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

Area of Science:

• Biotechnology
• Synthetic Biology
• Biochemistry

Background:

• β-Elemene, a pharmaceutically valuable sesquiterpene, faces production challenges due to inefficient plant extraction and chemical synthesis.
• Current methods for β-elemene production lack the efficiency required for large-scale pharmaceutical applications.

Purpose of the Study:

• To develop an efficient in vitro synthesis method for β-elemene using a hexa-enzyme cascade system.
• To improve the biosynthetic productivity of β-elemene from low-cost mevalonate.
• To engineer stable and reusable enzyme complexes for sustainable terpenoid production.

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 for β-elemene synthesis.
• Characterization of spatial co-localization within the engineered enzyme assemblies.

Main Results:

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

Conclusions:

• Bio-orthogonal multi-enzyme assembly engineering enables efficient in vitro biosynthesis of β-elemene.
• The developed strategy leverages the mevalonate pathway for sustainable engineered synthesis of terpenoid compounds.
• This approach offers a promising route for the efficient and scalable production of valuable terpenoids.