Biosynthesis, elicitation and roles of monocot terpenoid phytoalexins.

A long-standing goal in plant research is to optimize the protective function of biochemical agents that impede pest and pathogen attack. Nearly 40 years ago, pathogen-inducible diterpenoid production was described in rice, and these compounds were shown to function as antimicrobial phytoalexins. Using rice and maize as examples, we discuss recent advances in the discovery, biosynthesis, elicitation and functional characterization of monocot terpenoid phytoalexins. The recent expansion of known terpenoid phytoalexins now includes not only the labdane-related diterpenoid superfamily but also casbane-type diterpenoids and ß-macrocarpene-derived sequiterpenoids. Biochemical approaches have been used to pair pathway precursors and end products with cognate biosynthetic genes. The number of predicted terpenoid phytoalexins is expanding through advances in cereal genome annotation and terpene synthase characterization that likewise enable discoveries outside the Poaceae. At the cellular level, conclusive evidence now exists for multiple plant receptors of fungal-derived chitin elicitors, phosphorylation of membrane-associated signaling complexes, activation of mitogen-activated protein kinase, involvement of phytohormone signals, and the existence of transcription factors that mediate the expression of phytoalexin biosynthetic genes and subsequent accumulation of pathway end products. Elicited production of terpenoid phytoalexins exhibit additional biological functions, including root exudate-mediated allelopathy and insect antifeedant activity. Such findings have encouraged consideration of additional interactions that blur traditionally discrete phytoalexin classifications. The establishment of mutant collections and increasing ease of genetic transformation assists critical examination of further biological roles. Future research directions include examination of terpenoid phytoalexin precursors and end products as potential signals mediating plant physiological processes.

Comprehensive Engineering Strategies for Heterologous Production of Zealexin A1 in Saccharomyces cerevisiae.

Zealexin A1 is a nonvolatile sesquiterpene phytoalexin, which not only exhibits extensive antifungal and insecticidal activities but also has the ability to enhance the drought resistance of plants, and thus has potential applications in agricultural and food fields. In this study, the biosynthetic pathway of zealexin A1 was constructed in Saccharomyces cerevisiae for the first time, and the highest production of zealexin A1 reported to date was achieved. First, through screening of sesquiterpene synthases from various plants, BdMAS11 had a stronger (S)-ß-macrocarpene synthesis ability was obtained, and the heterologous synthesis of zealexin A1 was achieved by coexpressing BdMAS11 with cytochrome P450 oxygenase ZmCYP71Z18. Subsequently, after the site-directed mutagenesis of BdMAS11, fusion expression of farnesyl diphosphate synthase ERG20 and BdMAS11, and tailored truncation of BdMAS11 and ZmCYP71Z18, the strain coexpressing the manipulated BdMAS11 and original ZmCYP71Z18 produced 119.31 mg/L of zealexin A1 in shake-flask fermentation. Finally, the production of zealexin A1 reached 1.17 g/L through fed-batch fermentation in a 5 L bioreactor, which was 261.7-fold that of the original strain. This study lays the foundation for the industrial production of zealexin A1 and other terpenoids.

Characterization of CYP71Z18 indicates a role in maize zealexin biosynthesis.

Maize (Zea mays) produces zealexins as phytoalexins, with the inducible production of these antibiotics providing biochemical protection against fungal infection. However, the biosynthesis of these sesquiterpenoids has remained unclear. In particular, it is unclear how the olefinic precursor, (S)-ß-macrocarpene produced by the characterized maize sesquiterpene synthases TPS6/11, is further elaborated to form the bioactive zealexins. The first step is likely to be conversion of carbon-15 (C15) from a methyl group to a carboxylic acid by a cytochrome P450 mono-oxygenase (CYP). In this study, CYP71Z18, whose transcription is strongly induced by fungal infection, was found to catalyze oxidation of C15 in (S)-ß-macrocarpene, forming zealexin A1. The inducible transcription of CYP71Z18 matches that observed for TPS6/11 and the accumulation of zealexins, which is consistent with a role for CYP71Z18 in sesquiterpenoid phytoalexin production. This completes identification of zealexin A1 biosynthesis, and represents the initial CYP identified for the production of maize terpenoid phytoalexins.