Chromosome-level genome assembly of Cornus officinalis reveals the evolution of loganin biosynthesis

Abstract

Cornus officinalis is a traditional medicinal plant known for producing loganin, a bioactive iridoid glycoside with potential anticancer properties. However, the absence of a high-quality reference genome has limited insights into its biosynthetic pathways. Here, we present a chromosome-level genome assembly of C. officinalis with a size of 2.85 Gb. Comparative genomic analysis revealed that the genome expansion and longer gene structures, relative to other Cornales species, are primarily due to a recent expansion of transposable elements. In this study, we identified unique biosynthetic gene clusters coding multiple core enzymes, including loganin acid O-methyltransferase (LAMT), secologanin synthase (SLS), and cytochrome P450, all of which catalyze sequential steps leading to loganin formation. LAMT enzymes from C. officinalis capable of catalyzing the C-9 hydroxylation of loganin acid were identified, whereas the homolog (CoLMAT) was not found to possess this activity. Additionally, molecular docking studies revealed critical residues in CoLAMT that govern substrate positioning, providing insights into the mechanism of C-9 regioselective hydroxylation. Further characterization of 7-deoxyloganicacid hydroxylase, LAMT, and SLS enzymes allowed us to elucidate the complete biosynthetic pathway of major loganin derivatives in the medicinal plant C. officinalis. Finally, we introduced CoLAMT and its upstream genes into Nicotiana benthamiana and successfully achieved the de novo biosynthesis of a series of loganin derivatives. This work reveals key evolutionary and molecular mechanisms in loganin biosynthesis, providing insights into biotechnological applications in anticancer drug development.

Acknowledgements

This work was supported by the Project of the National Natural Science Foundation of China (nos. 32170370, and 32370396) and the 111 Project (no. B20050).

Authors contributions

D.Z. designed the experiments; X.Z., H.Z., J.W. performed the experiments; X.Z. and J.W. collected and analyzed the data; X.Z., H.Z., and D.Z. wrote the manuscript; D.Z. obtained funding and is responsible for this article.

Data availability

The gene sequence data and raw sequence data generated in this study have been deposited in the Genome Sequence Archive at the National Genomics Data Center as a BioProject number PRJCA041456.

Conflicts of interest statement

All authors did not have any conflict of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Cao B, Bai C, Zhang L. et al. Modeling habitat distribution of Cornus officinalis with Maxent modeling and fuzzy logics in China. J Plant Ecol. 2016; 9:742-51

[2] Li G, Zhang L, Bai C. Chinese Cornus officinalis: genetic resources, genetic diversity and core collection. Genet Resourc Crop Evol. 2012; 59:1659-71

[3] Zhang LJ, Li GS, Bai CK. et al. Evaluation and correlation analysis on quantitative traits of Cornus officinalis germplasm resources. J Plant Genet Resourc. 2012; 13:655-9

[4] Xue Y, Cao B, Liang H. et al. Environmental shifts have impor-tant impacts on the functional traits and bioactive products of medicinal crop Cornus officinalis. Ind Crop Prod. 2021; 162: 113304

[5] Yang L, Wang Z, Huang L. Isolation and structural character-ization of a polysaccharide FCAP1 from the fruit of Cornus officinalis. Carbohydr Res. 2010; 345:1909-13

[6] Chengke B, Yongmei W, Bo C. et al. De novo transcriptome assembly based on RNA-seq and dynamic expression of key enzyme genes in loganin biosynthetic pathway of Cornus offic-inalis. Tree Genet Genomes. 2018; 14:1-14

[7] Chun-Chu D. Research on major medical component in dif-ferent parts of Bothrocaryum controversum. J Anhui Agric Sci. 2009; 37:5490-1

[8] Kreiner J, Pang E, Lenon GB. et al. Saposhnikoviae divar-icata: a phytochemical, pharmacological, and pharmacoki-netic review. Chin J Nat Med. 2017; 15:255-64

[9] Khan S, Ahmad A, Ahmad I. A sensitive and rapid liquid chro-matography tandem mass spectrometry method for quanti-tative determination of 7-ethyl-10-hydroxycamptothecin (SN-38) in human plasma containing liposome-based SN-38 (LE-SN38). Biomed Chromatogr. 2003;17:493-9

[10] Han M, Luo W, Gu Q. et al. Isolation and characterization of a keratinolytic protease from a feather-degrading bacterium Pseudomonas aeruginosa C11. Afr J Microbiol Res. 2012; 6: 2211-21

[11] Wang Z, Li Y, Sun P. et al. A high-quality Buxus austro-yunnanensis (Buxales) genome provides new insights into karyotype evolution in early eudicots. BMC Biol. 2022; 20:216

[12] Austin CA, Shephard EA, Pike SF. et al. The effect of terpenoid compounds on cytochrome P-450 levels in rat liver. Biochem Pharmacol. 1988; 37:2223-9

[13] Sandmann G, Böger P, Sandmann G. et al. Interference of dimethazone with formation of terpenoid compounds. Zeitschrift für Naturforschung C. 1986; 41:729-32

[14] Dong NQ, Lin HX. Contribution of phenylpropanoid metabolism to plant development and plant-environment interactions FA. J Bot. 2021; 63:180-209

[15] Yang T, Li J, Wang HX. et al. A geraniol-synthase gene from Cinnamomum tenuipilum. Phytochemistry. 2005; 66:285-93

[16] Zonneveld BJM. Conifer genome sizes of 172 species, covering 64 of 67 genera, range from 8 to 72 picogram. Nord J Bot. 2012; 30:490-502

[17] Ikezawa N, Tanaka M, Nagayoshi M. et al. Molecular cloning and characterization of CYP719, a methylenedioxy bridge-forming enzyme that belongs to a novel P450 family Coptis japonica cells. J Biol Chem. 2003; 278:38557-65

[18] Solomon H. Iridoids and other monoterpenes in the Alzheimer’s brain: recent development and future prospects. Molecules. 2018; 23:117

[19] Huang J, Zhang Y, Dong L. et al. Ethnopharmacology, phy-tochemistry, and pharmacology of Cornus officinalis Sieb. J Ethnopharmacol. 2018; 213:280-301

[20] Wang L, Shi Y, Chang X. et al. DNA methylome analysis pro-vides evidence that the expansion of the tea genome is linked to TE bursts. Plant Biotechnol J. 2019; 17:826-35

[21] Dinda B, Chowdhury DR, Mohanta BC. Naturally occurring iridoids, secoiridoids and their bioactivity. An updated review, part 3. Chem Pharm Bull. 2009; 57:765-96

[22] Cao L, Wu Y, Liu KY. et al. Cornus officinalis vinegar alters the gut microbiota, regulating lipid droplet changes in nonalco-holic fatty liver disease model mice. Food Medicine Homology. 2024; 1:9420002

[23] Shi M, Gong H, Cui L. et al. Targeted metabolic engineering of committed steps improves anti-cancer drug camptothecin production in Ophiorrhiza pumila hairy roots. Ind Crop Prod. 2020; 148:112277

[24] Fischer, Meyer S, Claudel P. et al. Specificity of Ocimum basilicum geraniol synthase modified by its expression in dif-ferent heterologous systems. J Biol. 2013; 163:24-9

[25] Garza RM, Tran PN, Hampton RY. Geranylgeranyl pyrophosphate (GGPP) is a potent regulator of HRD-dependent HMG-CoA reductase degradation in yeast. J Biol Chem. 2009; 284:35368-80

[26] Salim V, Jones AD, DellaPenna D. Camptotheca acuminata 10-hydroxycamptothecin OO methyltransferase: an alkaloid biosynthetic enzyme co-opted from flavonoid metabolism. Plant J. 2018; 95:112-25

[27] Madhusudanan KP, Raj K, Bhaduri AP. Effect of alkali metal cationization and multiple alkali metal exchange on the collision-induced dissociation of loganic acid studied by elec-trospray ionization tandem mass spectrometry. Rapid Com-mun Mass Spectrom. 2020; 14:885-96

[28] Kang M, Fu R, Zhang P. et al. A chromosome-level Camp-totheca acuminata genome assembly provides insights into the evolutionary origin of camptothecin biosynthesis. Nat Commun. 2021; 12:3531

[29] Asada K, Salim V, Masada-Atsumi S. et al. A 7-deoxyloganetic acid glucosyltransferase contributes a key step in secolo-ganin biosynthesis in Madagascar periwinkle. Plant Cell. 2013; 25:4123-34

[30] Wahby S, Fashoyin-Aje L, Osgood CL. et al. FDA approval sum-mary: accelerated approval of Sacituzumab Govitecan-hziy for third-line treatment of metastatic triple-negative breast cancer. Am Assoc Cancer Res. 2021; 27:1850-4

[31] Croteau R, Ketchum REB, Long RM. et al. Taxol biosynthesis and molecular genetics. Phytochem Rev. 2006; 5:75-97

[32] Irmler S, Schröder G, St-Pierre B. et al. Indole alkaloid biosyn-thesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase. Plant J. 2000; 24:797-804

[33] Wildung MR, Croteau R. A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis. J Biol Chem. 1996; 271:9201-4

[34] Hsiang YH, Hertzberg RP, Hecht SM. et al. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 1985; 260: 14873-8

[35] Li J, Liu A, Najeeb U. et al. Genome-wide investigation and expression analysis of membrane-bound fatty acid desat-urase genes under different biotic and abiotic stresses in sunflower (Helianthus annuus L.). Int J Biol Macromol. 2021; 175:188-98

[36] Edwards ME, Dickson CA, Chengappa S. et al. Molecular char-acterisation of a membrane-bound galactosyltransferase of plant cell wall matrix polysaccharide biosynthesis. Plant J. 2010; 19:691-7

[37] Yang Y, Zhao H, Barrero RA. et al. Genome sequencing and analysis of the paclitaxel-producing endophytic fun-gus Penicillium aurantiogriseum NRRL 62431. BMC Genomics. 2014; 15:69-9

[38] Burge C, Karlin S. Prediction of complete gene structures in human genomic DNA. J Mol Biol. 1997; 268: 78-94

[39] Endress PK, Doyle JA. Reconstructing the ancestral angiosperm flower and its initial specializations. Am J Bot. 2009; 96:22-66

[40] Mckain MR, Tang H, McNeal JR. et al. A Phylogenomic assess-ment of ancient polyploidy and genome evolution across the Poales. Genome Biol Evol. 2016; 8:1150-64

[41] Xiaojun S, Yang L, Wang D. et al.1 K medicinal plant genome database: an integrated database combining genomes and metabolites of medicinal plants. Hortic Res. 2022;9:uhac075

[42] Attwood TK, Beck ME. PRINTS-a database of protein motif fingerprints. Nucleic Acids Res. 1994; 22:3590-6

[43] Jaillon O, Aury J-M, Noel B. et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature. 2007; 449:463-7

[44] Zhang W, Zhang Y, Qiu H. et al. Genome assembly of wild tea tree DASZ reveals pedigree and selection history of tea varieties. Nat Commun. 2020; 11:3719

[45] Dong C, Wang S, Zhang H. et al. Karyotype evolution of the Asterids insights from the first genome sequences of the fam-ily Cornaceae. DNA Res. 2023;30:dsac051

[46] He Z, Chao H, Zhou X. et al. A chromosome-level genome assembly provides insights into Cornus wilsoniana evolution, oil biosynthesis, and floral bud development. Hortic Res. 2023;10:uhad196

[47] Celedon JM, Bohlmann J. Oleoresin defenses in conifers: chemical diversity, terpene synthases and limitations of oleoresin defense under climate change. New Phytol. 2019; 224:1444-63

[48] Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980; 16:111-20

[49] Cossu RM, Casola C, Giacomello S. et al. LTR retrotransposons show low levels of unequal recombination and high rates of intraelement gene conversion in large plant genomes. Genome Biol Evol. 2017; 9:3449-62

[50] Shimizu Y, Rai A, Okawa Y. et al. Metabolic diversification of nitrogen-containing metabolites by the expression of a het-erologous lysine decarboxylase gene in Arabidopsis. Plant J. 2019; 100:505-21

[51] Rubin A. Plastocyanin and cytochrome f complex structures obtained by NMR, molecular dynamics, and alphafold 3 meth-ods compared to cryo-EM data. Int J Mol Sci. 2024; 25:11083

[52] Wang D, Wei J, Yuan X. et al. Transcriptome and comparative chloroplast genome analysis of Taxus yunnanensis individu-als with high and low paclitaxel yield. Heliyon. 2024;10:

[53] Moaine EB, Olivier P. Comparative genomic paleontology across plant kingdom reveals the dynamics of TE-driven genome evolution. Genome Biol Evol. 2013; 5:954-65

[54] Nowak MD, Russo G, Schlapbach R. et al. The draft genome of Primula veris yields insights into the molecular basis of heterostyly. Genome Biol. 2015; 16:12

[55] Song X, Wang J, Li N. et al. Deciphering the high-quality genome sequence of coriander that causes controversial feel-ings. Plant Biotechnol J. 2020; 18:1444-56

[56] Feldberg E, Porto JIR, Nakayama CM. et al. Karyotype evolu-tion in Curimatidae (Teleostei, Characiformes) from the Ama-zon region. Genome. 1993; 36:372-6

[57] Nystedt B, Street NR, Wetterbom A. et al. The Norway spruce genome sequence and conifer genome evolution. Nature. 2013; 497:579-84

[58] Niu S, Li J, Bo W. et al. The Chinese pine genome and methy-lome unveil key features of conifer evolution. Cell. 2022; 185: 204-217.e14

[59] Liu H, Wang X, Wang G. et al. The nearly complete genome of Ginkgo biloba illuminates gymnosperm evolution. Nat Plants. 2021; 7:748-56

[60] Medema MH, Cimermancic P, Sali A. et al. A systematic computational analysis of biosynthetic gene cluster evolu-tion: lessons for engineering biosynthesis. PLoS Comput Biol. 2014; 10:e1004016

[61] Goldsmith M, Tawfik DS. Enzyme engineering: reaching the maximal catalytic efficiency peak. Curr Opin Struct Biol. 2017; 47:140-50

[62] Bruice TC. A view at the millennium: the efficiency of enzy-matic catalysis. Acc Chem Res. 2002; 35:139-48

[63] Zhang X, Liu CJ. Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenyl-propanoids. Mol Plant. 2015; 8:17-27

[64] Bowles D, Lim EK, Poppenberger B. et al. Glycosyltrans-ferases of lipophilic small molecules. Annu Rev Plant Biol. 2006; 57:567-97

[65] Shuldiner AR, O’Connell JR, Bliden KP. et al. Association of cytochrome P 450 2C19 genotype with the antiplatelet effect and clinical efficacy of clopidogrel therapy. Jama. 2009; 302: 849-57

[66] Quiocho FA, Wilson DK, Vyas NK. Substrate specificity and affinity of a protein modulated by bound water molecules. Nature. 1989; 340:404-7

[67] Marais G, Kingsford C. A fast, lock-free approach for efficient parallel counting of occurrences of K-mers. Bioinformatics. 2011; 27:764-70

[68] Liu B, Shi Y, Yuan J. et al. Estimation of genomic characteristics by analyzing k-mer frequency in de novo genome projects. Quantitative Biology. 2013; 35:62-7

[69] Chin CS, Alexander DH, Marks P. et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequenc-ing data. Nat Methods. 2013; 10:563-9

[70] Chaisson MJ, Tesler G. Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory. BMC Bioinformatics. 2013; 13: 1-18

[71] Burton JN, Adey A, Patwardhan RP. et al. Chromosome-scale scaffolding of de novo genome assemblies based on chro-matin interactions. Nat Biotechnol. 2013; 31:1119-25

[72] Neva C, Robinson JT, Shamim MS. et al. Juicebox provides a visualization system for hi-C contact maps with unlimited zoom. Cell Systems. 2016; 3:99-101

[73] Charalampos L, Stephen K, Panagiotis N. et al. HiC-bench: comprehensive and reproducible hi-C data analysis designed for parameter exploration and benchmarking. BMC Genomics. 2017; 18:1-16

[74] Gary B. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999; 27:573-80

[75] Cybis GB, Valk M, Lopes SRC. Clustering and classification problems in genetics through U-statistics. J Stat Comput Simul. 2018; 88:1882-902

[76] Reference Genome Group of the Gene Ontology Consortium. The gene ontology’s reference genome project: a unified framework for functional annotation across species. PLoS Comput Biol. 2009; 5:e1000431

[77] Basu D. Randomization analysis of experimental data: the fisher randomization test. J Am Stat Assoc. 1980; 75: 575-82