Extreme promiscuity of a bacterial and a plant diterpene synthase enables combinatorial biosynthesis

• Published in: Metabolic engineering 2016-09, Vol.37, p.24-34
• Main Authors: Jia, Meirong, Potter, Kevin C., Peters, Reuben J.
• Format: Article
• Language: English
• Subjects: Alkyl and Aryl Transferases - chemistry; Alkyl and Aryl Transferases - genetics; Alkyl and Aryl Transferases - metabolism; Bacteria; biosynthesis; Biosynthetic Pathways - genetics; Diterpene synthases; Diterpenes; Diterpenes - chemistry; Diterpenes - metabolism; diterpenoids; Escherichia coli - enzymology; Escherichia coli - genetics; Metabolic engineering; Metabolic Engineering - methods; Metabolic Flux Analysis; Metabolic Networks and Pathways - genetics; olefin; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombination, Genetic - genetics; Substrate specificity
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2016.04.001

Diterpenes are widely distributed across many biological kingdoms, where they serve a diverse range of physiological functions, and some have significant industrial utility. Their biosynthesis involves class I diterpene synthases (DTSs), whose activity can be preceded by that of class II diterpene cyclases (DTCs). Here, a modular metabolic engineering system was used to examine the promiscuity of DTSs. Strikingly, both a bacterial and plant DTS were found to exhibit extreme promiscuity, reacting with all available precursors with orthogonal activity, producing an olefin or hydroxyl group, respectively. Such DTS promiscuity enables combinatorial biosynthesis, with remarkably high yields for these unoptimized non-native enzymatic combinations (up to 15mg/L). Indeed, it was possible to readily characterize the 13 unknown products. Notably, 16 of the observed diterpenes were previously inaccessible, and these results provide biosynthetic routes that are further expected to enable assembly of more extended pathways to produce additionally elaborated ‘non-natural’ diterpenoids. •Use of a wide variety of bicyclic class II diterpene cyclase products.•Considerable yields enabling structural elucidation of unknown diterpenes.•Novel biosynthetic access provided to 16 diterpenes.