In Vitro Multienzyme Pathway Assembly for Isoprenoids and Isoprenoid Precursors Production

Abstract

In vitro multienzymatic biosynthesis is a re-emerging, complementary method to cell-based biosynthesis. It is easily manipulated, bypasses cellular barrier, and ensures the stereoselectivity of the product. Therefore, it is our interest to explore the method to produce isoprenoids and its precursors, particularly, the key artemisinin precursor, amorpha-4,11-diene (AD). Various mathematical tools have been employed to guide the optimization process. Firstly, an optimal enzymatic composition was identified by Taguchi method, and a critical step, amorpha-4,11-diene synthase (Ads), was identified. Furthermore, insights from lin-log approximation led us to unravel the hitherto unrecognized inhibitions of Ads activity by ATP and pyrophosphate. Subsequently, the multienzymes were spatially co-immobilized and effectively recycled. The thesis further explored yeast whole-cell biotransformation to convert AD to dihydroartemisinic acid. Lastly, similar strategies were applied to the non-mevalonate pathway enzymes to produce an important intermediate, 2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC). The strategies applied are useful for other biosynthesis optimization in vitro.