Construct Phenylethanoid Glycosides Harnessing Biosynthetic Networks, Protein Engineering and One-Pot Multienzyme Cascades.

Phenylethanoid glycosides (PhGs) exhibit a multitude of structural variations linked to diverse pharmacological activities. Assembling various PhGs via multienzyme cascades represents a concise strategy over traditional synthetic methods. However, the challenge lies in identifying a comprehensive set of catalytic enzymes. This study explores biosynthetic PhG reconstruction from natural precursors, aiming to replicate and amplify their structural diversity. We discovered 12 catalytic enzymes, including four novel 6'-OH glycosyltransferases and three new polyphenol oxidases, revealing the intricate network in PhG biosynthesis. Subsequently, the crystal structure of CmGT3 (2.62 Å) was obtained, guiding the identification of conserved residue 144# as a critical determinant for sugar donor specificity. Engineering this residue in PhG glycosyltransferases (FsGT61, CmGT3, and FsGT6) altered their sugar donor recognition. Finally, a one-pot multienzyme cascade was established, where the combined action of glycosyltransferases and acyltransferases boosted conversion rates by up to 12.6-fold. This cascade facilitated the reconstruction of 26 PhGs with conversion rates ranging from 5-100 %, and 20 additional PhGs detectable by mass spectrometry. PhGs with extra glycosyl and hydroxyl modules demonstrated notable liver cell protection. This work not only provides catalytic tools for PhG biosynthesis, but also serves as a proof-of-concept for cell-free enzymatic construction of diverse natural products.

Insights into the missing apiosylation step in flavonoid apiosides biosynthesis of Leguminosae plants.

Apiose is a natural pentose containing an unusual branched-chain structure. Apiosides are bioactive natural products widely present in the plant kingdom. However, little is known on the key apiosylation reaction in the biosynthetic pathways of apiosides. In this work, we discover an apiosyltransferase GuApiGT from Glycyrrhiza uralensis. GuApiGT could efficiently catalyze 2″-O-apiosylation of flavonoid glycosides, and exhibits strict selectivity towards UDP-apiose. We further solve the crystal structure of GuApiGT, determine a key sugar-binding motif (RLGSDH) through structural analysis and theoretical calculations, and obtain mutants with altered sugar selectivity through protein engineering. Moreover, we discover 121 candidate apiosyltransferase genes from Leguminosae plants, and identify the functions of 4 enzymes. Finally, we introduce GuApiGT and its upstream genes into Nicotiana benthamiana, and complete de novo biosynthesis of a series of flavonoid apiosides. This work reports an efficient phenolic apiosyltransferase, and reveals mechanisms for its sugar donor selectivity.