Terpene Biosynthesis Pathway Research Articles

Exogenous glutathione (GSH/GSSG) promoted the synthesis of patchoulol and pogostone, main active components of Pogostemon cablin (Patchouli)

Glutathione redox pair (GSH/GSSG) is the main characterizing substance that regulates the redox balance in cells and affects several physiological processes, such as plant metabolism, antioxidants, and detoxification. Pogostemon cablin was treated with different concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG), and it was found that 2 mM GSH and 4 mM GSSG treatments promoted the synthesis of patchoulol and pogostone. A total of 6620 significant differentially expressed gene were identified by transcriptome analysis. KEGG analysis showed that DEGs were mainly enriched in metabolic pathways, secondary metabolite biosynthesis, carbon metabolism, and phytohormone signaling pathways; 388 DEGs in the 2 mM GSH (11d) group, and 254 DEGs in the 4 mM GSSG (11d) group, were related to the biosynthesis of secondary metabolites. Further analysis by Gene Set Enrichment Analysis (GSEA) revealed that there were 139 and 83 DEGs associated with terpene synthesis between the two treatment groups, respectively, and the expression of key genes in the synthesis pathway (11 HMGR, 4 DXR, 2 GPPS, and 8 PTS) was up-regulated, which may be the result of transcription factors regulating their expression. The present study preliminarily showed that glutathione redox pair induced the expression of genes related to the terpene biosynthesis pathway, thereby promoting the biosynthesis of patchoulol and pogostone, which provides a reference basis for the technique of regulating and controlling the content of medicinal constituents of patchouli.

Contents of paeoniflorin and albiflorin in two Korean landraces of Paeonia lactiflora and characterization of paeoniflorin biosynthesis genes in peony.

Paeoniflorin and albiflorin are monoterpene glycosides that exhibit various medicinal properties in Paeonia species. This study explored the terpene biosynthesis pathway and analyzed the distribution of these compounds in different tissues of two Korean landraces of Paeonia lactiflora to gain insights into the biosynthesis of monoterpene glycosides in P. lactiflora and their potential applications. Two Korean landraces, Hongcheon var. and Hwacheon var, of P. lactiflora were used for the analyses. Contents of the paeoniflorin and albiflorin were analyzed using HPLC. RNA was extracted, sequenced, and subjected to transcriptome analysis. Differential gene expression, KEGG, and GO analyses were performed. Paeoniflorin biosynthesis genes were isolated from the transcriptomes using the genes in Euphorbia maculata with the NBLAST program. Phylogenetic analysis of of 1-Deoxy-D-xylulose 5-phosphate synthase (DOXPS), geranyl pyrophosphate synthase (GPPS), and pinene synthase (PS) was carried out with ClustalW and MEGA v5.0. Analysis of paeoniflorin and albiflorin content in different tissues of the two P. lactiflora landraces revealed significant variation. Transcriptome analysis yielded 36,602 unigenes, most of which were involved in metabolic processes. The DEG analysis revealed tissue-specific expression patterns with correlations between landraces. The isolation of biosynthetic genes identified 173 candidates. Phylogenetic analysis of the key enzymes in these pathways provides insights into their evolutionary relationships. The sequencing and analysis of DOXPS, GPPS, PS revealed distinct clades and subclades, highlighting their evolutionary divergence and functional conservation. Our findings highlight the roots as the primary sites of paeoniflorin and albiflorin accumulation in P. lactiflora, underscoring the importance of tissue-specific gene expression in their biosynthesis. this study advances our understanding of monoterpene glycoside production and distribution in Paeonia, thereby guiding further plant biochemistry investigations.

A novel AP2/ERF transcription factor, NtERF10, positively regulates plant height in tobacco

Ethylene response factors have been shown to be involved in the effects of plant developmental processes and to regulate stress tolerance. The aim of this study was to recognize the regulatory mechanisms of ethylene response factors on tobacco plant height. In this study, a gene-edited mutant (ERF10-KO) and wild type (WT) were utilized as experimental materials. Transcriptome and metabolome analyses were used to investigate the regulatory mechanism of NtERF10 gene editing on plant height in tobacco. Here, through the analysis of differentially expressed genes (DEGs), 2051 genes were upregulated and 1965 genes were downregulated. We characterized the different ERF10-KO and WT plant heights and identified key genes for photosynthesis, the plant hormone signal transduction pathway and the terpene biosynthesis pathway. NtERF10 was found to affect the growth and development of tobacco by regulating the expression levels of the PSAA, PSBA, GLY17 and GGP3 genes. Amino acid metabolism was analyzed by combining analyses of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs). In addition, we found that members of the bHLH, NAC, MYB, and WRKY transcription factor families have vital roles in regulating plant height. This study not only provides important insights into the positive regulation of the ethylene response factor NtERF10 on plant height during plant growth and development but also provides new research ideas for tobacco molecular breeding.

Functional Characterization of PmDXR, a Critical Rate-Limiting Enzyme, for Turpentine Biosynthesis in Masson Pine (Pinus massoniana Lamb.).

As one of the largest and most diverse classes of specialized metabolites in plants, terpenoids (oprenoid compounds, a type of bio-based material) are widely used in the fields of medicine and light chemical products. They are the most important secondary metabolites in coniferous species and play an important role in the defense system of conifers. Terpene synthesis can be promoted by regulating the expressions of terpene synthase genes, and the terpene biosynthesis pathway has basically been clarified in Pinus massoniana, in which there are multiple rate-limiting enzymes and the rate-limiting steps are difficult to determine, so the terpene synthase gene regulation mechanism has become a hot spot in research. Herein, we amplified a PmDXR gene (GenBank accession no. MK969119.1) of the MEP pathway (methyl-erythritol 4-phosphate) from Pinus massoniana. The DXR enzyme activity and chlorophyll a, chlorophyll b and carotenoid contents of overexpressed Arabidopsis showed positive regulation. The PmDXR gene promoter was a tissue-specific promoter and can respond to ABA, MeJA and GA stresses to drive the expression of the GUS reporter gene in N. benthamiana. The DXR enzyme was identified as a key rate-limiting enzyme in the MEP pathway and an effective target for terpene synthesis regulation in coniferous species, which can further lay the theoretical foundation for the molecularly assisted selection of high-yielding lipid germplasm of P. massoniana, as well as provide help in the pathogenesis of pine wood nematode disease.

Endophyte-inoculated rhizomes of Paris polyphylla improve polyphyllin biosynthesis and yield: a transcriptomic analysis of the underlying mechanism.

Polyphyllin from Paris polyphylla var. yunnanensis exhibits anti-inflammatory, analgesic, antibacterial, and antiviral properties. However, the current production of polyphyllin can barely meet market demand. To improve the content of polyphyllin produced by P. polyphylla, two endophyte strains, Bacillus cereus LgD2 and Fusarium oxysporum TPB, were isolated from Paris fargesii Franch. and inoculated in the roots of P. polyphylla. Both symbiotic strains significantly promoted the accumulation of saponins in P. polyphylla. The content of polyphyllin in rhizomes of P. polyphylla treated with TPB with LgD2 strain was determined using High Performance Liquid Chromatography and the expressed genes were analyzed by RNA-seq. Gene Ontology and Kyoto Encyclopedia of Genes annotations were performed on the differentially expressed genes, a clustering tree of UDP-glycosyltransferase (UGT) and cytochrome P450 (CYP450) gene families was constructed, and UGT and CYP450 involved in the biosynthesis of polyphyllin were predicted using weighted correlation network analysis (WGCNA). RNA-seq and qRT-PCR analyses showed that endophytic inoculation did not promote polyphyllin accumulation by enhancing the upstream terpene biosynthesis pathway, but probably by up-regulating the downstream CYP450 and UGT genes associated with polyphyllin biosynthesis. Genomes enrichment analyses of differentially expressed genes indicated that inoculation with LgD2 and TPB played a positive role in promoting the defense against pathogenic bacteria, enhancing the biosynthesis of carbohydrates, attenuating the process of nitrogen metabolism, and maintaining the equilibrium of the redox reaction homeostasis, potentially indirectly enhancing the polyphyllin yield of P. polyphylla. By combining differentially expressed genes screening, WGCNA, and phylogenetic tree analyses, 17 CYP450 and 2 UGT candidate genes involved in the biosynthesis of polyphyllin I, polyphyllin II, polyphyllin VII, polyphyllin D, and polyphyllin H were identified. These results suggest that endophytes probably effectively promote the accumulation of polyphyllin by regulating key downstream genes in biosynthetic pathways. This study provides a new approach for investigating the regulatory mechanisms of endophytes that promote the production and accumulation of polyphyllin in P. polyphylla, providing a basis for further elucidating the mechanisms of plant-endophyte interactions.

Genome-Wide Identification, Characterization, and Expression Analysis of the Geranylgeranyl Pyrophosphate Synthase (GGPPS) Gene Family Reveals Its Importance in Chloroplasts of Brassica oleracea L.

GGPPS (geranylgeranyl pyrophosphate synthase) is a crucial enzyme in the terpene biosynthesis pathway. Terpenoids play essential roles in chlorophyll biosynthesis and the development of cabbage (Brassica oleracea L. var. capitata L.), a major cruciferous vegetable worldwide. However, limited information is available regarding B. oleracea GGPPS genes. In this study, we examined 10 BoGGPPS genes from the B. oleracea genome. The subcellular localization prediction suggests that BoGGPPS proteins are mainly expressed in chloroplasts and plastids. Similar BoGGPPS genes exhibited a similar structure and motif. Distribution, collinearity, and Ka/Ks analysis revealed multiple duplication events within the BoGGPPS gene family. Cabbage BoGGPPS may participate in light and hormone responses via analysis of cis-acting elements. Three-dimensional structure analysis demonstrated the abundance of α-helices and random coils among BoGGPPS members, suggesting their important functions. Based on qRT-PCR, we observed notable differences in the transcript levels of BoGGPPS genes between leaves and siliques. Bol028967 exhibited significantly higher transcript levels in WT (Wild-type) siliques compared to in Boas1 (Brassica oleracea albino silique 1), and subcellular localization analysis confirmed its expression in chloroplasts, implying its essential role in chloroplast synthesis. These findings lay the groundwork for further exploration and in-depth functional analysis of BoGGPPS genes and their relationship with terpenoids in the context of chlorophyll synthesis in B. oleracea.

Terpenoid VOC profiles and functional characterization of terpene synthases in diploid and tetraploid cytotypes of Chrysanthemum indicum L

Chrysanthemum indicum L. is a valuable medicinal plant with diploid and tetraploid forms that are widely distributed in central and southern China, and it contains abundant volatile organic compounds (VOCs). Despite the discovery of some terpene synthase (TPS) in C. indicum (i.e., CiTPS) in previous studies, many TPSs and their corresponding terpene biosynthesis pathways have yet to be discovered. In the present study, terpenoid VOCs in different tissues from two cytotypes of C. indicum were analyzed. We identified 52 types of terpenoid VOCs and systematically investigated the content and distribution of these compounds in various tissues. The two cytotypes of C. indicum exhibited different volatile terpenoid profiles. The content of monoterpenes and sesquiterpenes in the two cytotypes showed an opposite trend. In addition, four full-length candidate TPSs (named CiTPS5–8) were cloned from Ci-GD4x, and their homologous TPS genes were screened based on the genome data of Ci-HB2x. These eight TPSs displayed various tissue expression patterns and were discovered to produce 22 terpenoids, 5 of which are monoterpenes and 17 are sesquiterpenes. We further proposed corresponding terpene synthesis pathways, which can enable the establishment of an understanding of the volatile terpenoid profiles of C. indicum with different cytotypes. This knowledge may provide a further understanding of germplasm in C. indicum and may be useful for biotechnology applications of Chrysanthemum plants.

Genome-wide identification of the geranylgeranyl pyrophosphate synthase (GGPS) gene family involved in chlorophyll synthesis in cotton

BackgroundGeranylgeranyl pyrophosphate synthase (GGPS) is a structural enzyme of the terpene biosynthesis pathway that is involved in regulating plant photosynthesis, growth and development, but this gene family has not been systematically studied in cotton.ResultsIn the current research, genome-wide identification was performed, and a total of 75 GGPS family members were found in four cotton species, Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii. The GGPS genes were divided into three subgroups by evolutionary analysis. Subcellular localization prediction showed that they were mainly located in chloroplasts and plastids. The closely related GGPS contains a similar gene structure and conserved motif, but some genes are quite different, resulting in functional differentiation. Chromosome location analysis, collinearity and selection pressure analysis showed that many fragment duplication events occurred in GGPS genes. Three-dimensional structure analysis and conservative sequence analysis showed that the members of the GGPS family contained a large number of α-helices and random crimps, and all contained two aspartic acid-rich domains, DDxxxxD and DDxxD (x is an arbitrary amino acid), suggesting its key role in function. Cis-regulatory element analysis showed that cotton GGPS may be involved in light response, abiotic stress and other processes. A GGPS gene was silenced successfully by virus-induced gene silencing (VIGS), and it was found that the chlorophyll content in cotton leaves decreased significantly, suggesting that the gene plays an important role in plant photosynthesis.ConclusionsIn total, 75 genes were identified in four Gossypium species by a series of bioinformatics analysis. Gene silencing from GGPS members of G. hirsutum revealed that GGPS plays an important regulatory role in photosynthesis. This study provides a theoretical basis for the biological function of GGPS in cotton growth and development.

LcERF134 increases the production of monoterpenes by activating the terpene biosynthesis pathway in Litsea cubeba

Litsea cubeba, an aromatic species of the Lauraceae family, produces a diverse array of monoterpenes. The biosynthesis of monoterpenes is regulated by transcriptional factors (TFs), such as APETALA2/ethylene response factor (AP2/ERF). However, the regulatory mechanisms that control the AP2/ERF gene responsible for the biosynthesis of monoterpenes in L. cubeba have yet to be elucidated. Here, we identified an AP2/ERF gene, LcERF134, as an activator for the accumulation of citral and other monoterpenes. The expression level of LcERF134 was consistent with terpene synthase LcTPS42 in the pericarp. The transient overexpression of LcERF134 significantly increased monoterpene production in L. cubeba as well as the expression of rate-limiting genes involved in the monoterpene biosynthesis pathway. Furthermore, yeast one-hybrid, dual-luciferase and electrophoretic mobility shift assays demonstrated that LcERF134 activated the monoterpene biosynthesis pathway by directly binding to the GCC-box elements of the LcTPS42 and LcGPPS.SSU1 promoters. However, the overexpression of LcERF134 in tomatoes had no impact on the synthesis of monoterpenes, thus indicating that LcERF134 is a species-specific TF. Our research demonstrated that LcERF134 significantly increased the biosynthesis of monoterpenes by inducing the expression of LcTPS42 and LcGPPS.SSU1, thus offering insight into how to enhance the flavor of L. cubeba essential oil.

Transcriptome Analyses Revealed the Key Metabolic Genes and Transcription Factors Involved in Terpenoid Biosynthesis in Sacred Lotus.

As the largest group of structurally diverse metabolites, terpenoids are versatile natural compounds that act as metabolism mediators, plant volatiles, and ecological communicators. However, few terpenoid compounds have been identified in plant parts of sacred lotus (Nelumbo nucifera Gaertn.). To elucidate the molecular genetic basis of the terpene biosynthetic pathway, terpenes from different parts of the plant, including seeds (S), young leaves (YL), mature leaves (ML), white flowers (WF), yellow flowers (YF), and red flowers (RF), were identified by LC-MS/MS and the relative contents of the same terpenes in different parts were compared. The results indicate that all plant parts primarily consist of triterpenes, with only minor quantities of sesquiterpenes and diterpenes, and there were differences in the terpene content detected in different plant parts. To illustrate the biosynthesis of various terpenoids, RNA sequencing was performed to profile the transcriptomes of various plant parts, which generated a total of 126.95 GB clean data and assembled into 29,630 unigenes. Among these unigenes, 105 candidate unigenes are involved in the mevalonate (MVA) pathway, methyl-erythritol phosphate (MEP) pathway, terpenoid backbone biosynthesis pathway, and terpenoid synthases pathway. Moreover, the co-expression network between terpene synthase (TPS) and WRKY transcription factors provides new information for the terpene biosynthesis pathway.

Advances in Metabolic Engineering Paving the Way for the Efficient Biosynthesis of Terpenes in Yeasts.

Terpenes are a large class of secondary metabolites with diverse structures and functions that are commonly used as valuable raw materials in food, cosmetics, and medicine. With the development of metabolic engineering and emerging synthetic biology tools, these important terpene compounds can be sustainably produced using different microbial chassis. Currently, yeasts such as Saccharomyces cerevisiae and Yarrowia lipolytica have received extensive attention as potential hosts for the production of terpenes due to their clear genetic background and endogenous mevalonate pathway. In this review, we summarize the natural terpene biosynthesis pathways and various engineering strategies, including enzyme engineering, pathway engineering, and cellular engineering, to further improve the terpene productivity and strain stability in these two widely used yeasts. In addition, the future prospects of yeast-based terpene production are discussed in light of the current progress, challenges, and trends in this field. Finally, guidelines for future studies are also emphasized.

Transcriptome analysis reveals regulation mechanism of methyl jasmonate-induced terpenes biosynthesis in Curcuma wenyujin.

Curcuma wenyujin is the source plant of three traditional Chinese medicines, which have been widely used in clinical treatment over 1000 years. The content of terpenes, the major medicinal active ingredients, is relatively low in this plant. Studies have shown that MeJA can promote terpenes biosynthesis in plants. However, the mechanism underlying the effect of MeJA in C. wenyujin remains unclear. In this work, the transcriptome of C. wenyujin leaves with MeJA treatment was analyzed to elucidate the regulation mechanism of MeJA-mediated terpene biosynthesis. Based on the RNA-seq data, 7,246 unigenes were differentially expressed with MeJA treatment. Expression pattern clustering of DEGs revealed that unigenes, related to JA biosynthesis and signal transduction, responded to exogenous MeJA stimulation on the early stage and maintained throughout the process. Subsequently, unigenes related to terpene biosynthesis pathway showed a significant up-regulation with 6 h treatment. The analysis results suggested that MeJA induced the expression of JA biosynthesis genes (such as LOXs, AOSs, AOCs, OPRs, and MFPs) and JA signal transduction core genes (JAZs and MYCs) to activate JA signaling pathway. Meanwhile, downstream JA-responsive genes presented up-regulated expression levels such as AACT, HMGSs, HMGRs, DXSs, DXRs, MCTs, HDSs, and HDRs, thus promoting terpenes biosynthesis. The transcriptional expressions of these genes were validated by qRT-PCR. In addition, six CwTPS genes in response to MeJA were identified. With MeJA treatment, the expression levels of CwTPSs were increased as well as those of the transcription factors MYB, NAC, bZIP, WRKY, AP2/ERF, and HLH. These TFs might potentially regulate terpenes biosynthesis. These results provide insights for regulation mechanism of terpenes biosynthesis.

Expression of the Thaumatin-Like Protein-1 Gene (Bx-tlp-1) from Pine Wood Nematode Bursaphelenchus xylophilus Affects Terpene Metabolism in Pine Trees.

Pine wilt disease is a major forest disease worldwide, including in China, where it has severely damaged pine forest ecosystems, and the pathogen is pine wood nematode (Bursaphelenchus xylophilus). The thaumatin-like protein-1 gene (Bx-tlp-1) is a key gene associated with B. xylophilus pathogenicity, which is also responsive to α-pinene. In this study, an examination of Pinus massoniana seedlings infected by B. xylophilus revealed that monoterpene (sesquiterpene) levels peaked on days 15 and 27 (days 18 and 27). Meanwhile, P. massoniana Pm-tlp expression levels were high on days 3, 12, and 27, which were consistent with the expression of key enzymes genes in the terpene biosynthesis pathway. The functional similarity of B. xylophilus Bx-TLP-1 and P. massoniana Pm-TLP suggests Bx-TLP-1 and Pm-TLP may have similar roles in P. massoniana. There was also no secondary accumulation of terpenes in P. massoniana seedlings during B. xylophilus treated with dsRNA targeting Bx-tlp-1 (dsTLP1) infections, reflecting the decreased pathogenicity of B. xylophilus and the delayed disease progression in pine trees. And the results of micro-CT showed that the degree of cavitation for the trees inoculated with Bx-TLP-1 (0.3811 mm3) was greater than that for the trees inoculated with dsTLP1 PWNs (0.1204 mm3) on day 15 after inoculation. Results from this study indicated that B. xylophilus Bx-tlp-1 gene may induce the upregulated expression of related genes encoding enzymes in the terpene synthesis pathway of P. massoniana, resulting in the accumulation of terpenes, which also provided an insight to investigate the B. xylophilus pathogenicity in the future.

Elucidation of the essential oil biosynthetic pathways in Cinnamomum burmannii through identification of six terpene synthases

Cinnamomum burmannii is a traditional plant that has long been used as a spice, food preservative, and food flavoring. Essential oils in C. burmannii, which mainly consist of mono- and sesquiterpenes such borneol, linalool, and caryophyllene, have impressive pharmaceutical properties. Although the transcriptome-based discovery of (+)-bornyl diphosphate synthase (CbTPS1) from C. burmannii was reported in our previous study, the remaining terpene synthases (TPSs) corresponding to various terpene biosynthesis pathways remain unidentified. In this study, we report the results of RNA-sequencing of a borneol type plant and functional characterization of six additional full-length candidate TPS genes (named CbTPS2–7). Phylogenetic analysis revealed that CbTPS2 and CbTPS3 together with the previously identified CbTPS1 protein belong to the TPS-b subfamily, and enzyme assays using geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as substrates revealed that CbTPS1, CbTPS2 and CbTPS3 catalyze the formation of monoterpenes. CbTPS4, CbTPS5, and CbTPS6, which belong to the TPS-a clade, generated monoterpenes and sesquiterpenes. CbTPS7, which belongs to the TPS-g clade, showed linalool/nerolidol synthase activity. These CbTPSs identified in C. burmannii produced a total of 10 monoterpenes and 14 sesquiterpenes in an in vitro assay. These findings clarify the biosynthesis pathways of 13 monoterpenoids and 12 sesquiterpenoids in the leaf essential oil of C. burmannii and shed light on terpene biosynthesis in Cinnamomum.

Heterologous Production of Plant Terpenes in the Photosynthetic Bacterium Rhodobacter capsulatus.

Terpenes are one of the largest classes of secondary metabolites that occur in all kingdoms of life and offer diverse valuable properties for food and pharma industry including pleasant odor or taste as well as antimicrobial or anticancer activities. A multitude of terpene biosynthesis pathways are known, but their efficient biotechnological exploitation requires an adequate microorganism as host which can ideally provide an optimal supply with biosynthetic isoprene precursors. Rhodobacter capsulatus, a Gram-negative, facultative anaerobic, photosynthetic non-sulfur purple bacterium belonging to the α-proteobacteria represents such a host particularly suitable for terpene production. Here, we describe methods for the expression of terpene biosynthetic enzymes in R. capsulatus and the extraction of products for analysis. At the same time, we summarize the current strategies to adjust the biosynthetic precursor supply via isoprenoid biosynthetic pathways.

Fungal genomes: suffering with functional annotation errors

BackgroundThe genome sequence data of more than 65985 species are publicly available as of October 2021 within the National Center for Biotechnology Information (NCBI) database alone and additional genome sequences are available in other databases and also continue to accumulate at a rapid pace. However, an error-free functional annotation of these genome is essential for the research communities to fully utilize these data in an optimum and efficient manner.ResultsAn analysis of proteome sequence data of 689 fungal species (7.15 million protein sequences) was conducted to identify the presence of functional annotation errors. Proteins associated with calcium signaling events, including calcium dependent protein kinases (CDPKs), calmodulins (CaM), calmodulin-like (CML) proteins, WRKY transcription factors, selenoproteins, and proteins associated with the terpene biosynthesis pathway, were targeted in the analysis. Gene associated with CDPKs and selenoproteins are known to be absent in fungal genomes. Our analysis, however, revealed the presence of proteins that were functionally annotated as CDPK proteins. However, InterproScan analysis indicated that none of the protein sequences annotated as “calcium dependent protein kinase” were found to encode calcium binding EF-hands at the regulatory domain. Similarly, none of a protein sequences annotated as a “selenocysteine” were found to contain a Sec (U) amino acid. Proteins annotated as CaM and CMLs also had significant discrepancies. CaM proteins should contain four calcium binding EF-hands, however, a range of 2–4 calcium binding EF-hands were present in the fungal proteins that were annotated as CaM proteins. Similarly, CMLs should possess four calcium binding EF-hands, but some of the CML annotated fungal proteins possessed either three or four calcium binding EF-hands. WRKY transcription factors are characterized by the presence of a WRKY domain and are confined to the plant kingdom. Several fungal proteins, however, were annotated as WRKY transcription factors, even though they did not contain a WRKY domain.ConclusionThe presence of functional annotation errors in fungal genome and proteome databases is of considerable concern and needs to be addressed in a timely manner.

Transcriptome sequencing reveals terpene biosynthesis pathway genes accounting for volatile terpene of tree peony.

Transcriptomic and volatile component analyses showed that high expression levels of genes from the terpenoid backbone biosynthesis pathway and the monoterpene metabolic pathway can strengthen the floral fragrance of tree peony. Floral fragrance is a crucial ornamental trait whose improvement is one of the primary objectives of tree peony breeding. So far, exploration of the floral fragrance of tree peony has focused on the identification of its volatile components, but the molecular mechanisms responsible for their formation remain unclear. Here, we identified 128 volatile components from the petals of tree peony and found that they consisted primarily of terpenes, alcohols, and esters. Based on the distribution pattern of these major fragrance components, 24 tree peony cultivars were classified into 4 types: grassy scent (ocimene), woody scent (longifolene), lily of the valley scent (linalool), and fruity scent (2-ethyl hexanol). We used RNA-seq to explore the mechanistic basis of terpenoid metabolism in tree peony petals with various scents. The expression levels of AACT, HMGR, PMK, DXS, DXR, HDS, HDR, and GGPS, which encode key enzymes of terpenoid backbone biosynthesis, were upregulated in 'Huangguan' (strong fragrance) compared to 'Fengdan' (faint fragrance). Moreover, the transcript abundance of LIS and MYS, two monoterpene synthase genes, was also enhanced in petals of 'Huangguan' compared to those of 'Fengdan'. Together, these results demonstrate that differences in the expression of genes from the monoterpene synthesis and terpenoid backbone pathways are associated with differences in the fragrance of tree peony. This research provides crucial genetic resources for fragrance improvement and also lays a foundation for further clarification of the mechanisms that underlie tree peony fragrance.

Effect of Thidiazuron on Terpene Volatile Constituents and Terpenoid Biosynthesis Pathway Gene Expression of Shine Muscat (Vitis labrusca × V. vinifera) Grape Berries.

Volatile compounds are considered to be essential for the flavor and aroma quality of grapes. Thidiazuron (TDZ) is a commonly used growth regulator in grape cultivation that stimulates larger berries and prevents fruit drop. This study was conducted to investigate the effect of TDZ on the production of aroma volatiles and to identify the key genes involved in the terpene biosynthesis pathways that are affected by this compound. Treatment with TDZ had a negative effect on the concentration of volatile compounds, especially on monoterpenes, which likely impacts the sensory characteristics of the fruit. The expression analysis of genes related to the monoterpenoid biosynthesis pathways confirmed that treatment with TDZ negatively regulated the key genes DXS1, DXS3, DXR, HDR, VvPNGer and VvPNlinNer1. Specifically, the expression levels of the aforementioned genes were down-regulated in almost all berry development stages in the TDZ-treated samples. The novel results from the present study can be used to aid in the development of food products which maintain the flavor quality and sensory characteristics of grape. Furthermore, these findings can provide the theoretical basis that can help to optimize the utilization of TDZ for the field production of grapes at a commercial scale.

Phase-Separated Multienzyme Biosynthesis.

Liquid-liquid phase separation forms condensates that feature a highly concentrated liquid phase, a defined yet dynamic boundary, and dynamic exchange at and across the boundary. Phase transition drives the formation of dynamic multienzyme complexes in cells, for example, the purinosome, which forms subcellular macrobodies responsible for de novo purine biosynthesis. Here, we construct synthetic versions of multienzyme biosynthetic systems by assembling enzymes in protein condensates. A synthetic protein phase separation system using component proteins from postsynaptic density in neuronal synapses, GKAP, Shank, and Homer provides the scaffold for assembly. Three sets of guest proteins: a pair of fluorescent proteins (CFP and YFP), three sequential enzymes in menaquinone biosynthesis pathway (MenF, MenD, and MenH), and two enzymes in terpene biosynthesis pathway (Idi and IspA) are assembled via peptide-peptide interactions in the condensate. First, we discover that coassembly of CFP and YFP exhibited a broad distribution of the FRET signal within the condensate. Second, a spontaneous enrichment of the rate-limiting enzyme MenD in the condensate is sufficient to increase the 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate production rate by 70%. Third, coassembly of both Idi and IspA in the protein condensate increases the farnesyl pyrophosphate production rate by more than 50%. Altogether, we show here that phase separation significantly accelerates the efficiency of multienzyme biocatalysis.

Expanding the Isoprenoid Building Block Repertoire with an IPP Methyltransferase from Streptomyces monomycini

Many synthetic biology approaches aim at expanding the product diversity of enzymes or whole biosynthetic pathways. However, the chemical structure space of natural product forming routes is often restricted by the limited cellular availability of different starting intermediates. Although the terpene biosynthesis pathways are highly modular, their starting intermediates are almost exclusively the C5 units IPP and DMAPP. To amplify the possibilities of terpene biosynthesis through the modification of its building blocks, we identified and characterized a SAM-dependent methyltransferase converting IPP into a variety of C6 and C7 prenyl pyrophosphates. Heterologous expression in Escherichia coli not only extended the intracellular prenyl pyrophosphate spectrum with mono- or dimethylated IPP and DMAPP, but also enabled the biosynthesis of C11, C12, C16, and C17 prenyl pyrophosphates. We furthermore demonstrated the general high promiscuity of terpenoid biosynthesis pathways toward uncommon building blocks by the E. coli-based production of polymethylated C41, C42, and C43 carotenoids. Integration of the IPP methyltransferase in terpene synthesis pathways enables an expansion of the terpenoid structure space beyond the borders predetermined by the isoprene rule which indicates a restricted synthesis by condensation of C5 units.