Bioorthogonal chemistry-driven multi-enzyme strategy for efficient terpenoid biosynthesis.

Area of Science:

• Synthetic Biology
• Biotechnology
• Enzyme Engineering

Background:

• In vivo terpenoid biosynthesis faces limitations due to complex cellular environments and inefficient pathway flux.
• Developing efficient in vitro systems is crucial for producing high-value terpenoids sustainably.

Purpose of the Study:

• To develop an in vitro multi-enzyme cascade strategy for enhanced terpenoid synthesis.
• To create ordered cross-linked enzyme complexes (O-CLEs) using bioorthogonal chemistry for controlled spatial organization of enzymes.
• To demonstrate the general applicability and reusability of this strategy for producing various high-value terpenoids.

Main Methods:

• Assembled six enzymes from the mevalonate (MVA) pathway into O-CLEs via bioorthogonal protein pairing.
• Utilized multi-layer fluorescence labeling technology (MLFLT) to confirm enzyme arrangement within O-CLEs.
• Tested O-CLEs for β-farnesene production and demonstrated modularity by adapting for taxadiene biosynthesis.

Main Results:

• O-CLEs achieved a β-farnesene yield of 49.08 ± 1.91 mg/L, a 1.39-fold increase over free enzymes and a 3.03-fold increase over randomly cross-linked complexes.
• The O-CLEs retained 62 ± 9% of initial activity after six reuse cycles.
• The strategy was successfully extended to taxadiene biosynthesis by replacing β-farnesene synthase with taxadiene synthase.

Conclusions:

• The bioorthogonal chemistry-driven, ordered multi-enzyme immobilization strategy provides a robust and reusable platform for in vitro synthetic biology.
• This approach significantly enhances the production of high-value terpenoids compared to conventional methods.
• The modular design ensures general applicability for diverse terpenoid biosynthesis pathways.