Terpenoid Synthase Research Articles

Light-responsive transcription factors VvHYH and VvGATA24 mediate wax terpenoid biosynthesis in Vitis vinifera.

The cuticular wax that covers the surfaces of plants is the first barrier against environmental stresses and increasingly accumulates with light exposure. However, the molecular basis of light-responsive wax biosynthesis remains elusive. In grape (Vitis vinifera), light exposure resulted in higher wax terpenoid content and lower decay and abscission rates than controls kept in darkness. Assay for transposase-accessible chromatin with high-throughput sequencing and RNA-seq data were integrated to draw the chromatin accessibility and cis-elements regulatory map to identify the potential action sites. Terpenoid synthase 12 (VvTPS12) and 3-hydroxy-3-methylglutaryl-CoA reductase 2 (VvHMGR2) were identified as grape wax biosynthesis targets, while VvHYH and VvGATA24 were identified as terpenoid biosynthesis activators, as more abundant wax crystals and higher wax terpenoid content were observed in transiently overexpressed grape berries and Nicotiana benthamiana leaves. The interaction between VvHYH and the open chromatin of VvTPS12 was confirmed qualitatively using a dual luciferase assay and quantitatively using surface plasma resonance, with an equilibrium dissociation constant of 2.81 nm identified via the latter approach. Molecular docking simulation implied the structural nature of this interaction, indicating that 24 amino acid residues of VvHYH, including Arg106A, could bind to the VvTPS12 G-box cis-element. VvGATA24 directly bound to the open chromatin of VvHMGR2, with an equilibrium dissociation constant of 8.59 nm. Twelve amino acid residues of VvGATA24, including Pro218B, interacted with the VvHMGR2 GATA-box cis-element. Our work characterizes the mechanism underlying light-mediated wax terpenoid biosynthesis and provides gene targets for future molecular breeding.

Molecular Cloning and Expression Analysis of Geranyllinalool Synthase Gene (SgGES) from Salvia guaranitica Plants

Salvia guaranitica is considered one of the most significant medicinal and aromatic herbs in terms of nutritional and medical benefits due to its wealth of important active components. Among these compounds, terpenoids are the most prominent and abundant, particularly monoterpenes (C10), sesquiterpenes (C15), and diterpenes (C20). They are biologically advantageous to plants and perform a multitude of functions. The current study aimed to clone the S. guaranitica gene that encodes for geranyllinalool synthases (SgGES, EC: 4.2.3.144), with consideration for these features. The open reading frame of the 867-amino-acid protein encoded by SgGES consists of 2.721 base pairs. In addition, the SgGES protein has five domains that belong to the terpene synthase family, which are related to the terpene and terpenoid synthase domains. We manipulated and overexpressed the SgGES gene in Nicotiana tabacum to explore its function. When compared to the GUS control, the transgenic N. tabacum plants displayed an increase in leaf production and diameter when compared with the wild-type plants. Finally, analysis of transgenic plants using gas chromatography/mass spectrometry (GC-MS) showed that SgGES is responsible for producing various terpene species, especially diterpenes.

Endophytic bacteria with host-supportive genetic determinants in their genomes induce growth and antioxidant activity related gene functions in transcriptome of black rice (Oryza sativa L.)

Black rice is famed because of its high antioxidants content. The cooperation and contribution of endophytes towards growth and antioxidant properties of black rice has been experimented. The efficient endophytes were isolated from black rice, and Enterobacter sp., Kluyvera sp. and Bacillus subtilis were selected. The whole genome of these endophytes contained genes required for IAA production (trpAB.Z), phosphate solubilization (pqqAB), siderophore production (entABCD), colonization (fliAB.Z; cheAB.Z) and antioxidant activity (bglAB), which were further validated through biochemical and physiological analysis. The inoculation of these three effective endophytes resulted in enhancement of growth by 33.33%, 30.23% and 33.33% and antioxidant properties by 31.57%, 35.33% and 27.87% in Enterobacter, Kluyvera and B. subtilis treated plants respectively. The transcriptome of black rice plant, when colonized by these endophytes, resulted in 506 up-regulated and 332 down-regulated genes. Transcripts upregulated in response to endophytic colonization were mainly involved in plant growth, antioxidant properties, interactions, and synthesis of aroma compounds (terpenoid synthase). The overall patterns of functions encoded on the genome of endophytes and expressed in black rice plants were highly correlated. The strain specific augmentation through endophytism was used to achieve the desired properties in black rice.

Systematic identification of chemical forms of key terpene synthase in Cinnamomum camphora

Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.

Genome-wide identification of terpenoid synthase family genes in Gossypium hirsutum and functional dissection of its subfamily cadinene synthase A in gossypol synthesis.

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.

Rapid Detection of Potential Natural Food Preservatives and Identification of Artemisia Species via High-Sensitivity Photoionization Mass Spectrometry.

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.

Genome assembly of Musa beccarii shows extensive chromosomal rearrangements and genome expansion during evolution of Musaceae genomes.

Musa beccarii (Musaceae) is a banana species native to Borneo, sometimes grown as an ornamental plant. The basic chromosome number of Musa species is x = 7, 10, or 11; however, M. beccarii has a basic chromosome number of x = 9 (2n = 2x = 18), which is the same basic chromosome number of species in the sister genera Ensete and Musella. Musa beccarii is in the section Callimusa, which is sister to the section Musa. We generated a high-quality chromosome-scale genome assembly of M. beccarii to better understand the evolution and diversity of genomes within the family Musaceae. The M. beccarii genome was assembled by long-read and Hi-C sequencing, and genes were annotated using both long Iso-seq and short RNA-seq reads. The size of M. beccarii was the largest among all known Musaceae assemblies (∼570 Mbp) due to the expansion of transposable elements and increased 45S ribosomal DNA sites. By synteny analysis, we detected extensive genome-wide chromosome fusions and fissions between M. beccarii and the other Musa and Ensete species, far beyond those expected from differences in chromosome number. Within Musaceae, M. beccarii showed a reduced number of terpenoid synthase genes, which are related to chemical defense, and enrichment in lipid metabolism genes linked to the physical defense of the cell wall. Furthermore, type III polyketide synthase was the most abundant biosynthetic gene cluster (BGC) in M. beccarii. BGCs were not conserved in Musaceae genomes. The genome assembly of M. beccarii is the first chromosome-scale genome assembly in the Callimusa section in Musa, which provides an important genetic resource that aids our understanding of the evolution of Musaceae genomes and enhances our knowledge of the pangenome.

Expression of terpene synthase-related genes in parents and offspring of Flammulina filiformis based on differences in volatile aroma components

Flammulina filiformis (F. filiformis) is one of the four major edible types of fungus in the world and has been cultivated in China since 800 CE (Anno Domini). Some of the most essential criteria for evaluating the quality of F. filiformis are the types and contents of volatile components present. A focused study on screened the terpene synthase genes involved in the aroma of offspring and compared key terpenoids between parents and offspring, which is helpful for the development and application of F. filiformis. Firstly, the volatile aroma components of parent and offspring F. filiformis were extracted using two pretreatment procedures, and then were semi-quantified by an internal standard. Forty-eight, fifty-eight, and forty-eight volatile compounds were identified in parents and offspring of three different strains, and 15, 22, and 12 aroma compounds (OAVs ≥ 1) were further screened out via calculating their odor activity values (OAVs). Terpenoids, in particular linalool and eucalyptol, which contribute more to the aroma, result in the unique green and grassy aroma of the offspring. At last, the F. filiformis genome was resequenced and the coordinates of genes related to terpenoid synthase were determined. The results showed that Scaffolds, including scaffold3.t874 and scaffold9.t157 were connected to terpenoid synthesis of offspring (No. 61523). The variant genes g269 and g61 were related to terpenoid synthase sequences. This study provides a theoretical foundation for the cultivation of more diverse and unique varieties of F. filiformis.

Full-Length Transcriptome Sequencing Combined with RNA-Seq to Analyze Genes Related to Terpenoid Biosynthesis in Cinnamomum burmannii.

Cinnamomum burmannii is a cinnamomum plant rich in natural D-borneol. Natural D-borneol is a bicycle monoterpenoid compound widely used in the food, pharmaceutical, and cosmetic industries. Therefore, analyzing the biosynthesis mechanism of natural D-borneol in C. burmannii at the molecular level is helpful for directional breeding in the future and further development and utilization of C. burmannii and its related gene resources. In our study, 76 genes related to terpene metabolism were analyzed through third-generation sequencing and second-generation sequencing. Of these genes, 57 were associated with the synthesis of the terpenoid skeleton, and 19 belonged to terpenoid synthase, including four monoterpenoid synthases, seven sesquiterpenoid synthases, and eight diterpenoid synthases. Two genes in diterpenoid synthase were differentially expressed in high D-borneol and low D-borneol plants. It was speculated that these two genes might be related to D-borneol synthesis. How these two genes participate in the synthesis of D-borneol needs further study.

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 Biosynthesis and Pharmacological Effects of Cinnamomum camphora (L.) Presl Essential Oil

Cinnamomum camphora (L.) Presl essential oil (CCEO) is a volatile oil with aroma and is extracted from various tissues of Cinnamomumcamphora. It is traditionally used as a spice, preservative, as an anti-inflammatory and for sterilization. Terpenoids are the main active components in CCEO. Based on currently available research, considerable effort is still needed to study the biosynthesis and regulation of terpenoids in CCEO. In this review, the research progress related to terpenoid biosynthesis and bioactivity in CCEO in recent years is summarized, with the data compiled and presented mainly from online resources such as PubMed, Scopus and CNKI in China up to May 2022. The research advances related to key enzymes in the terpenoid biosynthesis pathway are mainly discussed. Previous studies have isolated some genes encoding key enzymes involved in terpenoid biosynthesis; however, among these genes, only a few TPS genes have been verified to catalyze the production of terpenoid synthases at the protein level. Most genes encoding key enzymes have been cloned and isolated, but no transgenic experiments have been carried out to verify gene function. In-depth study of the biosynthesis of terpenoids in CCEO may contribute to a better understanding of the differential accumulation of terpenoids in different types of C. camphora and provide reference for improving terpenoid content in CCEO.

Characterization of γ-Cadinene Enzymes in Ganoderma lucidum and Ganoderma sinensis from Basidiomycetes Provides Insight into the Identification of Terpenoid Synthases.

Enzymes boost protein engineering, directed evolution, and the biochemical industry and are also the cornerstone of metabolic engineering. Basidiomycetes are known to produce a large variety of terpenoids with unique structures. However, basidiomycetous terpene synthases remain largely untapped. Therefore, we provide a modeling method to obtain specific terpene synthases. Aided by bioinformatics analysis, three γ-cadinene enzymes from Ganoderma lucidum and Ganoderma sinensis were accurately predicted and identified experimentally. Based on the highly conserved amino motifs of the characterized γ-cadinene enzymes, the enzyme was reassembled as model 1. Using this model as a template, 67 homologous sequences of the γ-cadinene enzyme were screened from the National Center for Biotechnology Information (NCBI). According to the 67 sequences, the same gene structure, and similar conserved motifs to model 1, the γ-cadinene enzyme model was further improved by the same construction method and renamed as model 2. The results of bioinformatics analysis show that the conservative regions of models 1 and 2 are highly similar. In addition, five of these sequences were verified, 100% of which were γ-cadinene enzymes. The accuracy of the prediction ability of the γ-cadinene enzyme model was proven. In the same way, we also reanalyzed the identified Δ6-protoilludene enzymes in fungi and (−)-α-bisabolol enzymes in plants, all of which have their own unique conserved motifs. Our research method is expected to be used to study other terpenoid synthases with a similar or the same function in basidiomycetes, ascomycetes, bacteria, and plants and to provide rich enzyme resources.

Alternative metabolic pathways and strategies to high-titre terpenoid production in Escherichia coli.

Covering: up to 2021Terpenoids are a diverse group of chemicals used in a wide range of industries. Microbial terpenoid production has the potential to displace traditional manufacturing of these compounds with renewable processes, but further titre improvements are needed to reach cost competitiveness. This review discusses strategies to increase terpenoid titres in Escherichia coli with a focus on alternative metabolic pathways. Alternative pathways can lead to improved titres by providing higher orthogonality to native metabolism that redirects carbon flux, by avoiding toxic intermediates, by bypassing highly-regulated or bottleneck steps, or by being shorter and thus more efficient and easier to manipulate. The canonical 2-C-methyl-d-erythritol 4-phosphate (MEP) and mevalonate (MVA) pathways are engineered to increase titres, sometimes using homologs from different species to address bottlenecks. Further, alternative terpenoid pathways, including additional entry points into the MEP and MVA pathways, archaeal MVA pathways, and new artificial pathways provide new tools to increase titres. Prenyl diphosphate synthases elongate terpenoid chains, and alternative homologs create orthogonal pathways and increase product diversity. Alternative sources of terpenoid synthases and modifying enzymes can also be better suited for E. coli expression. Mining the growing number of bacterial genomes for new bacterial terpenoid synthases and modifying enzymes identifies enzymes that outperform eukaryotic ones and expand microbial terpenoid production diversity. Terpenoid removal from cells is also crucial in production, and so terpenoid recovery and approaches to handle end-product toxicity increase titres. Combined, these strategies are contributing to current efforts to increase microbial terpenoid production towards commercial feasibility.

An extremely promiscuous terpenoid synthase from the Lamiaceae plant Colquhounia coccinea var. mollis catalyzes the formation of sester-/di-/sesqui-/mono-terpenoids

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.

A Comparison of Two Monoterpenoid Synthases Reveals Molecular Mechanisms Associated With the Difference of Bioactive Monoterpenoids Between Amomum villosum and Amomum longiligulare.

The fruits of Amomum villosum and Amomum longiligulare are both used medicinally as Fructus Amomi the famous traditional Chinese medicine, however, the medicinal quality of A. villosum is better than that of A. longiligulare. Volatile terpenoids in the seeds, especially bornyl acetate and borneol, are the medicinal components of Fructus Amomi. The volatile terpenoids and transcriptome of developing seeds of A. villosum and A. longiligulare were compared in this study. The result revealed that the bornyl acetate and borneol contents were higher in A. villosum than in A. longiligulare. Additionally, six terpenoid synthase genes (AlTPS1–AlTPS6) were screened from the transcriptome of A. longiligulare, and AlTPS2 and AlTPS3 were found to share 98 and 83% identity with AvTPS2 and AvBPPS (bornyl diphosphate synthase) from A. villosum, respectively. BPPS is the key enzyme for the biosynthesis of borneol and bornyl acetate. Biochemical assays improved that AlTPS2 had an identical function to AvTPS2 as linalool synthase; however, AlTPS3 produced camphene as the major product and bornyl diphosphate (BPP) as the secondary product, whereas AvBPPS produced BPP as its major product. There was only one different amino acid between AlTPS3 (A496) and AvBPPS (G495) in their conserved motifs, and the site-directed mutation of A496G in DTE motif of AlTPS3 changed the major product from camphene to BPP. Molecular docking suggests that A496G mutation narrows the camphene-binding pocket and decreases the BPP-binding energy, thus increases the product BPP selectivity of enzyme. In addition, the expression level of AvBPPS was significantly higher than that of AlTPS3 in seeds, which was consistent with the related-metabolites contents. This study provides insight into the TPS-related molecular bases for the biosynthesis and accumulation differences of the bioactive terpenoids between A. villosum and A. longiligulare. BPPS, the key gene involved in the biosynthesis of the active compound, was identified as a target gene that could be applied for the quality-related identification and breeding of Fructus Amomi.

Assembly-Line Catalysis in Bifunctional Terpene Synthases.

The magnificent chemodiversity of more than 95 000 terpenoid natural products identified to date largely originates from catalysis by two types of terpene synthases, prenyltransferases and cyclases. Prenyltransferases utilize 5-carbon building blocks in processive chain elongation reactions to generate linear C5n isoprenoid diphosphates (n ≥ 2), which in turn serve as substrates for terpene cyclases that convert these linear precursors into structurally complex hydrocarbon products containing multiple rings and stereocenters. Terpene cyclization reactions are the most complex organic transformations found in nature in that more than half of the substrate carbon atoms undergo changes in chemical bonding during a multistep reaction sequence proceeding through several carbocation intermediates. Two general classes of cyclases are established on the basis of the chemistry of initial carbocation formation, and structural studies from our laboratory and others show that three fundamental protein folds designated α, β, and γ govern this chemistry. Catalysis by a class I cyclase occurs in an α domain, where a trinuclear metal cluster activates the substrate diphosphate leaving group to generate an allylic cation. Catalysis by a class II cyclase occurs in a β domain or at the interface of β and γ domains, where an aspartic acid protonates the terminal π bond of the substrate to yield a tertiary carbocation. Crystal structures reveal domain architectures of α, αβ, αβγ, βγ, and β.In some terpene synthases, these domains are combined to yield bifunctional enzymes that catalyze successive biosynthetic steps in assembly line fashion. Structurally characterized examples include bacterial geosmin synthase, an αα domain enzyme that catalyzes a class I cyclization reaction of C15 farnesyl diphosphate in one active site and a transannulation-fragmentation reaction in the other to yield C12 geosmin and C3 acetone products. In comparison, plant abietadiene synthase is an αβγ domain enzyme in which C20 geranylgeranyl diphosphate undergoes tandem class II-class I cyclization reactions to yield the tricyclic product. Recent structural studies from our laboratory show that bifunctional fungal cyclases form oligomeric complexes for assembly line catalysis. Bifunctional (+)-copalyl diphosphate synthase adopts (αβγ)6 architecture in which the α domain generates geranylgeranyl diphosphate, which then undergoes class II cyclization in the βγ domains to yield the bicyclic product. Bifunctional fusicoccadiene synthase adopts (αα)6 or (αα)8 architecture in which one α domain generates geranylgeranyl diphosphate, which then undergoes class I cyclization in the other α domain to yield the tricyclic product. The prenyltransferase α domain mediates oligomerization in these systems. Attached by flexible polypeptide linkers, cyclase domains splay out from oligomeric prenyltransferase cores.In this Account, we review structure-function relationships for these bifunctional terpene synthases, with a focus on the oligomeric systems studied in our laboratory. The observation of substrate channeling for fusicoccadiene synthase suggests a model for dynamic cluster channeling in catalysis by oligomeric assembly line terpenoid synthases. Resulting efficiencies in carbon management suggest that such systems could be particularly attractive for use in synthetic biology approaches to generate high-value terpenoid natural products.

Identification and Functional Characterization of the Gene Cluster Responsible for Fusaproliferin Biosynthesis in Fusarium proliferatum.

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.

Isolation, expression, and functional analysis of the geranylgeranyl pyrophosphate synthase (GGPPS) gene from Liriodendron tulipifera

Terpenoids are important secondary metabolites in plants and are involved in stress responses and pollinator attraction. Geranylgeranyl pyrophosphate synthase (GGPPS) is a key synthase in the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway of terpenoid synthesis, catalyzing the synthesis of diterpenoids. Liriodendron tulipifera is a nectar plant in North America. Little is known about the key genes involved in the biosynthetic pathways of terpenoids, the precursors of most compounds related to nectar, fragrance and coloring in flowers in L. tulipifera. In this study, the LtuGGPPS2 gene and its promoter (LtuGGPPS2-pro) were cloned from L. tulipifera. The results of sequence alignment showed that the LtuGGPPS2 gene is highly homologous to GGPPS genes of other plants. Subcellular localization analysis showed that the LtuGGPPS2 protein localizes to chloroplasts, suggesting that the LtuGGPPS2 gene is probably related to carotenoid and chlorophyll synthesis. Based on tissue expression profiles revealed by RT-qPCR, the expression level of the LtuGGPPS2 gene was highest in petals. These results were consistent with the changes in volatile and nonvolatile terpenoids in the flowers of L. tulipifera. GUS staining to examine the LtuGGPPS2 promoter indicated that it is responsive to hormones. Overexpression of the LtuGGPPS2 gene increased the carotenoid content and GGPPS enzyme activity in Arabidopsis thaliana, indicating that LtuGGPPS2 is the key terpenoid synthase in the flowers of L. tulipifera. Our findings lay a foundation for further functional analysis of the LtuGGPPS2 gene and deeper investigation of the terpenoid biosynthetic pathway in L. tulipifera.

A Sesquiterpene Synthase from the Endophytic Fungus Serendipita indica Catalyzes Formation of Viridiflorol.

Interactions between plant-associated fungi and their hosts are characterized by a continuous crosstalk of chemical molecules. Specialized metabolites are often produced during these associations and play important roles in the symbiosis between the plant and the fungus, as well as in the establishment of additional interactions between the symbionts and other organisms present in the niche. Serendipita indica, a root endophytic fungus from the phylum Basidiomycota, is able to colonize a wide range of plant species, conferring many benefits to its hosts. The genome of S. indica possesses only few genes predicted to be involved in specialized metabolite biosynthesis, including a putative terpenoid synthase gene (SiTPS). In our experimental setup, SiTPS expression was upregulated when the fungus colonized tomato roots compared to its expression in fungal biomass growing on synthetic medium. Heterologous expression of SiTPS in Escherichia coli showed that the produced protein catalyzes the synthesis of a few sesquiterpenoids, with the alcohol viridiflorol being the main product. To investigate the role of SiTPS in the plant-endophyte interaction, an SiTPS-over-expressing mutant line was created and assessed for its ability to colonize tomato roots. Although overexpression of SiTPS did not lead to improved fungal colonization ability, an in vitro growth-inhibition assay showed that viridiflorol has antifungal properties. Addition of viridiflorol to the culture medium inhibited the germination of spores from a phytopathogenic fungus, indicating that SiTPS and its products could provide S. indica with a competitive advantage over other plant-associated fungi during root colonization.

Structural insight on assembly-line catalysis in terpene biosynthesis

Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.