Plant metabolic clusters - from genetics to genomics

Abstract

Contents 771 I. 771 II. 772 III. 780 IV. 781 V. 786 786 References 786 SUMMARY: Plant natural products are of great value for agriculture, medicine and a wide range of other industrial applications. The discovery of new plant natural product pathways is currently being revolutionized by two key developments. First, breakthroughs in sequencing technology and reduced cost of sequencing are accelerating the ability to find enzymes and pathways for the biosynthesis of new natural products by identifying the underlying genes. Second, there are now multiple examples in which the genes encoding certain natural product pathways have been found to be grouped together in biosynthetic gene clusters within plant genomes. These advances are now making it possible to develop strategies for systematically mining multiple plant genomes for the discovery of new enzymes, pathways and chemistries. Increased knowledge of the features of plant metabolic gene clusters - architecture, regulation and assembly - will be instrumental in expediting natural product discovery. This review summarizes progress in this area.

Keywords: biosynthetic gene clusters; chromatin; genome mining; natural products; operons.

Fig. 1

Structures of the products of characterized clustered plant metabolic pathways. The structures are numbered according to Table 1.

Fig. 1

Structures of the products of characterized clustered plant metabolic pathways. The structures are numbered according to Table 1.

Fig. 2

Genomic organization of plant metabolic gene clusters. Examples that exemplify different cluster types and biosynthetic pathways are shown. The genes are indicated by arrows. The gene(s) for the first committed pathway step are indicated in red. Gene names are indicated above the clusters, and class of biosynthetic enzyme below. Abbreviations: OSC, oxidosqualene cyclase; DTS, diterpene synthase (class I and class II shown); IGPL, indole 3-glycerol phosphate lyase; DKS, diketone synthase; AT (BAHD), BAHD-acyltransferase; AT (SCPL), SCPL-acyltransferase; MT, methyltransferase; UGT, UDP-dependent sugar transferase, DHO, dehydrogenase/reductase; L/CT, lipase/carboxyltransferase; CES, carboxylesterase; DOX, dioxygenase; TA, transaminase; CYP, cytochrome P450. The maize DIMBOA pathway includes three genes that are not shown in the figure: Bx7, which is separated from the core cluster by an intervening region of 15 Mb; the sugar transferase gene Bx9, which is located on a different chromosome; and finally Bx6, which is not shown because its genomic location has not yet been established. [Correction added after online publication 26 April 2016; β-Diketone Hordeum vulgaris cluster has been corrected.]

Fig. 3

Benzylisoquinoline alkaloid biosynthesis in poppy. Poppy synthesizes a variety of different benzylisoquinoline alkaloids, including noscapine, codeine and morphine. The first committed step of the noscapine pathway (boxed) is the conversion of (S)-scoulerine to (S)-tetrahydrocolumbamine by PSMT1. The enzymes encoded by the genes within the noscapine cluster are indicated in red. The gene for one of the noscapine pathway steps (tetrahydroprotoberberine cis-N-methyltransferase; TNMT) is not represented within the cluster. Unlike the rest of the pathway genes, TNMT is present and expressed in HN1, HT1 and HM1 poppy varieties and so is likely to have other functions in addition to its role in noscapine biosynthesis (Winzer et al., 2012). The P450 oxidoreductase fusion protein STORR, which catalyses the first step in morphinan biosynthesis in poppy – the conversion of (S)-reticuline to (R)-reticuline – is indicated in purple.

Fig. 4

Types of cluster organization. (a) Archetypal cluster. The archetypal plant metabolic cluster is compact and contains contiguous pathway genes, including the gene encoding the enzyme for the first committed step in the pathway. (b) Core cluster plus peripheral gene(s). The genes for most of the metabolic pathway steps (including the gene for the first committed step) are clustered and contiguous, but there may be one or more peripheral genes encoding other pathway steps. These peripheral genes may be loosely linked to the core cluster (in cis) or may be unlinked (in trans). (c) Core cluster plus satellite subgroups. The bulk of the pathway genes, including the gene for the first committed step, are organized in a core cluster with small groups of two to three other genes present as satellite subgroups elsewhere in the genome. For the examples reported so far, these satellite subgroups contain genes for one or two different types of tailoring enzymes. (d) Core cluster plus peripheral gene for first step. In this scenario the core cluster encodes three or more different types of tailoring enzyme and the gene encoding the first committed step is elsewhere in the genome (either in cis or in trans). In each case the gene encoding the first committed pathway step is indicated in dark red and marked with an asterisk.

Fig. 5

Schematic representation of potential levels of gene cluster regulation. (a) Coregulated promotor motifs (black ovals) are located upstream of each cluster gene. (b) Distinct chromatin modifications facilitate the formation of readily accessible open chromatin structures (red flag) and closed condensed chromatin regions (grey flag). An open chromatin structure will allow transcription, whereas a closed structure will suppress it. (c) At the tertiary level, cis-acting regulatory elements (black ovals) associate to form chromatin hubs, looping out the intervening DNA and bringing the cluster genes together in close proximity. The genes close to the regulatory elements are expressed (coloured), whereas genes located further away are silent (grey). (d) Gene clusters may be located in discrete nuclear territories that are characterized by distinct chromosomal conformations.

Fig. 6

Origins of plant metabolic clusters. The scenarios shown are based on current knowledge of characterized plant metabolic clusters (see text). (a) De novo cluster assembly. The first step in this process is the recruitment of the gene for the first committed step in the pathway by gene duplication and neofunctionalization. This gene then ‘seeds’ the formation of a multigene cluster for a new metabolic pathway by an as yet unknown mechanism. (b) Formation of new metabolic clusters from a founder gene pair template. In this scenario, an ancestral gene pair (e.g. encoding a terpene synthase and a cytochrome P450 (CYP) enzyme) may give rise to different metabolic clusters for new natural product pathways as a result of independent events involving gene rearrangement and recruitment of new genes. This is exemplified by the Arabidopsis thaliana thalianol and marneral clusters (Field et al., 2011). These are both triterpene pathways, but the scaffolds are not the same and the subsequent modifications are different. For both (a) and (b) the process of cluster formation will be accompanied by refinement and optimization of individual pathway components and establishment of a functional gene neighbourhood that allows for coordinate pathway control. (c) Diversification of an ancestral founder cluster in syntenic regions of closely related taxonomic lineages. In these cases, these pathways may have a common scaffold but make slightly different compounds.

Fig. 7

Trait-based cluster discovery. Examples of compounds of agronomic/medicinal importance for which the underlying metabolic pathways were characterized bytrait-based cluster discovery are shown. The images of plants are reproduced with the kind permission of: (a) Paul Cristou, Institució Catalanade Recerca I Estudis, Lleida, Spain; (b) Anthony Pugh, Institute for Biological, Environmental and Rural Sciences, Aberystwyth, UK; (c) John Innes Centre Photography; (d) Sanwen Huang, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences; (e) Tanja Niggendijker/Creative Commons.

Fig. 8

Strategies used for identification of plant metabolic clusters. (a) Biochemical characterization of one or more pathway steps in the pregenome era. The existence of clustering may then become apparent once genome sequence data for the species of interest is available. (b) Investigation of induced or natural variation in a trait provides genetic evidence for a multigene locus. The relevant region is then further defined by mapping. The underlying cluster is identified by assembly and sequencing of bacterial artificial chromosome (BAC) contigs spanning the region (in the absence of genome sequence information) or by exploiting available genome sequence data in the region of the locus. (c) Discovery of metabolic clusters for new pathways by genome mining. Physical clustering of genes implicated in a specialized metabolic pathway in combination with transcriptomics data enables the delineation of new candidate metabolic gene clusters.