Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2012 Investigating labdane-related diterpenoids biosynthetic network in rice: characterization of cytochromes P450 and short-chain alcohol dehydrogenases/reductase Yisheng Wu Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons Recommended Citation Wu, Yisheng, "Investigating labdane-related diterpenoids biosynthetic network in rice: characterization of cytochromes P450 and short-chain alcohol dehydrogenases/reductase" (2012). Graduate Theses and Dissertations. 12680. https://lib.dr.iastate.edu/etd/12680 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Investigating labdane-related diterpenoids biosynthetic network in rice: Characterization of cytochromes P450 and short-chain alcohol dehydrogenases/reductase by Yisheng Wu A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Biochemistry Program of Study Committee: Reuben Peters, Major Professor Gustavo MacIntosh Thomas Bobik Bing Yang Mark Hargrove Iowa State University Ames, Iowa 2012 Copyright © Yisheng Wu , 2012. All rights reserved. ii TABLE OF CONTENT Chapter I: General Introduction 1 Overview and significance 1 Terpenoids 2 Rice phytoalexins biosynthesis network 4 Cytochromes P450 and metabolic engineering 7 Short-chain alcohol dehydrogenase/reductase 9 Reference 11 Figures 13 Chapter II: Functional characterization of wheat copalyl diphosphate synthases elucidates the early evolution of labdane-related diterpenoid metabolism in the cereals 17 Abstract 17 Introduction 18 Results 20 Discussion 24 Conclusion 25 Experimental 26 Acknowledgements 27 References 28 Figures and Tables 30 Chapter III: Parsing a multifunctional biosynthetic gene cluster from rice: iii Biochemical characterization of CYP71Z6 & 7 36 Abstract 36 Introduction 36 Results 42 Discussion 45 Acknowledgements 47 References 47 Figures 49 Supporting Information 53 Chapter IV: CYP76M6 and CYP76M8, two cytochromes P450 involved in oryzalexin D and E biosynthesis in rice 60 Abstract 60 Introduction 60 Results 62 Discussion 64 Material and methods 66 Acknowledgements 70 References 70 Figures 72 Supplementary data 77 Chapter V: Elucidating final oxygenation steps to produce oryzaelxin A, B and C: biochemical characterization of OsMAS, OsMASL1 and OsMASL2 78 iv Abstract 78 Introduction 79 Results 80 Discussion 83 Material and methods 86 Acknowledgements 88 References 88 Figures 91 Supplementary data 100 Chapter VI: Mutagenesis study in substrate recognition site changes substrate specificity 101 Abstract 101 Introduction 101 Results 103 Discussion 105 Material and methods 107 Acknowledgements 107 References 108 Figures 109 Chapter VII: Conclusion and future direction 119 Characterization of cytochromes P450 involved in rice labdane-related diterpenoids (LRD) production 119 v Characterization of short-chain dehydrogenase/reductase (SDR) involved in rice labdane-related diterpenoids (LRD) production 120 Mutagenesis analysis of functional characterized CYPs 121 Future directions 121 References 124 Figures 125 Acknowledgements 129 1 Chapter I: General Introduction Overview and significance Terpenoids are a large and diverse class of naturally occurring compounds with a range of biological functions in plants such as hormones (i.e. gibberellic acid) and phytoalexins against microbial infection. Terpenoids are also used as pharmaceuticals such as anti-malarial (i.e. artimisinin) and anti-cancer drug (i.e. taxol). Because of the biological and pharmaceutical importance, it is desirable to obtain terpenoids in large quantity. However, it is futile to isolate terpenoids directly from plant tissues due to their low abundance. Moreover, chemical synthesis is difficult because of their complex structure. For these reasons, an attractive alternative strategy is to engineer enzymes and pathways to produce functional terpenoids in a heterologous microbial system. Transferring pathways into microbial host requires good understanding of terpenoids biosynthetic pathways, most of which are only partially defined. Rice (Oryza sativa), one of the most important staple crops, can produce phytoalexins (Figure 1), which largely consist of labdane-related diterpenoids, when attacked by fungi and bacteria. Rice diterpene olefin biosynthetic pathways and the initial oxidative steps conducted by cytrochrome P450s (CYP) have been elucidated by our previous studies. In this dissertation we propose to use rice as a model system to further investigate diterpenoid biosynthesis pathways, with an emphasis on identifying downstream decorating oxygenases and oxidoreductase. This will further elucidate the complex diterpenoid network of rice, and elaborate our ability to engineer terpenoid biosynthesis pathways in microbial systems. 2 With defined enzymes and biosynthetic pathways in rice LRD phytoalexin biosynthesis, we are also interested in understanding how substrate specificity and product diversity are achieved by phylogenetically directed mutagenesis study. The mutants obtained around CYP’s substrate recognition sites (SRS) provide us information concerning how amino acids in SRS can affect substrate specificity and product outcome. These findings will eventually help us to rationally engineer the SRS to efficiently desired compounds. Terpenoids Terpenoids are one of the largest families of natural products with more than 50,000 have been discovered in nature with the majority found in plants. Consistent with their tremendous structural diversity, the functions of terpenoids are also varied. In plants, terpenoids function as primary structural and physiological molecules including membrane components (i.e. phytosterols), photosynthetic pigment (i.e. carotenoids), hormones (i.e. gibberellins), and electron carriers (i.e. ubiquinone)(1). In addition, many plants produce unique terpenoids repertoire that function as communicating and defense compounds, such as attractant for pollinators, herbivore repellents and antibiotics (1). Moreover, many terpenoids have pharmaceutical value for human such as anti-cancer drug taxol and anti-malaria drug artemisinin (1). Biosynthesis of all the terpenoids starts from five carbon isoprene unit: isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). IPP can be biosynthesized via mevalonate-dependent isoprenoid (MEV) pathway or mevalonate- independent methylerythritol phosphate (MEP) pathways (2, 3). In MEV pathway, three acetyl-coenzyme A (acetyl-coA) molecules condense successively to produce HMG-CoA, 3 which is then reduced to mevalonate, following by phosphorylation and decarboxylation reaction to produce IPP(3). MEV pathway operates in eukaryotes, archaebacteria, and cytosols of higher plants, whereas MEP pathway is used by many eubacteria, green algae, and chloroplast of higher plants, and is discovered more recently. MEP pathway starts from the condensation reaction between pyruvate and glyceraldehyde-3-phosphate to produce 1- deoxy-D-xylulose 5-phosphate (DXP), which is then rearranged to produce 2-C- methylerythrose-4-phosphate (MEP), followed by a series of complex reactions to produce IPP(3). IPP produced by both pathways can be converted to DMAPP by IPP isomerase(3). IPP and DMAPP are condensed to linear terpenoids precursor by prenyltransferase via ‘head to tail’ fashion. Depending on the number of isoprene units used in condensation reaction, the linear molecules can be geranyl diphosphate (GPP, C10), farnesyl diphosphate (FPP, C15), geranylgeranyl diphosphate (GGPP, C20) (Figure 2). The longer carbon chain precursors are produced, however, by head to head condensation of two FPPs or GGPPs. Linear terpene substrates will further undergo cyclization by terpene cyclases to provide tens of thousands of remarkable and unique terpene backbones(4). Two chemical strategies for initial carbocation formation are adapted by terpene cyclase. The first one is ionization- dependent mechanism in which metal such as Mg2+ triggers the departure of a pyrophosphate (PPi) leaving group(4). They are termed as class I terpene cyclase and have a signature ‘DDXXD/E’ motif(4). The other one is class II terpene cyclase with a ‘DXDD’ motif, which triggers protonation of a carbon-carbon double bond or epxoide ring(4). The diversity of super-family of terpenoids arises largely from the subsequent decoration reactions. Indisputably, most of the decoration reactions are initialized from oxygenation 4 reaction catalyzed by CYP inserting oxygen into terpene olefins to produce functional groups such as hydroxyl, which can further undergo transformations by adding a range of functional groups to it. Depending on the functional groups added, they can form more complex hybrid terpenoids products such as steviol glycoside (5, 6). Notably, the resulting molecules sometimes can undergo rearrangement of carbon backbone, adding complexity for terpenoids super-family. Rice phytoalexins biosynthesis network Because of their sessile nature, plants produce a broad range of compounds against biotic stress. Those compounds, named phytoalexins,