Structure-function studies of ultrahigh molecular weight isoprenes provide key insights into their biosynthesis.

Some plant trans-1,4-prenyltransferases (TPTs) produce ultrahigh molecular weight trans-1,4-polyisoprene (TPI) with a molecular weight of over 1.0 million. Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear. Here, we identified three novel Eucommia ulmoides TPT isoforms-EuTPT1, 3, and 5, which synthesized TPI in vitro without other components. Crystal structure analysis of EuTPT3 revealed a dimeric architecture with a central hydrophobic tunnel. Mutation of Cys94 and Ala95 on the central hydrophobic tunnel no longer synthesizd TPI, indicating that Cys94 and Ala95 were essential for forming the dimeric architecture of ultralong-chain TPTs and TPI biosynthesis. A spatiotemporal analysis of the physiological function of TPI in E. ulmoides suggested that it is involved in seed development and maturation. Thus, our analysis provides functional and mechanistic insights into TPI biosynthesis and uncovers biological roles of TPI in plants.

Two Eucommia farnesyl diphosphate synthases exhibit distinct enzymatic properties leading to end product preferences.

Farnesyl diphosphate synthase (FPS) is an essential enzyme in the biosynthesis of prenyl precursors for the production of primary and secondary metabolites, including sterols, dolichols, carotenoids and ubiquinones, and for the modification of proteins. Here we identified and characterized two FPSs (EuFPS1 and EuFPS2) from the plant Eucommia ulmoides. The EuFPSs had seven highly conserved prenyltransferase-specific domains that are critical for activity. Complementation and biochemical analyses using bacterially produced recombinant EuFPS isoforms showed that the EuFPSs had FPP synthesis activities both in vivo and in vitro. In addition to the typical reaction mechanisms of FPS, EuFPSs utilized farnesyl diphosphate (FPP) as an allylic substrate and participated in further elongation of the isoprenyl chain, resulting in the synthesis of geranylgeranyl diphosphate. However, despite the high amino acid similarities between the two EuFPS isozymes, their specific activities, substrate preferences, and final reaction products were different. The use of dimethylallyl diphosphate (DMAPP) as an allylic substrate highlighted the differences between the two enzymes: depending on the pH, the metal ion cofactor, and the cofactor concentration, EuFPS2 accumulated geranyl diphosphate as an intermediate product at a constant rate, whereas EuFPS1 synthesized little geranyl diphosphate. The reaction kinetics of the EuFPSs demonstrated that isopentenyl diphosphate and DMAPP were used both as substrates and as inhibitors of EuFPS activity. Taken together, the results indicate that the biosynthesis of FPP is highly regulated by various factors indispensable for EuFPS reactions in plants.

Gene coexpression network for trans-1,4-polyisoprene biosynthesis involving mevalonate and methylerythritol phosphate pathways in Eucommia ulmoides Oliver.

Eucommia ulmoides, a deciduous dioecious plant species, accumulates trans-1,4-polyisoprene (TPI) in its tissues such as pericarp and leaf. Probable TPI synthase (trans-isoprenyl diphosphate synthase (TIDS)) genes were identified by expressed sequence tags of this species; however, the metabolic pathway of TPI biosynthesis, including the role of TIDSs, is unknown. To understand the mechanism of TPI biosynthesis at the transcriptional level, comprehensive gene expression data from various organs were generated and TPI biosynthesis related genes were extracted by principal component analysis (PCA). The metabolic pathway was assessed by comparing the coexpression network of TPI genes with the isoprenoid gene coexpression network of model plants. By PCA, we dissected 27 genes assumed to be involved in polyisoprene biosynthesis, including TIDS genes, genes encoding enzymes of the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, and genes related to rubber synthesis. The coexpression network revealed that 22 of the 27 TPI biosynthesis genes are coordinately expressed. The network was clustered into two modules, and this was also observed in model plants. The first module was mainly comprised of MEP pathway genes and TIDS1 gene, and the second module, of MVA pathway genes and TIDS5 gene. These results indicate that TPI is likely biosynthesized by both the MEP and MVA pathways and that TIDS gene expression is differentially controlled by these pathways.

High-throughput and highly sensitive analysis method for polyisoprene in plants by pyrolysis-gas chromatography/mass spectrometry.

Natural polyisoprene is a biopolymer consisting of isoprene units (C(5)H(8)) that is used commercially in household, medical, and industrial materials. For the management of natural polyisoprene production, the selection of high-yield polyisoprene-producing trees, and an understanding of polyisoprene biosynthesis, a high-throughput and highly sensitive screening method for the quantification of polyisoprene is required. In this study, we examined pyrolysates from polyisoprenes, polyprenols, carotenoids, ubiquinone (CoQ-10), and sterols by pyrolysis gas chromatography/mass spectrometry (PyGC/MS) and determined that the amounts of isoprene and limonene released from polyprenols and polyisoprenes were dependent upon their molecular weights. Based on these results, we developed a relative quantification method for polyisoprene in leaves by direct analysis of 1 mg of leaves using PyGC/MS. This novel quantification method eliminated extraction steps and can be used in the measurement of polyisoprene contents in Eucommia ulmoides and Hevea brasiliensis.

Cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway genes of a natural-rubber producing plant, Hevea brasiliensis.

Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-(13)C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.

Overexpression of 1-Deoxy-D-xylulose-5-phosphate reductoisomerase gene in chloroplast contributes to increment of isoprenoid production.

Plants synthesize a large number of isoprenoid compounds that are of industrial, nutritional and medicinal importance. 1-Deoxy-D-xylulose reductoisomerase (DXR) catalyzes the first committed step of plastidial isoprenoid-precursor biosynthesis. In the present study, we generated transplastomic tobacco plants that overproduced DXR from Synechosystis sp. strain PCC6803. The transformants showed increase in the content of various isoprenoids such as chlorophyll a, beta-carotene, lutein, antheraxanthin, solanesol and beta-sitosterol, indicating that the DXR reaction is one of the key steps controlling isoprenoid level in tobacco leaves. A qualitative change in isoprenoid composition was also observed. The growth phenotype of the transplastomic plants was similar to that of wild-type plants. These results showed that plastid metabolic engineering is useful in manipulating the yield of isoprenoids in plants.

Quantification of trans-1,4-polyisoprene in Eucommia ulmoides by fourier transform infrared spectroscopy and pyrolysis-gas chromatography/mass spectrometry.

Commercial development of trans-1,4-polyisoprene from Eucommia ulmoides Oliver (EU-rubber) requires specific knowledge on selection of high-rubber-content lines and establishment of agronomic cultivation methods for achieving maximum EU-rubber yield. The development can be facilitated by high-throughput and highly sensitive analytical techniques for EU-rubber extraction and quantification. In this paper, we described an efficient EU-rubber extraction method, and validated that the accuracy was equivalent to that of the conventional Soxhlet extraction method. We also described a highly sensitive quantification method for EU-rubber by Fourier transform infrared spectroscopy (FT-IR) and pyrolysis-gas chromatography/mass spectrometry (PyGC/MS). We successfully applied the extraction/quantification method for study of seasonal changes in EU-rubber content and molecular weight distribution.

A high-throughput and solvent-free method for measurement of natural polyisoprene content in leaves by Fourier transform near infrared spectroscopy.

Commercial development of natural polyisoprene from polyisoprene-producing plants requires detailed knowledge on how to select high-polyisoprene-content lines and establish agronomic cultivation methods for achieving maximum polyisoprene yield. This development can be facilitated by a high-throughput quantification method for natural polyisoprene. In this paper, we describe the Fourier transform near infrared spectroscopy (FT-NIR) technique coupled with a partial least squares (PLS) regression model to quantify natural polyisoprene in Eucommia ulmoides leaves. PLS regression models are discussed with respect to linearity, root-mean-square error of estimation (RMSEE), and root-mean-square error of prediction (RMSEP). The best PLS regression model was obtained with second derivative NIR spectra in the region between 4000-6000 cm(-1) (R2Y, 0.95; RMSEE, 0.25; RMSEP, 0.37). This is the first report to employ FT-NIR analysis for high throughput and solvent-free quantification of natural polyisoprene in leaves.

Biosynthetic pathway for the C45 polyprenol, solanesol, in tobacco.

Feeding experiments were independently performed with [1-13C]deoxy-D-xylulose triacetate and (RS)-[2-13C]mevalonolactone in the tobacco plant. The labeling pattern for solanesol was elucidated to reveal that the isoprene moiety of solanesol would be derived from deoxy-xylulose. The result strongly suggests that tobacco solanesol is biosynthesized via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.