Laura K. Henry, Michael Gutensohn, Suzanne T. Thomas, Joseph P. Noel, and Natalia Dudareva

Significance

Terpenoids, one of the largest and most diverse classes of natural products, are found in all living organisms, where they play essential roles in growth and development, respiration and photosynthesis, and interactions with the environment. We discovered that a functional homolog of isopentenyl phosphate kinase (IPK), originally identified in archaebacteria, is unexpectedly present in plants and functions in their terpenoid metabolic network. Cytosolically localized, IPK phosphorylates isopentenyl phosphate (IP) and its isomer dimethylallyl phosphate (DMAP) to their corresponding diphosphates, IPP and DMAPP, the universal C5 building blocks of all natural terpenoids. IPK enhances terpenoid formation by returning IP/DMAP to the terpenoid biosynthetic network. This metabolite reactivation process offers a new approach for metabolic engineering of economically important terpenoids.

Abstract

Terpenoids, compounds found in all domains of life, represent the largest class of natural products with essential roles in their hosts. All terpenoids originate from the five-carbon building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which can be derived from the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways. The absence of two components of the MVA pathway from archaeal genomes led to the discovery of an alternative MVA pathway with isopentenyl phosphate kinase (IPK) catalyzing the final step, the formation of IPP. Despite the fact that plants contain the complete classical MVA pathway, IPK homologs were identified in every sequenced green plant genome. Here, we show that IPK is indeed a member of the plant terpenoid metabolic network. It is localized in the cytosol and is coexpressed with MVA pathway and downstream terpenoid network genes. In planta, IPK acts in parallel with the MVA pathway and plays an important role in regulating the formation of both MVA and MEP pathway-derived terpenoid compounds by controlling the ratio of IP/DMAP to IPP/DMAPP. IP and DMAP can also competitively inhibit farnesyl diphosphate synthase. Moreover, we discovered a metabolically available carbon source for terpenoid formation in plants that is accessible via IPK overexpression. This metabolite reactivation approach offers new strategies for metabolic engineering of terpenoid production.

See: http://www.pnas.org/content/112/32/10050.abstract

PNAS August 11, 2015 vol. 112 no. 32 10050-10055

Figure 1.  Position and potential role of IPK in the plant terpenoid metabolic network. Cytosolic and plastidial (highlighted in green) terpenoid metabolic pathways involved in the biosynthesis of sterols, sesquiterpenes, and monoterpenes in plants with individual enzymes depicted as boxes. The MVA pathway enzymes are highlighted in orange, the MEP pathway enzymes are highlighted in white, and enzymes involved in downstream terpenoid formation are highlighted in yellow. The enzymes with peroxisomal localization are depicted on a striped background. The unknown transporters involved in IPP and IP exchange across the plastid envelope membranes are shown in gray. Site of action of the MVA and MEP pathway-specific inhibitors (lovastatin and fosmidomycin, respectively), as well as feed-forward inhibition of FPPS by IP/DMAP, are indicated. The recently discovered MPD acting in the alternative MVA pathway in R. castenholzii and H. volcanii is shown on a gray background. Dashed lines in sterol biosynthesis represent multiple enzymatic steps. Abbreviations: AACT, aceto-acetyl-CoA thiolase; CMK, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase; DXS, 1-deoxy-d-xylulose 5-phosphate synthase; GA-3P, d-glyceraldehyde 3-phosphate; HDR, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HYD1, C-8,7 sterol isomerase; MCT, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase; MDS, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; MK, mevalonate kinase; Phos, phosphatase(s); SQS, squalene synthase; TPS, terpene synthases (including monoterpene synthases and sesquiterpene synthases).