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. 2020 Jun 1;27(3):dsaa013.
doi: 10.1093/dnares/dsaa013.

Genome assembly of Chiococca alba uncovers key enzymes involved in the biosynthesis of unusual terpenoids

Kin H Lau  1 , Wajid Waheed Bhat  2 , John P Hamilton  1 , Joshua C Wood  1 , Brieanne Vaillancourt  1 , Krystle Wiegert-Rininger  1 , Linsey Newton  1 , Britta Hamberger  2 , Daniel Holmes  3 , Bjoern Hamberger  2   4 , C Robin Buell  1   4   5
Affiliations

Genome assembly of Chiococca alba uncovers key enzymes involved in the biosynthesis of unusual terpenoids

Kin H Lau et al. DNA Res. .

Abstract

Chiococca alba (L.) Hitchc. (snowberry), a member of the Rubiaceae, has been used as a folk remedy for a range of health issues including inflammation and rheumatism and produces a wealth of specialized metabolites including terpenes, alkaloids, and flavonoids. We generated a 558 Mb draft genome assembly for snowberry which encodes 28,707 high-confidence genes. Comparative analyses with other angiosperm genomes revealed enrichment in snowberry of lineage-specific genes involved in specialized metabolism. Synteny between snowberry and Coffea canephora Pierre ex A. Froehner (coffee) was evident, including the chromosomal region encoding caffeine biosynthesis in coffee, albeit syntelogs of N-methyltransferase were absent in snowberry. A total of 27 putative terpene synthase genes were identified, including 10 that encode diterpene synthases. Functional validation of a subset of putative terpene synthases revealed that combinations of diterpene synthases yielded access to products of both general and specialized metabolism. Specifically, we identified plausible intermediates in the biosynthesis of merilactone and ribenone, structurally unique antimicrobial diterpene natural products. Access to the C. alba genome will enable additional characterization of biosynthetic pathways responsible for health-promoting compounds in this medicinal species.

Keywords: Chiococca alba; 10×; alkaloid; genome; linked reads; terpene synthase.

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Figures

Figure 1
Figure 1
Chiococca alba. (A) Chiococca alba (snowberry) with fruit (left) and flower (right). (B) Chemical diversity of C. alba terpenoids. Representative monoterpene, seco-iridoid glucoside Alboside I, triterpene Chiococcasaponin IV, diterpene kaurane-type ent-kaurenoic acid, nor-kaurane-type 1-hydroxy-18-nor-kaur-4, 16-dien-3-one,ent-manoyl oxide-type Ribenone, plausibly nor-seco-pimarane-type Merilactone.
Figure 2
Figure 2
Synteny between C. alba and C. canephora.(A) Syntenic regions between C. alba scaffolds (ca) and C. canephora Chromosome 9. (B) Expanded view of syntenic regions between C. alba scaffolds 265 (ca265) and 327 (ca327). The position of the C. canephora NMT genes on Chromosome 9 is denoted with a red triangle. (C) Zoomed in view of C. canephora (Cc) syntenic region encoding caffeine biosynthetic pathway genes (Cc09_g06950, Cc09_g06960, Cc09_g06970, and Cc09_g07000; red triangles) and C. alba scaffold 265 (Ca).
Figure 3
Figure 3
Gene counts of orthogroups containing C. alba genes predicted to be involved in terpene synthesis. Orthogroups were classified as mono-, sesqui-, or diTPSs by collapsing the TPS annotation for C. alba gene members. OG0000211 contained both sesqui- and mono-TPS genes.
Figure 4
Figure 4
Phylogenetic tree of TPSs. Maximum likelihood phylogenetic tree of candidate TPSs from C. alba and reference TPSs. The bifunctional ent-CPP/ent-kaurene synthase from Physcomitrella patens is used as an outgroup. Reference names and amino acid sequences are available in Supplementary Tables S15 and S16. Bootstrap values of 1,000 replicates are given on nodes; scale bar represents site changes.
Figure 5
Figure 5
Proposed reaction pathways en route to merilactone, ribenone, and other C. alba diterpenes. (A) Following the common framework of labdane biosynthesis in angiosperms, pairs of monofunctional Classes II and I diTPSs were identified that produce distinct diterpene scaffolds in C. alba. First, CaTPS1 and CaTPS2 transform the central precursor GGPP (0) into the bicyclic prenyl diphosphate intermediates ent-CPP (1) and ent-LPP (2), respectively. Second, Class I diTPSs CaTPS3/CaTPS4 act on 1 to produce ent-kaurene (3), precursor to ent-CPP derived general [Gibberellic acid GA12; (9)] and specialized metabolites (10–13) in C. alba. CaTPS3/CaTPS4 also use 2 as substrate to produce (13R)-ent-manoyl oxide (4). We hypothesize 4 to be the immediate precursor of Ribenone (7) via sequential oxidation at C3 to Ribenol (6), presumably by a cytochrome P450 enzyme. CaTPS5 catalysed the formation of 13-epi-dolabradiene (5), a possible committed step in the biosynthesis of the C19 diterpene, merilactone (8). (B) FPKM values of functionally characterized C. alba diTPSs. Three leaf samples (L1–L3) and two root samples (R1 and R2) from independent plants were used for the study.
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