Enhanced Lutein Production in Chlamydomonas reinhardtii by Overexpression of the Lycopene Epsilon Cyclase Gene.

Chlamydomonas reinhardtii is a well-established microalgal model species with a shorter doubling time, which is a promising natural source for the efficient production of high-value carotenoids. In the microalgal carotenoid biosynthetic pathway, lycopene is converted either into ß-carotene by lycopene ß-cyclase or into α-carotene by lycopene ε-cyclase (LCYE) and lycopene ß-cyclase. In this study, we overexpressed the LCYE gene in C. reinhardtii to estimate its effect on lycopene metabolism and lutein production. Chlamydomonas transformants (CrLCYE#L1, #L5, and #L6) produced significantly increased amounts of lutein per culture (up to 2.6-fold) without a decrease in cell yields. Likewise, the expression levels of LCYE gene in transformants showed a significant increase compared with that of the wild-type strain. These results suggest that LCYE overexpression enhances the conversion of lycopene to α-carotene, which in turn improves lutein productivity. Interestingly, their ß-carotene productivity appeared to increase slightly rather than decrease. Considering that the inhibition of the lycopene cyclization steps often induces higher expression in genes upstream of metabolic branches, this result implies that the redirection from ß-carotene to α-carotene by LCYE overexpression might also enhance upstream gene expression, thereby leading to auxiliary ß-carotene production.

Musashi2 promotes the progression of pancreatic cancer through a novel ISYNA1-p21/ZEB-1 pathway.

Our previous studies found overexpression of Musashi2 (MSI2) conduced to the progression and chemoresistance of pancreatic cancer (PC) by negative regulation of Numb and wild type p53 (wtp53). Now, we further investigated the novel signalling involved with MSI2 in PC. We identified inositol-3-phosphate synthase 1 (ISYNA1) as a novel tumour suppressor regulated by MSI2. High MSI2 and low ISYNA1 expression were prevalently observed in 91 PC tissues. ISYNA1 expression was negatively correlated with MSI2 expression, T stage, vascular permeation and poor prognosis in PC patients. What's more, patients expressed high MSI2 and low ISYNA1 level had a significant worse prognosis. And in wtp53 Capan-2 and SW1990 cells, ISYNA1 was downregulated by p53 silencing. ISYNA1 silencing promoted cell proliferation and cell cycle by inhibiting p21 and enhanced cell migration and invasion by upregulating ZEB-1. However, MSI2 silencing upregulated ISYNA1 and p21 but downregulated ZEB-1, which can be rescued by ISYNA1 silencing. Moreover, reduction of cell migration and invasion resulting from MSI2 silencing was significantly reversed by ISYNA1 silencing. In summary, MSI2 facilitates the development of PC through a novel ISYNA1-p21/ZEB-1 pathway, which provides new gene target therapy for PC.

The Chalcone Isomerase Family in Cotton: Whole-Genome Bioinformatic and Expression Analyses of the Gossypium barbadense L. Response to Fusarium Wilt Infection.

Chalcone isomerase (CHI) is a key component of phenylalanine metabolism that can produce a variety of flavonoids. However, little information and no systematic analysis of CHI genes is available for cotton. Here, we identified 33 CHI genes in the complete genome sequences of four cotton species (Gossypium arboretum L., Gossypium raimondii L., Gossypium hirsutum L., and Gossypium barbadense L.). Cotton CHI proteins were classified into two main groups, and whole-genome/segmental and dispersed duplication events were important in CHI gene family expansion. qRT-PCR and semiquantitative RT-PCR results suggest that CHI genes exhibit temporal and spatial variation and respond to infection with Fusarium wilt race 7. A preliminary model of CHI gene involvement in cotton evolution was established. Pairwise comparison revealed that seven CHI genes showed higher expression in cultivar 06-146 than in cultivar Xinhai 14. Overall, this whole-genome identification unlocks a new approach to the comprehensive functional analysis of the CHI gene family, which may be involved in adaptation to plant pathogen stress.

Co-expression of squalene epoxidases with triterpene cyclases boosts production of triterpenoids in plants and yeast.

Triterpene cyclases catalyze the first committed step in triterpene biosynthesis, by forming mono- to pentacyclic backbone structures from oxygenated C30 isoprenoid precursors. Squalene epoxidase precedes this cyclization by providing the oxygenated and activated substrate for triterpene biosynthesis. Three squalene epoxidases from Cucurbita pepo (CpSEs) were isolated and shown to have evolved under purifying selection with signs of sites under positive selection in their N- and C-termini. They all localize to the Endoplasmic Reticulum (ER) and produce 2,3-oxidosqualene and 2,3:22,23-dioxidosqualene when expressed in a yeast erg1 (squalene epoxidase) erg7 (lanosterol synthase) double mutant. Co-expression of the CpSEs with four different triterpene cyclases, either transiently in Nicotiana benthamiana or constitutively in yeast, showed that CpSEs boost triterpene production. CpSE2 was the best performing in this regard, which could reflect either increased substrate production or superior channeling of the substrate to the triterpene cyclases. Fluorescence Lifetime Imaging Microscopy (FLIM) analysis with C. pepo cucurbitadienol synthase (CpCPQ) revealed a specific interaction with CpSE2 but not with the other CpSEs. When CpSE2 was transformed into C. pepo hairy root lines, cucurbitacin E production was increased two folds compared to empty vector control lines. This study provides new insight into the importance of SEs in triterpene biosynthesis, suggesting that they may facilitate substrate channeling, and demonstrates that SE overexpression is a new tool for increasing triterpene production in plants and yeast.

Expression of heterologous lycopene ß-cyclase gene in flax can cause silencing of its endogenous counterpart by changes in gene-body methylation and in ABA homeostasis mechanism.

Previously we described flax plants with expression of Arabidopsis lycopene ß-cyclase (lcb) gene in which decreased expression of the endogenous lcb and increased resistance to fungal pathogen was observed. We suggested that co-suppression was responsible for the change. In this study we investigated the molecular basis of the observed effect in detail. We found that methylation changes in the Lulcb gene body might be responsible for repression of the gene. Treatment with azacitidine (DNA methylation inhibitor) confirmed the results. Moreover, we studied how the manipulation of carotenoid biosynthesis pathway increased ABA level in these plants. We suggest that elevated ABA levels may be responsible for the increased resistance of the flax plants to pathogen infection through activation of chitinase (PR gene).

Design and Selection of a Synthetic Feedback Loop for Optimizing Biofuel Tolerance.

Feedback control allows cells to dynamically sense and respond to environmental changes. However, synthetic controller designs can be challenging because of implementation issues, such as determining optimal expression levels for circuit components within a feedback loop. Here, we addressed this by coupling rational design with selection to engineer a synthetic feedback circuit to optimize tolerance of Escherichia coli to the biojet fuel pinene. E. coli can be engineered to produce pinene, but it is toxic to cells. Efflux pumps, such as the AcrAB-TolC pump, can improve tolerance, but pump expression impacts growth. To address this, we used feedback to dynamically regulate pump expression in response to stress. We developed a library with thousands of synthetic circuit variants and subjected it to three types of pinene treatment (none, constant, and varying pinene). We were able to select for strains that were biofuel tolerant without a significant growth cost in the absence of biofuel. Using next-generation sequencing, we found common characteristics in the designs and identified controllers that dramatically improved biofuel tolerance.

Differential expression of carotenoid biosynthetic pathway genes in two contrasting tomato genotypes for lycopene content.

Tomato (Solanum lycopersicum L.) is one of the model plant to study carotenoid biosynthesis. In the present study, the fruit carotenoid content were quantified at different developmental stages for two contrasting genotypes, viz. IIHR-249-1 and IIHR-2866 by UPLC. Lycopene content was high in IIHR-249-1 (19.45 mg/100 g fresh weight) compared to IIHR-2866 (1.88 mg/100 g fresh weight) at the ripe stage. qPCR was performed for genes that are involved in the carotenoid biosynthetic pathway to study the difference in lycopene content in fruits of both the genotypes. The expression of Phytoene synthase (PSY) increased by 36-fold and Phytoene desaturase (PDS) increased by 14-fold from immature green stage to ripe stage in IIHR-249-1. The expression of Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta cyclase (CYC-B) decreased gradually from the initial stage to the ripe stage in IIHR-249-1. IIHR 249-1 showed 3- and 1.8-fold decrease in gene expression for Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta-cyclase (CYC-B) .The F2 hybrids derived from IIHR-249-1 and IIHR-2866 were analysed at the ripe stage for lycopene content. The gene expression of Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta-cyclase (CYC-B) in high and low lycopene lines from F2 progenies also showed the decrease in transcript levels of both the genes in high lycopene F2 lines. We wish to suggest that the differential expression of lycopene beta-cyclases can be used in marker-assisted breeding.

Tissue-specific production of limonene in Camelina sativa with the Arabidopsis promoters of genes BANYULS and FRUITFULL.

MAIN CONCLUSION: Arabidopsis promoters of genes BANYULS and FRUITFULL are transcribed in Camelina. They triggered the transcription of limonene synthase and induced higher limonene production in seeds and fruits than CaMV 35S promoter. Camelina sativa (Camelina) is an oilseed crop of relevance for the production of biofuels and the plant has been target of a recent and intense program of genetic manipulation aimed to increase performance, seed yield and to modify the fatty acid composition of the oil. Here, we have explored the performance of two Arabidopsis thaliana (Arabidopsis) promoters in triggering transgene expression in Camelina. The promoters of two genes BANYULS (AtBAN pro ) and FRUITFULL (AtFUL pro ), which are expressed in seed coat and valves of Arabidopsis, respectively, have been chosen to induce the expression of limonene synthase (LS) from Citrus limon. In addition, the constitutive CaMV 35S promoter was utilized to overexpress LS in Camelina . The results of experiments revealed that AtBAN pro and AtFUL pro are actively transcribed in Camelina where they also retain specificity of expression in seeds and valves as previously observed in Arabidopsis. LS induced by AtBAN pro and AtFUL pro leads to higher limonene production in seeds and fruits than when the CaMV 35S was used to trigger the expression. In conclusion, the results of experiments indicate that AtBAN pro and AtFUL pro can be successfully utilized to induce the expression of the transgenes of interest in seeds and fruits of Camelina.

Elevation of lutein content in tomato: a biochemical tug-of-war between lycopene cyclases.

Lutein is becoming increasingly important in preventive medicine due to its possible role in maintaining good vision and in preventing age-related maculopathy. Average daily lutein intake in developed countries is often below suggested daily consumption levels, and lutein supplementation could be beneficial. Lutein is also valuable in the food and feed industries and is emerging in nutraceutical and pharmaceutical markets. Currently, lutein is obtained at high cost from marigold petals, and synthesis alternatives are thus desirable. Tomato constitutes a promising starting system for production as it naturally accumulates high levels of lycopene. To develop tomato for lutein synthesis, the tomato Red Setter cultivar was transformed with the tomato lycopene ε-cyclase-encoding gene under the control of a constitutive promoter, and the HighDelta (HD) line, characterised by elevated lutein and δ-carotene content in ripe fruits, was selected. HD was crossed to the transgenic HC line and to RS(B) with the aim of converting all residual fruit δ-carotene to lutein. Fruits of both crosses were enriched in lutein and presented unusual carotenoid profiles. The unique genetic background of the crosses used in this study permitted an unprecedented analysis of the role and regulation of the lycopene cyclase enzymes in tomato. A new defined biochemical index, the relative cyclase activity ratio, was used to discern post-transcriptional regulation of cyclases, and will help in the study of carotenoid biosynthesis in photosynthetic plant species and particularly in those, like tomato, that have been domesticated for the production of food, feed or useful by-products.

Carotenogenic gene expression and carotenoid accumulation in three varieties of Cucurbita pepo during fruit development.

The control of gene expression is a crucial regulatory mechanism in carotenoid accumulation of fruits and flowers. We investigated the role of transcriptional regulation of nine genes involved in the carotenoid biosynthesis pathway in three varieties of Cucurbita pepo with evident differences in fruit color. The transcriptional levels of the key genes involved in the carotenoid biosynthesis were higher in flower-, leaf-, and fruit skin tissues than flesh tissues. This correlated with higher concentration of carotenoid content in these tissues. The differential expression among the colored and white cultivars detected for some genes, such as LCYe, in combination with other regulatory mechanisms, could explain the large differences found in terms of carotenoid content among the three varieties. These results are a first step to elucidate carotenogenesis in C. pepo and demonstrate that, in general, regulation of the pathway genes is a critical factor that determines the accumulation of these compounds.

Levels of lycopene ß-cyclase 1 modulate carotenoid gene expression and accumulation in Daucus carota.

Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene ß-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and ß-carotene through the cyclization of lycopene. Carotenoids are produced in both carrot (Daucus carota) leaves and reserve roots, and high amounts of α-carotene and ß-carotene accumulate in the latter. In some plant models, the presence of different isoforms of carotenogenic genes is associated with an organ-specific function. D. carota harbors two Lcyb genes, of which DcLcyb1 is expressed in leaves and storage roots during carrot development, correlating with an increase in carotenoid levels. In this work, we show that DcLCYB1 is localized in the plastid and that it is a functional enzyme, as demonstrated by heterologous complementation in Escherichia coli and over expression and post transcriptional gene silencing in carrot. Transgenic plants with higher or reduced levels of DcLcyb1 had incremented or reduced levels of chlorophyll, total carotenoids and ß-carotene in leaves and in the storage roots, respectively. In addition, changes in the expression of DcLcyb1 are accompanied by a modulation in the expression of key endogenous carotenogenic genes. Our results indicate that DcLcyb1 does not possess an organ specific function and modulate carotenoid gene expression and accumulation in carrot leaves and storage roots.

Isolation and characterization of a muskmelon cDNA encoding Lycopene Beta-cyclase.

Lycopene Beta-cyclase (LCY-B) is thought to play a critical role in Beta-carotene synthesis in fruit. A full-length cDNA clone encoding Lycopene Beta-cyclase was isolated from muskmelon (Cucumis melo L.) by RT-PCR and RACE. The clone, designated CmLcyb1, contains 1871 nucleotides, with an open reading frame of 1512 nucleotides. The deduced 504-amino-acid sequence showed high identities with other plant Lycopene Beta-cyclases. Real time quantitative RT-PCR analysis indicated that CmLcyb1 was expressed in all tissues and organs of muskmelon inbred M01-3 with white mesocarp and, 'Homoka', an orange mesocarp cultivar. The expression levels of CmLcyb1 in roots, stems, leaves and flowers in the two genotypes differed little. The expression level was highest in mature fruit of 'Homoka' and was much higher than that in mature fruit of M01-3. Moreover, the mRNA level of CmLcyb1 was very low in fruits before fruit-size fixation and increased dramatically in the size-fixed fruits of these two genotypes. The mRNA levels of CmLcyb1 during fruit development of 'Homoka' were all higher than those of M01-3. Interestingly, Beta-carotene content showed almost the same change trend as mRNA levels during fruit development in these two genotypes, suggesting that Beta-carotene accumulation may be linked to the CmLcyb1 transcript level in muskmelon fruit.

Polymorphism and expression of isoflavone synthase genes from soybean cultivars.

Isoflavones are synthesized by isoflavone synthases via the phenylpropanoid pathway in legumes. We have cloned two isoflavone synthase genes, IFS1 and IFS2, from a total of 18 soybean cultivars. The amino acid residues of the proteins that differed between cultivars were dispersed over the entire coding region. However, amino acid sequence variation did not occur in conserved domains such as the ERR triad region, except that one conserved amino acid was changed in the IFS2 protein of the GS12 cultivar (R374G) and the IFS1 proteins of the 99M06 and Soja99s65 cultivars (A109T, F105I). In three cultivars (99M06, 99M116, and Simheukpi), most of amino acid changes were such that the difference between the amino acid sequences of IFS1 and IFS2 was reduced. The expression profiles of three enzymes that convert naringenin to the isoflavone, genistein, chalcone isomerase (CHI), isoflavone synthase (IFS) and flavanone 3-hydroxylase (F3H) were examined. In general, IFS mRNA was more abundant in etiolated seedlings than mature plants whereas the levels of CHI and F3H mRNAs were similar in the two stages. During seed development, IFS was expressed a little later than CHI and F3H but expression of these three genes was barely detectable, if at all, during later seed hardening. In addition, we found that the levels of CHI, F3H, and IFS mRNAs were under circadian control. We also showed that IFS was induced by wounding and by application of methyl jasmonate to etiolated soybean seedlings.

cDNA isolation and functional expression of myrcene synthase from Perilla frutescens.

cDNA cloning of a monoterpene synthase from Perilla frutescens whose steam-distilled oil contains 92.9% perillaketone, was performed by the PCR method using primers designed based on limonene synthase. The full-length nucleotide sequence of this cDNA consisted of 1978 bp including a 1827-bp translational region encoding a deduced protein of 608 amino acids, which was similar to that of limonene synthase from P. frutescens (85% identity). Functional expression of this clone in Escherichia coli yielded an active monoterpene synthase enzyme, which converted geranyl diphosphate into 53.8% myrcene, 20.9% sabinene, 19.8% linalool and 5.5% limonene. As for the extraction of reaction products, we performed SPME (solid phase micro extraction) as well as conventional solvent extraction, and compared these two extraction methods.

A novel sex-specific and inducible monoterpene synthase activity associated with a pine bark beetle, the pine engraver, Ips pini.

Ecological interactions of conifers and coniferophagous bark beetles are determined in part by terpenoids (isoprenoids), which are major defense metabolites of conifer oleoresin. Curiously, similar compounds are important aggregation pheromones of conifer-attacking bark beetles. Terpene synthases are responsible for generating the enormous variety of terpenoid carbon skeletons found in nature. These catalysts convert short-chain prenyl diphosphates to a diverse assortment of hemiterpene, monoterpene, sesquiterpene, and diterpene natural products. While terpene synthases have frequently been characterized from plant and microbial sources, they have not yet been described in animals. Here we report the discovery of a monoterpene synthase activity in an insect, the pine engraver, Ips pini (Say) (Coleoptera: Scolytidae). Cell-free assays of I. pini revealed that geranyl diphosphate (GDP) is converted to the acyclic monoterpene myrcene in whole-body extracts from males, but not females. Furthermore, the monoterpene synthase activity in males can be induced by prior treatment with juvenile hormone III (JH III) or by feeding on phloem from the host trees, Jeffrey pine (Pinus jeffreyi Grev. & Balf.) or red pine (Pinus resinosa Ait.). The sex-specificity and endocrine induction of this activity argue for its involvement in the biosynthesis of monoterpenoid pheromones mediated by enzymes from insect tissue. This discovery is the first example of a monoterpene synthase in the Metazoa and evokes exciting new questions about the origin, evolution, and occurrence of terpene synthases.

cDNAs for the synthesis of cyclic carotenoids in petals of Gentiana lutea and their regulation during flower development.

cDNAs encoding lycopene epsilon -cyclase, lycopene beta-cyclase, beta-carotene hydroxylase and zeaxanthin epoxidase were isolated from a Gentiana lutea petal cDNA library. The function of all cDNAs was analyzed by complementation in Escherichia coli. Transcript levels during different stages of flower development of G. lutea were determined and compared to the carotenoid composition. Expression of all genes increased by a factor of up to 2, with the exception of the lycopene epsilon -cyclase gene. The transcript amount of the latter was strongly decreased. These results indicate that during flower development, carotenoid formation is enhanced. Moreover, metabolites are shifted away from the biosynthetic branch to lutein and are channeled into beta-carotene and derivatives.

Preliminary X-ray crystallographic analysis of tRNA pseudouridine 55 synthase from the thermophilic eubacterium Thermotoga maritima.

Thermotoga maritima TruB, an enzyme responsible for the formation of pseudouridine in tRNA, has been purified and crystallized by the hanging-drop vapour-diffusion method in 100 mM citrate pH 3.5, 200 mM Li(2)SO(4), 20% glycerol, 13% PEG 8000. Crystals display orthorhombic symmetry, with unit-cell parameters a = 47.39, b = 83.88, c = 98.72 A, and diffract to 2.0 A resolution using synchrotron radiation. A solution was obtained by molecular replacement using part of the recently published crystal structure of Escherichia coli TruB bound to a synthetic RNA.

Molecular cloning, functional expression and characterization of d-limonene synthase from Agastache rugosa.

We cloned the gene of d-limonene synthase (ArLMS) from Agastache rugosa (Labiatae). The function of ArLMS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. ArLMS consisted of 2077 nucleotides including 1839 bp of coding sequence that encodes a protein of 613 amino acids. This protein has a 60 kDa molecular weight, which is identical to that of d-limonene synthase from Schizonepeta tenuifolia (Labiatae). The deduced amino acid sequence of ArLMS shows high homology with the known d- and l-limonene synthases from Labiatae plants. Here, we discussed the amino acid residues responsible for the stereochemical regulation in limonene biosynthesis.

Molecular cloning, functional expression and characterization of d-limonene synthase from Schizonepeta tenuifolia.

Limonene is one of the most simple cyclic monoterpenes, and two enantiomers, d- and l-limonene occur due to the chiral carbon at 4-position. Cyclization of GPP into limonene is catalyzed by the limonene synthase, and some l-limonene synthase cDNAs have already been cloned from several species of plants, mainly from Labiatae family. However, the d-limonene synthase gene has not yet been obtained, therefore, no information is available on the molecular mechanism of stereochemical regulation in limonene formation. To resolve this, we cloned the d-limonene synthase gene (dLMS) from Schizonepeta tenuifolia (Labiatae) by a reverse genetic approach, and we found that both d- and l-limonene synthase share similar features such as a transit peptide, an arginine rich domain, and a metal cation binding site in their structures. Here, we report on the cloning of dLMS, and the putative stereochemical regulation mechanism is discussed based on the comparison of the deduced amino acid sequence of dLMS with those of known l-limonene synthases.

Regulation by two CatR proteins that differ in binding affinity to catB promoters expressing two cat gene clusters.

We isolated the two LysR-type regulatory proteins CatR1 and CatR2, which regulate the expression of cat1 and cat2 gene clusters, respectively, required for catechol degradation in the bacterium Frateuria sp. ANA-18. In a gel mobility shift assay using CatR1 and the DNA fragment containing the catB1 promoter region, the formation of two complexes, complex 1-1 (C1-1) and complex 1-2 (C1-2), was observed in the presence of cis,cis-muconate. On the other hand, CatR2 and the DNA fragment containing the catB2 promoter region formed only complex 2-2 (C2-2) at a lower concentration of cis,cis-muconate than that at which C1-1 and C1-2 were formed. As the concentration of cis,cis-muconate decreased, the production of the muconate cycloisomerase isozyme MC II encoded by catB2 decreased as well as that of MC I encoded by catB1. However, the amount of MC II synthesized was larger than that of MC I at low concentrations. On the basis of these results, we concluded that the catB2 promoter was activated at low concentrations of cis,cis-muconate.