Development of in vitro lycopene biosynthesis from geranyl pyrophosphate employing cell‑free protein synthesis

Abstract

Lycopene is a compound classified as carotenoid, also known as tetraterpenoids, and its high antioxidative capabilities make demand in pharmaceutical and nutrient fields. For these reasons, much research on microbial production of lycopene has been developed and reported for more than two decades. Nevertheless, a standardized in vitro biosynthesis method for lycopene synthesis has not been reported to date. The major reasons of the absence of this method lie on the poor solubility of hydrophobic intermediates (geranylgeranyl pyrophosphate [GGPP] and phytoene), and the difficulty of employing membrane-binding enzyme, phytoene desaturase (CrtI) into in vitro reactions. In this study, we developed a standard method of in vitro biosynthesis of lycopene from geranyl pyrophosphate using four enzymes, namely farnesyl pyrophosphate synthase (IspA), GGPP synthase (CrtE), phytoene synthase (CrtB), phytoene desaturase (CrtI), and liposome-the key material, which can provide both hydrophobic area and a lipid membrane for the membrane-binding enzyme CrtI. Moreover, we performed a screening of the in vitro lycopene synthetic pathway using cell-free protein synthesis system, which verifies the applicability of our system as a tool for screening the lycopene synthesis pathway.