Evaluation of Solutions Containing Fluoride, Sodium Trimetaphosphate, Xylitol, and Erythritol, Alone or in Different Associations, on Dual-Species Biofilms.

Although the association of polyols/polyphosphates/fluoride has been demonstrated to promote remarkable effects on dental enamel, little is known on their combined effects on biofilms. This study assessed the effects of solutions containing fluoride/sodium trimetaphosphate (TMP)/xylitol/erythritol on dual-species biofilms of Streptococcus mutans and Candida albicans. Biofilms were grown in the continuous presence of these actives alone or in different associations. Quantification of viable plate counts, metabolic activity, biofilm biomass, and extracellular matrix components were evaluated. Overall, fluoride and TMP were the main actives that significantly influenced most of the variables analyzed, with a synergistic effect between them for S. mutans CFUs, biofilm biomass, and protein content of the extracellular matrix (p < 0.05). A similar trend was observed for biofilm metabolic activity and carbohydrate concentrations of the extracellular matrix, although without statistical significance. Regarding the polyols, despite their modest effects on most of the parameters analyzed when administered alone, their co-administration with fluoride and TMP led to a greater reduction in S. mutans CFUs and biofilm biomass compared with fluoride alone at the same concentration. It can be concluded that fluoride and TMP act synergistically on important biofilm parameters, and their co-administration with xylitol/erythritol significantly impacts S. mutans CFUs and biomass reduction.

Rheological parameters of mixed Brazilian Cerrado fruits sugar-free preserves: the effect of body agents.

The aim of this study was to evaluate the effect of the bodying agents (erythritol, sorbitol, xylitol, and polydextrose) and their mixtures on the preparation of mixed Brazilian Cerrado fruit preserves (marolo, soursop, and sweet passion fruit). Mixture design was used for product optimization and the preserves were evaluated by texture profile, stress relaxation test, and uniaxial compression test. The research data were analyzed using regression equations on SAS software. The results indicated that the rheological parameters were affected by the body agents. Erythritol should not be used as an isolated component because it provoked changes in the properties of the final product (harder and brittle preserves); xylitol and sorbitol made preserves more elastic, cohesive, and more fragile; and polydextrose showed a synergistic effect with erythritol for rupture deformation and lesser effects for hardness, adhesiveness, and gumminess.

The effect of toothpaste with reduced concentration of fluoride-containing sodium trimetaphosphate and polyols on initial enamel erosion.

OBJECTIVES: This in vitro study evaluated the efficacy of toothpaste containing fluoride (F), sodium trimetaphosphate (TMP), and xylitol and erythritol (XE) to inhibit or repair initial enamel erosion lesions. MATERIALS AND METHODS: Bovine enamel blocks (n = 120) were selected according to surface hardness (SH) and randomly divided into 5 experimental groups (n = 24 blocks/group): Placebo (no F, TMP, XE); 1100 ppm F; 16% xylitol + 4% erythritol (XE); 200 ppm F + 0.2% TMP (200 ppm F/TMP); and 200 ppm F + 0.2% TMP + 16% xylitol + 4% erythritol (200 ppm F/TMP/XE). The sound and softened blocks were immersed in toothpaste slurry in human saliva for 2 min. The blocks were then submitted to 4 erosive challenges in citric acid (0.75%, pH 3.5), each challenge for 1 min, with stirring. The SH of the blocks was determined after treatment (t) and after the 4 erosive challenges. In addition, the precipitates were analyzed by scanning electron microscopy (SEM). Variables were submitted to a two-way repeated-measures analysis of variance followed by a Student-Newman-Keuls test (p < 0.05). RESULTS: Toothpaste containing 200 ppm F/TMP/XE led to the highest protective and repair effect compared to the other groups (p < 0.001). The protective and repair effect was XE > 200 ppm F/TMP > 1100 ppm F > placebo (p < 0.001). All groups produced precipitates with a thicker layer for XE and TMP groups. CONCLUSIONS: Toothpaste containing 200 ppm F, TMP, and polyols demonstrated a superior protective and repair effect in initial enamel erosive lesions in vitro. CLINICAL RELEVANCE: Toothpaste containing F and polyols could be an advantage in patients affected by dental erosion, due to its higher preventive potential, mainly in individuals who frequently drink acidic beverages. However, future studies are needed to confirm these results.

In situ evaluation of 200 ppm fluoride toothpaste content trimetaphosphate, xylitol and erythritol on enamel demineralization and dental biofilm.

OBJECTIVE: To evaluate the effect of low-fluoride (F-) toothpaste and sodium trimetaphosphate (TMP) associated with xylitol and erythritol (XE) on enamel demineralization and biofilm composition. METHODS: This crossover double-blind in situ study consisted of five phases (seven days each), in which 14 volunteers wore oral appliances containing four enamel bovine blocks. The cariogenic challenge was performed by exposure to a 30% sucrose solution (6x/day). The toothpaste treatments (3x/day) were as follows: placebo (no F-/TMP/XE); 200 ppm F- (NaF) (200F); 1,100 ppm F- (1100F); 16% Xylitol and 4% Erythritol (XE); and 200 ppm F-, 0.2% TMP, 16% xylitol, and 4% erythritol (200F-TMP-XE). Percentage of surface hardness loss (%SH) and integrated loss of subsurface hardness (ΔKHN), and calcium (Ca2+), phosphate (PO43-), and F- on enamel and biofilm were determined; as well as insoluble extracellular polysaccharide (EPS). RESULTS: XE and 1100F groups showed no significant difference for %SH and ΔKHN values (p = 0.220 and p = 0.886), and the 200F-TMP-XE group had the lowest mineral loss (p < 0.001). Ca2+ and PO43- in the enamel showed the highest values (p < 0.001) for the 200F-TMP-XE group. Higher values of F- in the enamel and biofilm were observed for the 1100F group (p < 0.001). There was no difference for Ca2+ (p = 1.00) and EPS (p =0.918) values between XE and 200-TMP-XE groups in the biofilm, but their values were higher and lower than the 1100F (p = 0.002 and p = 0.029), respectively. CONCLUSIONS: 200F-TMP-XE promoted a greater protective effect against enamel demineralization and significantly affected the composition of biofilm formed in situ compared to 1100F toothpaste. CLINICAL SIGNIFICANCE: Low-F- toothpaste containing TMP and polyols can be considered an effective and safe measure to improve the oral health of individuals, especially patients with high caries activity.

Potential for Use of Erythritol as a Socially Transferrable Ingested Insecticide for Ants (Hymenoptera: Formicidae).

Ants are significant structural and agricultural pests, generating a need for human-safe and effective insecticides for ant control. Erythritol, a sugar alcohol used in many commercial food products, reduces survival in diverse insect taxa including fruit flies, termites, and mosquitos. Erythritol also decreases longevity in red imported fire ants; however, its effects on other ant species and its ability to be transferred to naïve colony members at toxic doses have not been explored. Here, we show that erythritol decreases survival in Tetramorium immigrans Santschi (Hymenoptera: Formicidae) in a concentration-dependent manner. Access to ad-libitum water reduced the toxic effects of erythritol, but worker mortality was still increased over controls with ad-lib water. Foraging T. immigrans workers transferred erythritol at lethal levels to nest mates that had not directly ingested erythritol. Similar patterns of mortality following erythritol ingestion were observed in Formica glacialis Wheeler (Hymenoptera: Formicidae), Camponotus subarbatus Emery (Hymenoptera: Formicidae), and Camponotus chromaiodes Bolton (Hymenoptera: Formicidae). These findings suggest that erythritol may be a highly effective insecticide for several genera of ants. Erythritol's potential effectiveness in social insect control is augmented by its spread at lethal levels through ant colonies via social transfer (trophallaxis) between workers.

Erythritol Ingestion Causes Concentration-Dependent Mortality in Eastern Subterranean Termites (Blattodea: Rhinotermitidae).

Damage from termite infestations is economically significant and control can be costly when requiring the widespread use of conventional insecticides. Erythritol, a polyalcohol sweetener that is safe for human consumption, causes increased mortality when ingested by some insects, indicating potential as a safe alternative insecticide. Here, we investigated the applicability of erythritol as a novel toxicant method of termite control. Eastern subterranean termites, Reticulitermes flavipes Kollar (Blattodea: Rhinotermitidae), were fed paper foods treated with increasing concentrations of erythritol and were assessed for mortality and bait consumption. Termite survival to 8 d (the duration of the experiment) significantly decreased as erythritol treatment concentration increased, indicating that the lethal effects of erythritol were concentration-dependent. Termites consumed erythritol-treated paper at all concentrations and did not display avoidance in choice assays, suggesting that erythritol may be practical for use as an ingestible bait. These results provide a basis for further development of erythritol as a safe alternative method of termite control.

Efeito de dentifrícios com reduzida concentração de fluoreto contendo trimetafosfato de sódio e polióis na erosão inicial do esmalte / Effect of low-fluoride toothpaste containing sodium trimetaphosphate and polyols on initial enamel erosion

Este estudo in vitro avaliou o efeito de dentifrícios contendo fluoreto (F), trimetafosfato de sódio (TMP) e/ou xilitol e eritritol (XE) em inibir ou reparar lesão erosiva inicial do esmalte. Blocos de esmalte bovino (n=120) foram selecionados por dureza de superfície (SH) inicial e divididos em 5 grupos compostos por dentifrícios (24 blocos/grupo): Placebo (sem F, TMP e XE); 1100 ppm F; 16% xilitol + 4% eritritol (XE); 200 ppm F + 0,2% TMP (200 ppm F/TMP); e 200 ppm F + 0,2% TMP + 16% xilitol + 4% eritritol (200 ppm F/TMP/XE). Para a análise do efeito protetor, blocos hígidos (n=60) foram imersos 1x/2 minutos em suspensão de dentifrícios/saliva humana. Em seguida, os blocos foram submetidos a 4 desafios erosivos com ácido cítrico (0,75%, pH 3,5) de 1 minuto cada, sob agitação. A seguir, a SH foi determinada pós-tratamento (t) e após o 1º, 2º, 3º e 4º desafios ácidos erosivos (d) para o cálculo da porcentagem de variação da SH (%SH). Para a análise do efeito reparador, esmalte previamente erodido (n=60) foi tratado e submetido aos mesmos desafios erosivos descritos anteriormente. A %SH de recuperação (R) e %SHt foram calculadas, bem como, a diferença entre a %SHR e %SHd obtendo-se o Δ%SH para cada desafio. Experimento adicional foi realizado para análise da deposição de precipitados por Microscopia Eletrônica de Varredura (MEV) em esmalte hígido e erodido. Os dados foram submetidos à análise de variância de medidas repetidas a dois critérios, seguida pelo teste de Student-Newman-Keuls (p<0,05). Os resultados mostraram que o maior efeito protetor e reparador foi produzido pelo dentifrício 200 ppm F/TMP/XE quando comparado aos demais grupos (p<0,001). Os grupos 1100 ppm F e 200 ppm F/TMP apresentaram similar efeito protetor para o 1º, 2º e 3º desafios (p>0,05), e menor quando comparados ao XE (p<0,001). O efeito protetor e reparador foi: XE>200 ppm F/TMP>1100 ppm F>Placebo (p<0,001). Na MEV observou-se deposição de precipitado no esmalte para todos os grupos, formando uma camada mais espessa e homogênea nos grupos contendo XE e/ou TMP. Concluiu-se que dentifrício contendo 200 ppm F, TMP e polióis apresenta efeito protetor e reparador superior quando comparado a um dentifrício 1100 ppm F em lesões erosivas iniciais no esmalte(AU)

This in vitro study evaluated the effect of toothpaste containing fluoride (F), sodium trimetaphosphate (TMP) and/or xylitol and erythritol (XE) in inhibit or repair initial enamel erosion lesions. Bovine enamel blocks (n=120) were selected by initial surface hardness (SH) and divided into 5 groups of toothpastes (n=24 blocks/group): Placebo (no F, TMP or XE); 1100 ppm F; 16% xylitol + 4% erythritol (XE); 200 ppm F + 0.2% TMP (200 ppm F/TMP); and 200 ppm F + 0.2% TMP + 16% xylitol + 4% erythritol (200 ppm F/TMP/XE). For the analysis of the protective effect, sound blocks (n=60) were immersed in toothpaste slurry in human saliva once for 2 minutes. Hereafter, the blocks were submitted to 4 erosive challenges in citric acid (0.75%, pH 3.5) by 1 minute, under stirring. Then, the SH was determined after treatment (t) and after the 1st, 2nd, 3rd and 4th erosive acid challenges (d) to calculate the percentage of change of the SH (%SH). For the analysis of the repair effect, eroded enamel (n=60) were treated and submitted to erosive challenges as describe previously. The recovery (R) of %SH and %SHt were calculated, as well as the difference between the %SHR and %SHd obtaining the Δ%SH for each challenge. Additional experimental was performed to analysis the deposition of precipitates by Scanning Electronic Microscopy (SEM) on sound and eroded enamel. Variables were submitted to two-way repeated measures analysis of variance followed by StudentNewman-Keuls test (p<0.05). The results showed that the highest protective and repair effect was produced by the 200 ppm F/TMP/XE toothpaste when compared to the other groups (p<0.001). The 1100 ppm F and 200 ppm F/TMP groups had similar protective effect for the 1st, 2nd and 3rd challenges (p>0.05), and lower when compared to XE (p<0.001). The protective and repair effect was: XE>200 ppm F/TMP>1100 ppm F>Placebo (p<0.001). There were deposition of precipitates on enamel for all groups, with a thicker layer and homogeneous for XE and/or TMP groups. It was concluded that toothpaste containing 200 ppm F, TMP and polyols has superior protective and repair effect when compared to 1100 ppm F toothpaste in initial enamel erosive lesions(AU)

A novel osmotic pressure strategy to improve erythritol production by Yarrowia lipolytica from glycerol.

Polyethylene glycol (PEG) is polymer that was used to replace NaCl (reference media) as an osmotic stress agent for the synthesis of erythritol by the osmophilic yeast Yarrowia lipolytica. Two strains, the wild-type strain IMUFRJ 50682 and the lab strain W29, were grown in the presence of PEG of different molecular weights. For strain IMUFRJ 50682, the erythritol titer was increased by 40% in the presence of PEG2000 as compared to the reference media (with NaCl). A similar increase was also observed for strain W29, except that it occurred in the presence of PEG6000. Moreover, in those experimental conditions neither strain produced mannitol, in contrast to the control medium. These results highlight that PEG could be used to increase erythritol productivity and to simultaneously inhibit mannitol synthesis, representing a good substitute for NaCl as an osmotic stress agent.

Atividade antimicrobiana de Stevia rebaudiana Bertoni e de adoçantes não calóricos sobre bactérias cariogênicas: estudo in vitro / Antimicrobial activity of Stevia rebaudiana Bertoni and non-caloric sweeteners on cariogenic bacteria: in vitro study

Objetivo: avaliar a atividade antimicrobiana in vitro da planta Stevia rebaudiana Bertoni e de adoçantes não calóricos sobre o crescimento de Streptococcus mutanse Lactobacillus casei, micro-organismos cariogênicos presentes na cavidade bucal. Materiais e método: o estudo foi realizado utilizando as cepas padrões de S.mutans (UA159) e L. casei (ATCC7469). Foram avaliados diferentes compostos não calóricos substitutos dasacarose nas concentrações de 1%, 5% e 10%: eritritol(ER), Fit Sucralose® (SU), Stevita® (ST), solução de Steviarebaudiana Bertoni (SSr) e, como controle positivo,digluconato de clorexidina (DC). A análise do efeito inibitório desses compostos no crescimento das bactériasfoi feita por meio da técnica de difusão em ágar. Resultado:observou-se que existe um efeito inibitório decrescimento de ambos os micro-organismos por parte da SSr e do ER, enquanto os demais adoçantes testa dosnão tiveram efeito inibitório sobre esses micro-organismos.Conclusão: os resultados demonstram que SSR eER apresentam efeito inibidor no crescimento das cepastestadas de S. mutans e L. casei. (AU)

Objective: The study evaluated the in vitro antimicrobial activity of the Stevia rebaudiana Bertoni plant and non-caloric sweeteners on the growth of Streptococcus mutans and Lactobacillus casei, which are cariogenic microorganisms present in the oral cavity. Materials and method: The study was conducted using the standard strains of S. mutans (UA159) and L. casei (ATCC7469). Different non-caloric compounds were evaluated at concentrations of 1%, 5%, and 10%: erythritol (ER), Fit Sucralose™ (SU), Stevita™ (ST), Stevia rebaudiana Bertoni solution (SSr), and chlorhexidine digluconate (CD) as positive control. The inhibitory effect of these compounds on the growth of bacteria were analyzed by the agar diffusion technique. Result: There was a growth inhibition effect for both microorganisms by SSr and ER, whereas the other sweeteners tested had no inhibitory effect on the microorganisms. Conclusion: The results showed that SSr and ER present an inhibitory effect on the growth the strains tested of S. mutans and L. casei. (AU)

A role for ß,ß-xanthophylls in Arabidopsis UV-B photoprotection.

Plastidial isoprenoids, such as carotenoids and tocopherols, are important anti-oxidant metabolites synthesized in plastids from precursors generated by the methylerythritol 4-phosphate (MEP) pathway. In this study, we found that irradiation of Arabidopsis thaliana plants with UV-B caused a strong increase in the accumulation of the photoprotective xanthophyll zeaxanthin but also resulted in slightly higher levels of γ-tocopherol. Plants deficient in the MEP enzymes 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase showed a general reduction in both carotenoids and tocopherols and this was associated with increased DNA damage and decreased photosynthesis after exposure to UV-B. Genetic blockage of tocopherol biosynthesis did not affect DNA damage accumulation. In contrast, lut2 mutants that accumulate ß,ß-xanthophylls showed decreased DNA damage when irradiated with UV-B. Analysis of aba2 mutants showed that UV-B protection was not mediated by ABA (a hormone derived from ß,ß-xanthophylls). Plants accumulating ß,ß-xanthophylls also showed decreased oxidative damage and increased expression of DNA-repair enzymes, suggesting that this may be a mechanism for these plants to decrease DNA damage. In addition, in vitro experiments also provided evidence that ß,ß-xanthophylls can directly protect against DNA damage by absorbing radiation. Together, our results suggest that xanthophyll-cycle carotenoids that protect against excess illumination may also contribute to protection against UV-B.

Strategy of oxygen transfer coefficient control on the L-erythrulose fermentation by newly isolated Gluconobacter kondonii

Background: The effect of diverse oxygen transfer coefficient on the L-erythrulose production from meso-erythritol by a newly isolated strain, Gluconobacter kondonii CGMCC8391 was investigated. In order to elucidate the effects of volumetric mass transfer coefficient (K La) on the fermentations, baffled and unbaffled flask cultures, and fed-batch cultures were developed in present work. Results: With the increase of the K La value in the fed-batch culture, L-erythrulose concentration, productivity and yield were significantly improved, while cell growth was not the best in the high K La. Thus, a two-stage oxygen supply control strategy was proposed, aimed at achieving high concentration and high productivity of L-erythrulose. During the first 12 h, Klawas controlled at 40.28 h-1 to obtain high value for cell growth, subsequently K La was controlled at 86.31 h-1 to allow for high L-erythrulose accumulation. Conclusions: Under optimal conditions, the L-erythrulose concentration, productivity, yield and DCW reached 207.9 ± 7.78 g/L, 6.50 g/L/h, 0.94 g/g, 2.68 ± 0.17 g/L, respectively. At the end of fermentation, the L-erythrulose concentration and productivity were higher than those in the previous similar reports.

Enhanced production of erythritol and mannitol by Yarrowia lipolytica in media containing surfactants

Abstract Various chemical compounds, including surfactants, when introduced to culture media may increase the permeability of cellular membranes and thereby affect the quantity of metabolites excreted by cells. The aim of the present study was to evaluate the impact of detergents including Triton X-100, Span 20 and Tween 80 on erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 in a shake-flask experiment, batch and fed-batch cultures. When Span 20 was added to a fed-batch culture with glycerol as a carbon source (300 g L-1), erythritol production increased by 15% compared to the culture without the surfactant where it reached 142 g L-1 after 5 days, which corresponded to 0.47 g g-1 yield and productivity of 1.1 g L-1 h-1. Therefore, it was concluded that Span 20 considerably enhanced the production of this polyol from glycerol.

Enhanced production of erythritol and mannitol by Yarrowia lipolytica in media containing surfactants

Various chemical compounds, including surfactants, when introduced to culture media may increase the permeability of cellular membranes and thereby affect the quantity of metabolites excreted by cells. The aim of the present study was to evaluate the impact of detergents including Triton X-100, Span 20 and Tween 80 on erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 in a shake-flask experiment, batch and fed-batch cultures. When Span 20 was added to a fed-batch culture with glycerol as a carbon source (300 g L-1), erythritol production increased by 15% compared to the culture without the surfactant where it reached 142 g L-1 after 5 days, which corresponded to 0.47 g g-1 yield and productivity of 1.1 g L-1 h-1. Therefore, it was concluded that Span 20 considerably enhanced the production of this polyol from glycerol.(AU)

Enhanced production of erythritol and mannitol by Yarrowia lipolytica in media containing surfactants.

Various chemical compounds, including surfactants, when introduced to culture media may increase the permeability of cellular membranes and thereby affect the quantity of metabolites excreted by cells. The aim of the present study was to evaluate the impact of detergents including Triton X-100, Span 20 and Tween 80 on erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 in a shake-flask experiment, batch and fed-batch cultures. When Span 20 was added to a fed-batch culture with glycerol as a carbon source (300gL(-1)), erythritol production increased by 15% compared to the culture without the surfactant where it reached 142gL(-1) after 5 days, which corresponded to 0.47gg(-1) yield and productivity of 1.1gL(-1)h(-1). Therefore, it was concluded that Span 20 considerably enhanced the production of this polyol from glycerol.

De novo transcriptome sequencing in Bixa orellana to identify genes involved in methylerythritol phosphate, carotenoid and bixin biosynthesis.

BACKGROUND: Bixin or annatto is a commercially important natural orange-red pigment derived from lycopene that is produced and stored in seeds of Bixa orellana L. An enzymatic pathway for bixin biosynthesis was inferred from homology of putative proteins encoded by differentially expressed seed cDNAs. Some activities were later validated in a heterologous system. Nevertheless, much of the pathway remains to be clarified. For example, it is essential to identify the methylerythritol phosphate (MEP) and carotenoid pathways genes. RESULTS: In order to investigate the MEP, carotenoid, and bixin pathways genes, total RNA from young leaves and two different developmental stages of seeds from B. orellana were used for the construction of indexed mRNA libraries, sequenced on the Illumina HiSeq 2500 platform and assembled de novo using Velvet, CLC Genomics Workbench and CAP3 software. A total of 52,549 contigs were obtained with average length of 1,924 bp. Two phylogenetic analyses of inferred proteins, in one case encoded by thirteen general, single-copy cDNAs, in the other from carotenoid and MEP cDNAs, indicated that B. orellana is closely related to sister Malvales species cacao and cotton. Using homology, we identified 7 and 14 core gene products from the MEP and carotenoid pathways, respectively. Surprisingly, previously defined bixin pathway cDNAs were not present in our transcriptome. Here we propose a new set of gene products involved in bixin pathway. CONCLUSION: The identification and qRT-PCR quantification of cDNAs involved in annatto production suggest a hypothetical model for bixin biosynthesis that involve coordinated activation of some MEP, carotenoid and bixin pathway genes. These findings provide a better understanding of the mechanisms regulating these pathways and will facilitate the genetic improvement of B. orellana.

Cloning and expression analysis of terpenoid metabolism-related gene NsyCMS in Nicotiana sylvestris.

Terpenoids constitute the main class of secondary metabolites produced in plants with industrial, pharmacological, and agricultural interests. Nicotiana sylvestris has been widely adopted as a diploid model system in plant biology for studies of terpenoid biosynthesis. In this paper, we report the isolation and analysis of the 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (CMS) gene of the MEP (methylerythritol 4-phosphate) pathway from N. sylvestris. We used homologous-based cloning with a RACE method to obtain the full-length coding sequence of the NsCMS. Then, the physical and chemical properties, function, and three-dimensional structure of the NsyCMS protein were predicted. Fluorogenic quantitative PCR was used to conduct an expression analysis at different developmental stages of various tissues of the NsyCMS. The sequence of the NsyCMS consists of a 954-bp open reading frame and encodes a predicted protein of 317 amino acids, with a molecular weight of approximately 49.6 kDa and pi of 6.92. The in vivo localization of the encoded protein was cytoplasmic with no signal peptide, whereas 2 transmembrane regions were found in NsyCMS. The conserved domains of typical 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, aminotransferase, and pyridoxal phosphate-dependent transferase were found in NsyCMS. Differential expression patterns of the NsyCMS were observed throughout the different developmental stages and tissues. NsyCMS messenger RNA was expressed in all tissues, with the highest level of expression in the seedling leaves. NsyMK was expressed at a higher level in the resettling roots. The results from our study set the foundation for exploring the terpenoid biosynthetic pathways in N. sylvestris.

Novel bioassay for the discovery of inhibitors of the 2-C-methyl-D-erythritol 4-phosphate (MEP) and terpenoid pathways leading to carotenoid biosynthesis.

The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.

Primary and secondary organics in the tropical Amazonian rainforest aerosols: chiral analysis of 2-methyltetraols.

This work presents the application of a new method to facilitate the distinction between biologically produced (primary) and atmospherically produced (secondary) organic compounds in ambient aerosols based on their chirality. The compounds chosen for this analysis were the stereomers of 2-methyltetraols, (2R,3S)- and (2S,3R)-methylerythritol, (l- and d-form, respectively), and (2S,3S)- and (2R,3R)-methylthreitol (l- and d-form), shown previously to display some enantiomeric excesses in atmospheric aerosols, thus to have at least a partial biological origin. In this work PM10 aerosol fractions were collected in a remote tropical rainforest environment near Manaus, Brazil, between June 2008 and June 2009 and analysed. Both 2-methylerythritol and 2-methylthreitol displayed a net excess of one enantiomer (either the l- or the d-form) in 60 to 72% of these samples. These net enantiomeric excesses corresponded to compounds entirely biological but accounted for only about 5% of the total 2-methyltetrol mass in all the samples. Further analysis showed that, in addition, a large mass of the racemic fractions (equal mixtures of d- and l-forms) was also biological. Estimating the contribution of secondary reactions from the isomeric ratios measured in the samples (=ratios 2-methylthreitol over 2-methylerythritol), the mass fraction of secondary methyltetrols in these samples was estimated to a maximum of 31% and their primary fraction to a minimum of 69%. Such large primary fractions could have been expected in PM10 aerosols, largely influenced by biological emissions, and would now need to be investigated in finer aerosols. This work demonstrates the effectiveness of chiral and isomeric analyses as the first direct tool to assess the primary and secondary fractions of organic aerosols.

Biotechnological and in situ food production of polyols by lactic acid bacteria.

Polyols such as mannitol, erythritol, sorbitol, and xylitol are naturally found in fruits and vegetables and are produced by certain bacteria, fungi, yeasts, and algae. These sugar alcohols are widely used in food and pharmaceutical industries and in medicine because of their interesting physicochemical properties. In the food industry, polyols are employed as natural sweeteners applicable in light and diabetic food products. In the last decade, biotechnological production of polyols by lactic acid bacteria (LAB) has been investigated as an alternative to their current industrial production. While heterofermentative LAB may naturally produce mannitol and erythritol under certain culture conditions, sorbitol and xylitol have been only synthesized through metabolic engineering processes. This review deals with the spontaneous formation of mannitol and erythritol in fermented foods and their biotechnological production by heterofermentative LAB and briefly presented the metabolic engineering processes applied for polyol formation.

Metabolic routes affecting rubber biosynthesis in Hevea brasiliensis latex.

The cytosolic mevalonate (MVA) pathway in Hevea brasiliensis latex is the conventionally accepted pathway which provides isopentenyl diphosphate (IPP) for cis-polyisoprene (rubber) biosynthesis. However, the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway may be an alternative source of IPP since its more recent discovery in plants. Quantitative RT-PCR (qRT-PCR) expression profiles of genes from both pathways in latex showed that subcellular compartmentalization of IPP for cis-polyisoprene synthesis is related to the degree of plastidic carotenoid synthesis. From this, the occurrence of two schemes of IPP partitioning and utilization within one species is proposed whereby the supply of IPP for cis-polyisoprene from the MEP pathway is related to carotenoid production in latex. Subsequently, a set of latex unique gene transcripts was sequenced and assembled and they were then mapped to IPP-requiring pathways. Up to eight such pathways, including cis-polyisoprene biosynthesis, were identified. Our findings on pre- and post-IPP metabolic routes form an important aspect of a pathway knowledge-driven approach to enhancing cis-polyisoprene biosynthesis in transgenic rubber trees.