RESUMEN

ObjectiveBioinformatics methods were used to systematically identify the Salvia miltiorrhiza terpenoid synthase （SmTPS） gene family members and predict their functions from the perspective of the genome. MethodThe genome and transcriptome data of S. miltiorrhiza， Arabidopsis thaliana， and tomato were obtained from the national genomics data center （NGDC）， national center for biotechnology information （NCBI）， the Arabidopsis information resource （TAIR）， and tomato functional genomics database （TFGD）， and the whole genome identification and bioinformatics analysis of the SmTPS gene family member were carried out with the help of Perl language programming， Tbtools， and other bioinformatics tools. ResultA total of 52 TPS gene family members were identified， and they were distributed on eight chromosomes of S. miltiorrhiza. Their coding amino acid number was 207-822 aa. The isoelectric points were 4.76-9.16. The molecular mass was 24.11-94.81 kDa， and all members are hydrophilic proteins. Gene structure analysis showed that there were significant differences in the number of introns among different subfamilies. The number of introns in 72.6% of TPS-a， b， and g subfamilies was 6， and that in 88.9% of TPS-c and e/f subfamilies was more than 10. Protein motifs were conserved among TPS subfamilies. The analysis of promoter cis-acting elements showed that all promoters of the SmTPSs contained a large number of light-responsive elements， and most of them had hormone-responsive elements. Gene expression analysis showed that SmTPS gene family members exhibited tissue-specific expression， and 24 of them responded to exogenous methyl jasmonate. ConclusionBased on the published S. miltiorrhiza genome， 52 SmTPS gene family members were identified， and their functions were predicted based on the phylogenetic analysis and expression patterns. This paper provides reference information for the further biosynthesis pathway and regulatory mechanism analysis of terpenoids in S. miltiorrhiza.

RESUMEN

Plant terpenoid synthase (TPS) family genes participate in metabolite synthesis, hormones, gossypol, etc. Here, we genome-widely identified TPS family genes in 12 land plant species. Four hundred and thirty TPS-related genes were divided into seven subfamilies. The TPS-c in Bryophytes was suggested to be the earliest subfamily, followed by the TPS-e/f and TPS-h presence in ferns. TPS-a, the largest number of genes, was derived from monocotyledonous and dicotyledonous plants. Collinearity analysis showed that 38 out of the 76 TPS genes in G. hirsutum were collinear within G. arboreum and G. raimondii. Twenty-one GhTPS-a genes belong to the cadinene synthase (GhCDN) subfamily and were divided into five groups, A, B, C, D, and E. The special cis-elements in the promoters of 12 GhCDN-A genes suggested that the JA and ethylene signaling pathways may be involved in their expression regulation. When 12 GhCDN-A genes were simultaneously silenced through virus-induced gene silencing, the glandular color of GhCDN-A-silenced plants was lighter than that of the control, supported by a gossypol content decrease based on HPLC testing, suggesting that GhCDN-A subgroup genes participate in gossypol synthesis. According to RNA-seq analysis, gossypol synthesis-related genes and disease-resistant genes in the glandular variety exhibited upregulated expression compared to the glandless variety, whereas hormone signaling-related genes were downregulated. All in all, these results revealed plant TPS gene evolution rules and dissected the TPS subfamily, GhCDN-A, function in gossypol synthesis in cotton.

RESUMEN

Natural food preservatives are being sought extensively as a safe alternative to chemical food preservatives. This study aimed to identify potential natural preservatives from herbs using single-photon ionization time-of-flight mass spectrometry (SPI-TOF-MS). Five Artemisia species and four other herbs were analyzed, and the random forest (RF) algorithm was used to simulate olfaction and distinguish the Artemisia species by identifying the characteristic peaks of volatile terpenoids (VTPs). Results showed that the terpenoid synthase (TPS) gene family was expanded in Artemisia species, potentially contributing to the increased production of VTPs, which have potential as natural preservatives and specifically identify these species. The limits of detections (LODs) for principle VTPs in Artemisia species were as low as 22-39 parts-per-trillion-by-volume (pptv) using SPI-TOF-MS. This study highlights the potential for headspace mass spectrometry to be used in the development of natural preservatives and the identification of plant species.

RESUMEN

Sesquiterpenyl epoxy-cyclohexenoids (SECs) that depend on a polyketide synthase-terpenoid synthase (PKS-TPS) pathway are widely distributed in plant pathogenic fungi. However, the biosynthesis and function of the acetylated SECs still remained cryptic. Here, we identified that AOL_s00215g 273 (273) was responsible for the acetylation of SECs in Arthrobotrys oligospora via the construction of Δ273, in which the acetylated SECs were absent and major antibacterial nonacetylated SECs accumulated. Mutant Δ273 displayed increased trap formation, and nematicidal and antibacterial activities but decreased fungal growth and soil colonization. Glutamine, a key precursor for NH3 as a trap inducer, was highly accumulated, and biologically active phenylpropanoids and antibiotics were highly enriched in Δ273. The decreased endocytosis and increased autophagosomes, with the most upregulated genes involved in maintaining DNA and transcriptional stability and pathways related to coronavirus disease and exosome, suggested that lack of 273 might result in increased virus infection and the acetylation of SECs played a key role in fungal diverse antagonistic ability.

Asunto(s)

RESUMEN

Atractylodes macrocephala Koidz. (AMK) is widely used in traditional Chinese medicine owing to its pharmacological activity. Here, we aimed to characterize the differentially expressed genes (DEGs) of one- and three-year growth (OYG and TYG) rhizomes of AMK, combined with endophytic bacterial diversity analysis using high-throughput RNA sequencing. A total of 114 572 unigenes were annotated using six public databases. In all, 3570 DEGs revealed a clear difference, of which 936 and 2634 genes were upregulated and downregulated, respectively. The results of KEGG pathway analysis indicated that DEGs corresponding to terpenoid synthesis gene were downregulated in TYG rhizomes. In addition, 414 424 sequences corresponding to the 16S rRNA gene were divided into 1267 operational taxonomic units (OTUs). Moreover, the diversity of endophytic bacteria changed with species in the OYG (773) and TYG (1201) rhizomes at the OTU level, and Proteobacteria, Actinobacteria, and Bacteroidetes were the dominant phyla. A comparison of species differences among different growth years revealed that some species were significantly different, such as Actinomycetes, Variovorax, and Cloacibacterium. Interestingly, the decrease in the function-related metabolism of terpenoids and polyketides was correlated with the low expression of terpene synthesis genes in TYG rhizomes, as assessed using PICRUSt2. These data provide a scientific basis for elucidating the mechanisms underlying metabolite accumulation and endophytic bacterial diversity in relation to the growth years in AMK.

Asunto(s)

RESUMEN

Essential oil of Cinnamomum burmannii is rich in monoterpenes and sesquiterpenes and is widely used in cosmetics and medicines. Knowledge about the enzymes that catalyze the formation of monoterpenes and sesquiterpenes in C. burmannii is insufficient. Therefore, anatomy observation of C. burmannii at the four developmental stages (7 days, CBS1; 14 days, CBS2; 21 days, CBS3, and 28 days, CBS4) were conducted to elucidate the origins of essential oil production. Twelve full-length transcriptomes of C. burmannii leaves at the four stages were generated using Oxford Nanopore Technologies. GC-MS analysis revealed 15 monoterpene and sesquiterpenes dramatically increased from CBS1 to CBS4. A weighted correlation network analysis (WGCNA) in association and differentially expressed genes across four developmental stages were performed. A total of 44 differentially expressed genes (DEGs) were involved in terpenoid syntheses during leaf development. Among them, the DEGs of the mevalonate acid (MVA) pathway were predominantly expressed at CBS1, while those of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway showed increased expression from CBS2 to CBS4. Besides, fourteen genes were associated with monoterpene synthesis and nine with sesquiterpene synthesis. Functions of these DEGs were further predicted with regard to gene expression profile and phylogenetic relationship with those characterized in previous studies. In addition, 922 long noncoding RNAs (lncRNAs) were detected, of which twelve were predicted to regulate monoterpene and sesquiterpene biosynthesis. The present study provided new insights the molecular mechanisms of monoterpenoid and sesquiterpenoid syntheses of C. burmannii.

RESUMEN

Terpenoids are the largest class of natural products with complex structures and extensive bioactivities; their scaffolds are generated by diverse terpenoid synthases (TPSs) from a limited number of isoprenoid diphosphate precursors. Promiscuous TPSs play important roles in the evolution of terpenoid chemodiversity, but they remain largely unappreciated. Here, an extremely promiscuous terpenoid synthase (CcTPS1) of the TPS-b subfamily was cloned and functionally characterized from a leaf-specific transcriptome of the Lamiaceae plant Colquhounia coccinea var. mollis. CcTPS1 is the first sester-/di-/sesqui-/mono-TPS identified from the plant kingdom, accepting C25/C20/C15/C10 diphosphate substrates to generate a panel of sester-/di-/sesqui-/mono-terpenoids. Engineered Escherichia coli expressing CcTPS1 produced three previously unreported terpenoids (two sesterterpenoids and a diterpenoid) with rare cyclohexane-containing skeletons, along with four sesquiterpenoids and one monoterpenoid. Their structures were elucidated by extensive nuclear magnetic resonance spectroscopy. Nicotiana benthamiana transiently expressing CcTPS1 also produced the diterpenoid and sesquiterpenoids, demonstrating the enzyme's promiscuity in planta. Its highly leaf-specific expression pattern combined with detectable terpenoid products in leaves of C. coccinea var. mollis and N. benthamiana expressing CcTPS1 suggested that CcTPS1 was mainly responsible for diterpenoid and sesquiterpenoid biosynthesis in plants. CcTPS1 expression and the terpenoid products could be induced by methyl jasmonate, suggesting their possible role in plant-environment interaction. CcTPS1 was localized to the cytosol and may differ from mono-TPSs in subcellular compartmentalization and substrate tolerance. These findings will greatly aid our understanding of plant TPS evolution and terpenoid chemodiversity; they also highlight the enormous potential of transcriptome mining and heterologous expression for the exploration of unique enzymes and natural products hidden in plants.

Asunto(s)

RESUMEN

The emerging mycotoxin fusaproliferin is produced by Fusarium proliferatum and other related Fusarium species. Several fungi from other taxonomic groups were also reported to produce fusaproliferin or the deacetylated derivative, known as siccanol or terpestacin. Here, we describe the identification and functional characterization of the Fusarium proliferatum genes encoding the fusaproliferin biosynthetic enzymes: a terpenoid synthase, two cytochrome P450s, a FAD-oxidase and an acetyltransferase. With the exception of one gene encoding a CYP450 (FUP2, FPRN_05484), knock-out mutants of the candidate genes could be generated, and the production of fusaproliferin and intermediates was tested by LC-MS/MS. Inactivation of the FUP1 (FPRN_05485) terpenoid synthase gene led to complete loss of fusaproliferin production. Disruption of a putative FAD-oxidase (FUP4, FPRN_05486) did not only affect oxidation of preterpestacin III to terpestacin, but also of new side products (11-oxo-preterpstacin and terpestacin aldehyde). In the knock-out strains lacking the predicted acetyltransferase (FUP5, FPRN_05487) fusaproliferin was no longer formed, but terpestacin was found at elevated levels. A model for the biosynthesis of fusaproliferin and of novel derivatives found in mutants is presented.

Asunto(s)

RESUMEN

Species within the mint family, Lamiaceae, are widely used for their culinary, cultural, and medicinal properties due to production of a wide variety of specialized metabolites, especially terpenoids. To further our understanding of genome diversity in the Lamiaceae and to provide a resource for mining biochemical pathways, we generated high-quality genome assemblies of four economically important culinary herbs, namely, sweet basil (Ocimum basilicum L.), sweet marjoram (Origanum majorana L.), oregano (Origanum vulgare L.), and rosemary (Rosmarinus officinalis L.), and characterized their terpenoid diversity through metabolite profiling and genomic analyses. A total 25 monoterpenes and 11 sesquiterpenes were identified in leaf tissue from the 4 species. Genes encoding enzymes responsible for the biosynthesis of precursors for mono- and sesqui-terpene synthases were identified in all four species. Across all 4 species, a total of 235 terpene synthases were identified, ranging from 27 in O. majorana to 137 in the tetraploid O. basilicum. This study provides valuable resources for further investigation of the genetic basis of chemodiversity in these important culinary herbs.

Asunto(s)

RESUMEN

Cordyceps militaris, a valuable edible and medicinal fungus, has attracted increasing attention because of its various bioactive ingredients. However, the biosynthetic pathway of C. militaris carotenoids is still unknown due to lack of transcriptome information. To uncover genes related to the biosynthesis of C. militaris carotenoids, the transcriptomes of mycelia CM10_D cultured under dark conditions and mycelia CM10_L cultured under light exposure conditions were sequenced. Compared with mycelia CM10_D, 866 up-regulated genes and 856 down-regulated genes were found in mycelia CM10_L. Gene ontology (GO) analysis of differentially expressed genes (DEGs) indicated that DEGs were mainly classified into the "metabolic process," "membrane," and "catalytic activity" terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs suggested that DEGs were mainly enriched in "metabolic pathways," "MAPK signaling pathway-yeast," and "biosynthesis of secondary metabolites." In addition, the carotenoid content of the Cmtns gene deletion mutant (ΔCmtns) was significantly lower than that of the wild-type C. militaris CM10, while the carotenoid content of the complementary strain (ΔCmtns-c) of the Cmtns gene was not significantly different from that of C. militaris CM10, suggesting that the Cmtns gene significantly affected the biosynthesis of carotenoids in C. militaris. These results potentially pave the way for revealing the biosynthetic pathway of carotenoids and improving carotenoids production in C. militaris.

RESUMEN

Antodia cinnamomea, a precious brown-rot fungus endemic to Taiwan, has pharmaceutical applications due to its diverse array of metabolites. The terpenoids found in A. cinnamomea contribute to its most important bioactivities. We identified several terpenoid compounds in A. cinnamomea and revealed that their content in mycelium and fruiting body were significantly different. Using next-generation sequencing and an in-house transcriptome database, we identified several terpene synthase (TPS) candidates. After sequence analysis and functional characterization, 10 out of 12 candidates were found to have single or multiple terpene synthesis functions. Most of the terpenoid compounds were found to confer important bioactivities. RT-PCR results showed a positive correlation between terpene synthase expression pattern and terpenoid content. In addition, we identified several modification enzyme candidates that may be involved in the postmodification of terpenoid compounds with a genomic DNA scaffold, and a putative genetic network.

Asunto(s)

RESUMEN

Terpenoid fragrances are powerful mediators of ecological interactions in nature and have a long history of traditional and modern industrial applications. Plants produce a great diversity of fragrant terpenoid metabolites, which make them a superb source of biosynthetic genes and enzymes. Advances in fragrance gene discovery have enabled new approaches in synthetic biology of high-value speciality molecules toward applications in the fragrance and flavor, food and beverage, cosmetics, and other industries. Rapid developments in transcriptome and genome sequencing of nonmodel plant species have accelerated the discovery of fragrance biosynthetic pathways. In parallel, advances in metabolic engineering of microbial and plant systems have established platforms for synthetic biology applications of some of the thousands of plant genes that underlie fragrance diversity. While many fragrance molecules (eg, simple monoterpenes) are abundant in readily renewable plant materials, some highly valuable fragrant terpenoids (eg, santalols, ambroxides) are rare in nature and interesting targets for synthetic biology. As a representative example for genomics/transcriptomics enabled gene and enzyme discovery, we describe a strategy used successfully for elucidation of a complete fragrance biosynthetic pathway in sandalwood (Santalum album) and its reconstruction in yeast (Saccharomyces cerevisiae). We address questions related to the discovery of specific genes within large gene families and recovery of rare gene transcripts that are selectively expressed in recalcitrant tissues. To substantiate the validity of the approaches, we describe the combination of methods used in the gene and enzyme discovery of a cytochrome P450 in the fragrant heartwood of tropical sandalwood, responsible for the fragrance defining, final step in the biosynthesis of (Z)-santalols.

Asunto(s)

RESUMEN

Plants frequently experience heat ramps of various severities, but how and to what degree plant metabolic activity recovers from mild and severe heat stress is poorly understood. In this study, we exposed the constitutive terpene emitter, Solanum. lycopersicum leaves to mild (37 and 41 °C), moderate (46 °C) and severe (49 °C) heat ramps of 5 min. and monitored foliage photosynthetic activity, lipoxygenase pathway volatile (LOX), and mono- and sesquiterpene emissions and expression of two terpene synthase genes, ß-phellandrene synthase and (E)-ß-caryophyllene/α-humulene synthase, through a 24 h recovery period upon return to pre-stress conditions. Leaf monoterpene emissions were dominated by ß-phellandrene and sesquiterpene emissions by (E)-ß-caryophyllene, and thus, these two terpene synthase genes were representative for the two volatile terpene classes. Photosynthetic characteristics partly recovered under moderate heat stress, and very limited recovery was observed under severe stress. All stress treatments resulted in elicitation of LOX emissions that declined during recovery. Enhanced mono- and sesquiterpene emissions were observed immediately after the heat treatment, but the emissions decreased even to below the control treatment during recovery between 2-10 h, and raised again by 24 h. The expression of ß-phellandrene and (E)-ß-caryophyllene synthase genes decreased between 2-10 h after heat stress, and recovered to pre-stress level in mild heat stress treatment by 24 h. Overall, this study demonstrates a highly sensitive heat response of terpenoid synthesis that is mainly controlled by gene level responses under mild stress, while severe stress leads to non-recoverable declines in foliage physiological and gene expression activities.

RESUMEN

The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (-)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3-carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (-)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene, whereas phenylalanine promotes (-)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects.

Asunto(s)