Yeast Surface Display for In Vitro Biosynthetic Pathway Reconstruction

Area of Science:

• Biotechnology
• Synthetic Biology
• Enzyme Engineering

Background:

• Enzyme immobilization is crucial for in vitro metabolic pathway reconstruction.
• Conventional enzyme isolation methods can be costly and time-consuming.
• Yeast surface display (YSD) offers a promising alternative for enzyme immobilization.

Purpose of the Study:

• To demonstrate the feasibility of using YSD-immobilized enzymes for in vitro metabolic pathway reconstruction.
• To produce geraniol via a one-pot cascade reaction using YSD-immobilized enzymes.
• To optimize conditions for efficient geraniol biosynthesis.

Main Methods:

• Enzymes for the mevalonate pathway were displayed on the yeast surface.
• A one-pot cascade reaction system was established using YSD-immobilized enzymes.
• Optimization of catalytic components, cofactor regeneration, and byproduct removal was performed.

Main Results:

• A complex multi-enzymatic system for geraniol production was successfully reconstituted in vitro.
• The YSD-immobilized cascade enzymes achieved a final geraniol yield of 7.55 mg L-1 after seven cycles.
• The study demonstrated an economical approach for enzyme enrichment and application.

Conclusions:

• Yeast surface display is an effective strategy for immobilizing enzymes for in vitro applications.
• YSD-immobilized enzymes enable the efficient reconstruction of complex biosynthetic pathways.
• This approach provides a cost-effective alternative for enzyme characterization and chemical biosynthesis.