene are inefficient.
• There is a need for improved in vitro production strategies.

Purpose of the Study:

• To develop an efficient in vitro synthesis of β-elemene from mevalonate.
• To engineer a hexa-enzyme cascade system for enhanced biosynthetic productivity.
• To utilize bio-orthogonal protein pairs for enzyme complex assembly.

Main Methods:

• Construction of orderly cross-linked hexa-enzyme complexes using bio-orthogonal protein pairs (Tag/Catcher, RIAD/RIDD2, K/Q-Tag).
• Spontaneous one-step co-purification and immobilization of six enzymes.
• Spatial co-localization confirmation of the organized enzyme assemblies.

Main Results:

• Achieved a β-elemene titer of 160.08 mg/L, a significant increase from 27.54 mg/L using free enzymes.
• Demonstrated favorable operational stability, retaining ~70% activity over six reuse cycles.
• Facilitated easy recovery of enzyme complexes via centrifugation.

Conclusions:

• Bio-orthogonal multi-enzyme assembly engineering enables efficient in vitro biosynthesis of β-elemene.
• This strategy offers a sustainable approach for engineered synthesis of terpenoid compounds.
• The mevalonate pathway is a viable route for engineered terpenoid synthesis.