Yeast Surface Display forIn VitroBiosynthetic Pathway Reconstruction

The enzymes immobilized through yeast surface display (YSD) can be used inin vitrometabolic pathway reconstruction as alternatives to the enzymes isolated or purified through conventional biochemistry methods. They can be easily prepared by growing and collecting yeast cells harboring display constructs. This may provide an economical method for enriching certain enzymes for biochemistry characterization and application. Herein, we took the advantage of one-pot cascade reactions catalyzed by YSD-immobilized enzymes in the mevalonate pathway to produce geraniolin vitro. YSD-immobilized enzymes of 10 cascade reactions for geraniol production, together with optimization of catalytic components, cofactor regeneration, and byproduct removal, achieved a final yield of 7.55 mg L^-1after seven cycles. This study demonstrated that it is feasible to reconstitute a complex multi-enzymatic system for the chemical biosynthesisin vitroby exploiting YSD-immobilized cascade enzymes.