The chromosome-scale genome of Phoebe bournei reveals contrasting fates of terpene synthase (TPS)-a and TPS-b subfamilies

Terpenoids,including aromatic volatile monoterpenoids and sesquiterpenoids,function in defense against pathogens and herbivores.Phoebe trees are remarkable for their scented wood and decay resistance.Unlike other Lauraceae species investigated to date,Phoebe species predominantly accumulate sesquiterpenoids instead of monoterpenoids.Limited genomic data restrict the elucidation of terpenoid variation and functions.Here,we present a chromosome-scale genome assembly of a Lauraceae tree,Phoebe bournei,and identify 72 full-length terpene synthase(TPS)genes.Genome-level comparison shows pervasive lineage-specific duplication and contraction of TPS subfamilies,which have contributed to the extreme terpenoid variation within Lauraceae species.Although the TPS-a and TPS-b subfamilies were both expanded via tandem duplication in P.bournei,more TPS-a copies were retained and constitutively expressed,whereas more TPS-b copies were lost.The TPS-a genes on chromosome 8 functionally diverged to synthesize eight highly accumulated sesquiterpenes in P.bournei.The essential oil of P.bournei and its main component,b-caryophyllene,exhibited antifungal activities against the three most widespread canker pathogens of trees.The TPS-a and TPS-b subfamilies have experienced contrasting fates over the evolution of P.bournei.The abundant sesquiterpenoids produced by TPS-a proteins contribute to the excellent pathogen resistance of P.bournei trees.Overall,this study sheds light on the evolution and adaptation of terpenoids in Lauraceae and provides valuable resources for boosting plant immunity against pathogens in various trees and crops.

Molecular Basis for Sesterterpene Diversity Produced by Plant Terpene Synthases

Class I terpene synthase(TPS)generates bioactive terpenoids with diverse backbones.Sesterterpene synthase(sester-TPS,C25),a branch of class I TPSs,was recently identified in Brassicaceae.However,the catalytic mechanisms of sester-TPSs are not fully understood.Here,we first identified three nonclustered functional sester-TPSs(AtTPS06,AtTPS22,and AtTPS29)in Arabidopsis thaliana.AtTPS06 utilizes a type-B cyclization mechanism,whereas most other sester-TPSs produce various sesterterpene backbones via a type-A cyclization mechanism.We then determined the crystal structure of the AtTPS18–FSPP complex to explore the cyclization mechanism of plant sester-TPSs.We used structural comparisons and site-directed mutagenesis to further elucidate the mechanism:(1)mainly due to the outward shift of helix G,plant sester-TPSs have a larger catalytic pocket than do mono-,sesqui-,and di-TPSs to accommodate GFPP;(2)type-A sester-TPSs have more aromatic residues(five or six)in their catalytic pocket than classic TPSs(two or three),which also determines whether the type-A or type-B cyclization mechanism is active;and(3)the other residues responsible for product fidelity are determined by interconversion of AtTPS18 and its close homologs.Altogether,this study improves our understanding of the catalytic mechanism of plant sester-TPS,which ultimately enables the rational engineering of sesterterpenoids for future applications.

The chromosome-level genome of double-petal phenotype jasmine provides insights into the biosynthesis of floral scent

Jasmine(Jasminum sambac Aiton)is a well-known cultivated plant species for its fragrant flowers used in the perfume industry and cosmetics.However,the genetic basis of its floral scent is largely unknown.In this study,using PacBio,Illumina,10×Genomics and highthroughput chromosome conformation capture(Hi-C)sequencing technologies,a high-quality chromosome-level reference genome for J.sambac was obtained,exploiting a double-petal phenotype cultivar‘Shuangbanmoli’(JSSB).The results showed that the final assembled genome of JSSB is 580.33 Mb in size(contig N50=1.05 Mb;scaffold N50=45.07 Mb)with a total of 39618 predicted protein-coding genes.Our analyses revealed that the JSSB genome has undergone an ancient whole-genome duplication(WGD)event at 91.68 million years ago(Mya).It was estimated that J.sambac diverged from the lineage leading to Olea europaea and Osmanthus fragrans about 28.8 Mya.On the basis of a combination of genomic,transcriptomic and metabolomic analyses,a range of floral scent volatiles and genes were identified involved in the benzenoid/phenylpropanoid and terpenoid biosynthesis pathways.The results provide new insights into the molecular mechanism of its fragrance biosynthesis in jasmine.

Functional characterization of(E)-β-caryophyllene synthase from lima bean and its up-regulation by spider mites and alamethicin

（E）-β-Caryophyllene is a sesquiterpene compound widely distributed in plants and functions in plant defence. However, little is known about the sequence and function of （E）-β-caryophyllene synthase in lima bean （Phaseolus lunatus）. Here, we report a new full-length cDNA （PICAHS） encoding （E）-β-caryophyllene synthase, a possible key enzyme of plant defence. The cDNA of PICAHS contains an open reading frame of 1 761 bp, encoding a protein of 586 amino acids with a predicted mass of 67.95 kDa. The deduced amino acid sequence shows 52% identity with sesquiterpene synthase MtCAHS of Med- icago truncatula. Based on phylogenetic analysis, PICAHS is classified as the terpene synthases （TPS）-a subfamily. The recombinant enzyme, expressed in Escherichia coil, catalysed the formation of a major product （E）-β-caryophyllene （82%） and a minor product a-humulene （18%） from farnesyl dJphosphate. Real-time quantitative PCR （qRT-PCR） analysis found that the PICAHS transcript was significantly up-regulated in leaves after treatment with spider mites and alamethicin （ALA）, suggesting its ecological function in plant defence.

A likely autotetraploidization event shaped the Chinese mahogany(Toona sinensis)genome

Chinese mahogany(Toona sinensis) is of considerable medical and economic importance, and its genome has been deciphered. However, the process underlying its polyploidy is unclear, and the chromosomal evolutionary trajectory is poorly understood. Here, by reanalysing the T.sinensis genome, we found evidence of a tetraploidization event(T. sinensis special tetraploidization, TST) that occurred approximately 15-17 million years ago(MYA) after the core eudicot-common hexaploidization(ECH or gamma) event. We characterized the synonymous nucleotide substitution rates(Ks values) of collinear genes and found that T. sinensis genes affected by the TST evolve at a slower rate than Acer yangbiense genes. Furthermore, we identified homologous genes related to polyploidization and speciation and constructed multiple alignments with different reference genomes. Notably, the significant balance of gene retention and loss characterized in the two TST-derived subgenomes suggests an autopolyploid nature of the TST. Moreover, we deduced the chromosomal karyotypes of the two subgenomes and identified 7chromosomal fusions that have shaped the T. sinensis genome;more information is available on a newly constructed karyotype platform(http://www.cgrpoee.top/Toona_sinensis/index.html). The T. sinensis genome preserves the ancestral chromosome structure of dicotyledons well and could serve as a good reference for understanding genomic changes in other Meliaceae and Sapindales plants. In addition, we verified that tandem duplication and the ECH have promoted the expansion of terpene synthase(TPS) genes;conversely, the TST seems to have inhibited expansion of these genes. This present effort has clarified the polyploidy events of the T. sinensis genome, filled gaps in the history of karyotype evolution, and laid a solid foundation for further genomic studies in the Meliaceae research community and beyond.

Genomics-driven derivatization of the bioactive fungal sesterterpenoid variecolin:Creation of an unnatural analogue with improved anticancer properties

A biosynthetic gene cluster for the bioactive fungal sesterterpenoids variecolin(1)and variecolactone(2)was identified in Aspergillus aculeatus ATCC 16872.Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB.Intriguingly,the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues(5-7).Analysis of the compounds’anticancer activity in vitro and in vivo revealed that although 5 and 1 had comparable activities,5 was associated with significantly reduced toxic side effects in cancer-bearing mice,indicating its potentially broader therapeutic window.Our study describes the first tests of variecolin and its analogues in animals and demonstrates the utility of synthetic biology for creating molecules with improved biological activities.

Norditerpenoids biosynthesized by variediene synthase-associated P450 machinery along with modifications by the host cell Aspergillus oryzae

The chemical diversity of terpenoids is typically established by terpene synthase-catalyzed cyclization and diversified by post-tailoring modifications.Fungal bifunctional terpene synthase(BFTS)associated P450 enzymes have shown significant catalytic potentials through the development of various new terpenoids with different biological activities.This study discovered the BFTS and its related gene cluster from the plant endophytic fungus Didymosphaeria variabile 17020.Heterologous expression of the BFTS in Saccharomyces cerevisiae resulted in the characterization of a major product diterpene variediene(1),along with two new minor products neovariediene and neoflexibilene.Further heterologous expression of the BFTS and one cytochrome P450 enzyme VndE(CYP6138B1)in Aspergillus oryzae NSAR1 led to the identification of seven norditerpenoids(19 carbons)with a structurally unique 5/5 bicyclic ring system.Interestingly,in vivo experiments suggested that the cyclized terpene variediene(1)was modified by VndE along with the endogenous enzymes from the host cell A.oryzae through serial chemical conversions,followed by multi-site hydroxylation via A.oryzae endogenous enzymes.Our work revealed that the two-enzymes biosynthetic system and host cell machinery could produce structurally unique terpenoids.

A chromosome-scale genome assembly of Artemisia argyi reveals unbiased subgenome evolution and key contributions of gene duplication to volatile terpenoid diversity

Artemisia argyi Le´vl.et Vant.,a perennial Artemisia herb with an intense fragrance,is widely used in traditional medicine in China and many other Asian countries.Here,we present a chromosome-scale genome assembly of A.argyi comprising 3.89 Gb assembled into 17 pseudochromosomes.Phylogenetic and comparative genomic analyses revealed that A.argyi underwent a recent lineage-specificwhole-genomeduplication(WGD)event after divergence fromArtemisia annua,resulting in two subgenomes.Wedeciphered the diploid ancestral genome of A.argyi,and unbiased subgenome evolution was observed.The recent WGD led to a large number of duplicated genes in the A.argyi genome.Expansion of the terpene synthase(TPS)gene family through various types of gene duplication may have greatly contributed to the diversity of volatile terpenoids in A.argyi.In particular,we identified a typical germacrene D synthase gene cluster within the expanded TPS gene family.The entire biosynthetic pathways of germacrenes,(+)-borneol,and(+)-camphor were elucidated in A.argyi.In addition,partial deletion of the amorpha-4,11-diene synthase(ADS)gene and loss of function of ADS homologs may have resulted in the lack of artemisinin production in A.argyi.Our study provides newinsights into the genome evolution of Artemisia and lays a foundation for further improvement of the quality of this important medicinal plant.

Engineering Nannochloropsis oceanica for the production of diterpenoid compounds

Photosynthetic microalgae like Nannochloropsis hold enormous potential as sustainable,light-driven biofactories for the production of high-value natural products such as terpenoids.Nannochloropsis oceanica is distinguished as a particularly robust host with extensive genomic and transgenic resources available.Its capacity to grow in wastewater,brackish,and sea waters,coupled with advances in microalgal metabolic engineering,genome editing,and synthetic biology,provides an excellent opportunity.In the present work,we demonstrate how N.oceanica can be engineered to produce the diterpene casbene—an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids.Casbene accumulated after stably expressing and targeting the casbene synthase from Daphne genkwa(DgTPS1)to the algal chloroplast.The engineered strains yielded production titers of up to 0.12 mg g^(−1) total dry cell weight(DCW)casbene.Heterologous overexpression and chloroplast targeting of two upstream rate-limiting enzymes in the 2-C-methyl-d-erythritol 4-phosphate pathway,Coleus forskohlii 1-deoxy-d-xylulose-5-phosphate synthase and geranylgeranyl diphosphate synthase genes,further enhanced the yield of casbene to a titer up to 1.80 mg g^(−1) DCW.The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae.

Plant Terpenoids： Biosynthesis and Ecological Functions

Among plant secondary metabolites terpenolds are a structurally most diverse group; they function as phytoalexins In plant direct defense, or as signals In Indirect defense responses which involves herbivores and their natural enemies. In recent years, more and more attention has been paid to the Investigation of the ecological role of plant terpenolds. The biosynthesis pathways of monoterpenes, sesquiterpenes, and diterpenes Include the synthesis of C5 precursor isopentenyl diphosphate （IPP） and Its allylic isomer dlmethylallyl dlphosphate （DMAPP）, the synthesis of the immediate diphosphate precursors, and the formation of the diverse terpenoids. Terpene synthases （TPSs） play a key role In volatile terpene synthesis. By expression of the TPS genes, significant achievements have been made on metabolic engineering to Increase terpenoid production. This review mainly summarizes the recent research progress In elucidating the ecological role of terpenoids and characterization of the enzymes Involved in the terpenold biosynthesis. Spatial and temporal regulations of terpenoids metabolism are also discussed.

Rice gene expression profiles responding to larval feeding of the striped stem borer at the 1st to 2nd instar stage

The objective of this study was to identify rice genes that are in response to the striped stem borer （SSB） （Chilo suppressalis Walker） feeding at the first to second larval stage. Using combined suppression subtractive hybridization （SSH） and dot blot approaches, we analyzed the induced defense genes that took place during the first 72 h of infesting intact rice （Oryza sativa L.） plants in sheath tissues with SSB larvae. By sequencing the whole SSH library, 39 expressed sequence tags involved in disease stress, insect stress or other stress responses were identified to be up-regulated by SSB larvae feeding. Among these genes, rice allene oxide cyclase （AOC）, terpene synthase （TPS） and four proteinase inhibitor （PI） genes were up-regulated by SSB larvae feeding. Real-time quantitative reverse transcription polymerase chain reaction analysis showed that four rice PI genes were already up-regulated at 6 h, and reached peaks between 6 h to 12 h. In addition, the transcription ofgene involving in jasmonate signaling pathway such as allene oxide cyclase （AOC） concerning rice early defense response to SSB feeding was activated after rice feeding by SSB for 2 h. Although the expression office terpene synthase （TPS） gene, involved in the biosynthesis ofmonoterpenes or diterpenes, was already up-regulated at 7 h, a significant increase in the expression was delayed until 12 h and reached its peak at 24 h. The present study identified six SSB-response genes and their expression patterns, which provides evidence and information to understand insect stress-response in plants.