CYP76M7 is an ent-cassadiene C11alpha-hydroxylase defining a second multifunctional diterpenoid biosynthetic gene cluster in rice

Abstract

Biosynthetic gene clusters are common in microbial organisms, but rare in plants, raising questions regarding the evolutionary forces that drive their assembly in multicellular eukaryotes. Here, we characterize the biochemical function of a rice (Oryza sativa) cytochrome P450 monooxygenase, CYP76M7, which seems to act in the production of antifungal phytocassanes and defines a second diterpenoid biosynthetic gene cluster in rice. This cluster is uniquely multifunctional, containing enzymatic genes involved in the production of two distinct sets of phytoalexins, the antifungal phytocassanes and antibacterial oryzalides/oryzadiones, with the corresponding genes being subject to distinct transcriptional regulation. The lack of uniform coregulation of the genes within this multifunctional cluster suggests that this was not a primary driving force in its assembly. However, the cluster is dedicated to specialized metabolism, as all genes in the cluster are involved in phytoalexin metabolism. We hypothesize that this dedication to specialized metabolism led to the assembly of the corresponding biosynthetic gene cluster. Consistent with this hypothesis, molecular phylogenetic comparison demonstrates that the two rice diterpenoid biosynthetic gene clusters have undergone independent elaboration to their present-day forms, indicating continued evolutionary pressure for coclustering of enzymatic genes encoding components of related biosynthetic pathways.

Figure 1.

Functional Map of Rice Diterpenoid Biosynthesis. The cyclases and corresponding reactions are indicated, along with the downstream natural products, where known. Thicker arrows indicate enzymatic reactions specifically involved in GA metabolism; dashed arrows indicate multiple enzymatic reactions.

Figure 2.

Schematic of Rice Diterpenoid Biosynthetic Gene Cluster. Black boxes represent genes induced by chitin elicitation, while white boxes represent those not induced (Okada et al., 2007), with the arrowheads representing the direction of transcription. Note that no other genes appear to be present in these regions.

Figure 3.

Hydroxylation of ent-Cassadiene by CYP76M7. (A) Gas chromatography (GC)-MS chromatogram of extract from E. coli engineered for production of ent-cassadiene and coexpressing CYP76M7 and rice CPR1 (1, ent-cassadiene; 2, 11α-hydroxy-ent-cassadiene). (B) Mass spectra for peak 1 (ent-cassadiene). (C) Mass spectra for peak 2 (11α-hydroxy-ent-cassadiene).

Figure 4.

Kinetic Plot for CYP76M7 Activity. The measured initial rates from duplicate assays (data points are the average and errors bars represent the sd) and fit to the Michaelis-Menton equation (R² = 0.96) are shown.

Figure 5.

Reaction Catalyzed by CYP76M7 and Its Putative Relationship to Phytocassane Biosynthesis. The dashed arrow indicates the multiple biosynthetic steps leading to the mixture of phytocassanes A to E.

Figure 6.

Molecular Phylogenetic Analysis of the Coclustered Rice Diterpenoid Biosynthetic Enzymatic Gene Families. (A) Phylogenetic tree for cereal CPSs. (B) Phylogenetic tree for Os-KSL. (C) Phylogenetic tree for CYP71Z, CYP76M, and CYP99A subfamilies from rice. The genes in the rice diterpenoid biosynthetic gene clusters are indicated by the corresponding chromosomal number (i.e., Chr. 2 or 4) in parentheses. The scale bars indicate the expected substitutions per site for the given branch length.

Figure 7.

Hypothetical Assembly Process for the Observed Current Rice Diterpenoid Biosynthetic Gene Clusters Depicted in Figure 2. Each box represents a gene in the cluster. In those cases where duplication has been postulated to occur after cluster assembly, a precursor gene is indicated (i.e., Os-KSL5/6 as the precursor to the current Os-KSL5 and 6, with “x” designating the precursor to the various CYP subfamily members found in the clusters).