Model‐assisted identification of metabolic engineering strategies for Jatropha curcas lipid pathways

Efficient approaches to increase plant lipid production are necessary to meet current industrial demands for this important resource. While Jatropha curcas cell culture can be used for in vitro lipid production, scaling up the system for industrial applications requires an understanding of how growth conditions affect lipid metabolism and yield. Here we present a bottom‐up metabolic reconstruction of J. curcas supported with labeling experiments and biomass characterization under three growth conditions. We show that the metabolic model can accurately predict growth and distribution of fluxes in cell cultures and use these findings to pinpoint energy expenditures that affect lipid biosynthesis and metabolism. In addition, by using constraint‐based modeling approaches we identify network reactions whose joint manipulation optimizes lipid production. The proposed model and computational analyses provide a stepping stone for future rational optimization of other agronomically relevant traits in J. curcas.     

In vitro somatic embryogenesis of Texas ebony (Ebenopsis ebano [Berland.] Barneby & J.W. Grimes)

In Vitro Cellular & Developmental Biology - Plant - Texas ebony (Ebenopsis ebano [Berland.] Barneby & J.W. Grimes) is a member of the Fabaceae that is native to Mexico. Its wood has...     

E. coli outbreak in a neonate intensive care unit in a general hospital in Mexico City

Nosocomial infections are a major cause of morbidity and mortality among neonates admitted to neonatal intensive care units (NICUs). The aim of this paper was to describe an outbreak of Escherichia coli among infants admitted to the NICU of the General Hospital “Dr. Manuel Gea Gonzalez” in May of 2008. The isolated E. coli strains were identified using standard biochemical methods. The susceptibilities of these strains were analysed by determining their minimal inhibitory concentrations. Following this, their molecular relationships to each other were assessed by pulsed field gel electrophoresis (PFGE) analysis and corroborated by serology. Twelve E. coli strains were isolated from blood, urine, or indwelling catheter samples from five cases of preterm infants within a 3-day period. Patients were admitted to the NICU of the general hospital and, during the outbreak, developed sepsis caused by E. coli. For four of the patients, the average age was 23 days, while one patient was a 3-month-old infant. Prior to sepsis, the infants had received assisted ventilation and hyperalimentation through a central venous catheter. Two profiles were observed by PFGE; profile A was identified as the outbreak’s cause and an outcome of cross-infection, while profile B showed genetic differences but serologically it was identified as part of the same serotype. We conclude that E. coli colonised the patients through horizontal transmission. A focal source of the microorganism in this outbreak was not identified, but cross-transmission through handling was the most probable route.     

Constrained Unfolding of a Helical Peptide: Implicit versus Explicit Solvents

Steered Molecular Dynamics (SMD) has been seen to provide the potential of mean force (PMF) along a peptide unfolding pathway effectively but at significant computational cost, particularly in all-atom solvents. Adaptive steered molecular dynamics (ASMD) has been seen to provide a significant computational advantage by limiting the spread of the trajectories in a staged approach. The contraction of the trajectories at the end of each stage can be performed by taking a structure whose nonequilibrium work is closest to the Jarzynski average (in naive ASMD) or by relaxing the trajectories under a no-work condition (in full-relaxation ASMD—namely, FR-ASMD). Both approaches have been used to determine the energetics and hydrogen-bonding structure along the pathway for unfolding of a benchmark peptide initially constrained as an α-helix in a water environment. The energetics are quite different to those in vacuum, but are found to be similar between implicit and explicit solvents. Surprisingly, the hydrogen-bonding pathways are also similar in the implicit and explicit solvents despite the fact that the solvent contact plays an important role in opening the helix.     

Mathematical Modeling-Guided Evaluation of Biochemical,Developmental, Environmental,and Genotypic Determinants of Essential Oil Composition and Yield in Peppermint Leaves

We have previously reported the use of a combination of computational simulations and targeted experiments to build a first generation mathematical model of peppermint (Mentha × piperita) essential oil biosynthesis. Here, we report on the expansion of this approach to identify the key factors controlling monoterpenoid essential oil biosynthesis under adverse environmental conditions. We also investigated determinants of essential oil biosynthesis in transgenic peppermint lines with modulated essential oil profiles. A computational perturbation analysis, which was implemented to identify the variables that exert prominent control over the outputs of the model, indicated that the essential oil composition should be highly dependent on certain biosynthetic enzyme concentrations [(+)-pulegone reductase and (+)-menthofuran synthase], whereas oil yield should be particularly sensitive to the density and/or distribution of leaf glandular trichomes, the specialized anatomical structures responsible for the synthesis and storage of essential oils. A microscopic evaluation of leaf surfaces demonstrated that the final mature size of glandular trichomes was the same across all experiments. However, as predicted by the perturbation analysis, differences in the size distribution and the total number of glandular trichomes strongly correlated with differences in monoterpenoid essential oil yield. Building on various experimental data sets, appropriate mathematical functions were selected to approximate the dynamics of glandular trichome distribution/density and enzyme concentrations in our kinetic model. Based on a χ² statistical analysis, simulated and measured essential oil profiles were in very good agreement, indicating that modeling is a valuable tool for guiding metabolic engineering efforts aimed at improving essential oil quality and quantity.The essential oil distilled from peppermint (Mentha × piperita) leaves is used in numerous consumer products (e.g. chewing gum, toothpaste, and mouthwash), as a flavor in the confectionary and pharmaceutical industries, and as a source of active ingredients for aromatherapy. Peppermint oil consists primarily of p-menthane-type monoterpenes, with smaller amounts of other monoterpenes and very minor quantities of sesquiterpenes (Rohloff, 1999). The essential oil is synthesized and accumulated in specialized anatomical structures called peltate glandular trichomes (Gershenzon et al., 1989; McCaskill et al., 1992). These trichomes contain secretory cells, arranged in an eight-celled disc, which are responsible for the synthesis of the oil. Nascent essential oil is secreted into an emerging cavity formed by the separation of a preformed layer of cuticular material (Amelunxen, 1965). Over the last two decades, the entire complement of genes and enzymes involved in the peppermint monoterpenoid essential oil biosynthetic pathway has been characterized (for review, see Croteau et al., 2005).Transgenic peppermint plants have been generated in efforts aimed at modulating essential oil yield and composition. Mahmoud and Croteau (2001) reported that, by overexpressing the gene encoding 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), oil yield increases (compared with wild-type plants) of up to 50% were observed. Antisense suppression of the (+)-menthofuran synthase (MFS) gene led to a dramatic decrease in the amounts of the undesirable side product (+)-menthofuran (elite transgenic line designated MFS7a; Mahmoud and Croteau, 2001). A slight increase in overall monoterpene yields was reported for transgenic plants with increased expression levels of the gene encoding (−)-limonene synthase (LS; Diemer et al., 2001), whereas only negligible effects on yield were detected in an independent study (Krasnyansky et al., 1999). Transgenic plants overexpressing the gene coding for (−)-limonene 3-hydroxylase (L3H) did not accumulate increased levels of the recombinant protein, and the composition and yield of the essential oils were the same as in wild-type controls; however, cosuppression of the L3H gene resulted in a vastly increased accumulation of the intermediate (−)-limonene, without notable effects on oil yield (elite transgenic line designed L3H20; Mahmoud et al., 2004).Mathematical modeling can be a powerful tool to support metabolic engineering efforts, including those performed with peppermint. Stoichiometric modeling only requires knowledge of the topology of reactions in the pathway and inputs/outputs. This is a particularly useful approach to determine flux distributions and the systemic characteristics of metabolic networks (for review, see Llaneras and Picó, 2008). When experimental designs supporting metabolic and isotopic steady state are employed, isotope labeling data can be utilized for the development of quantitative flux maps of metabolic pathways (for review, see Libourel and Shachar-Hill, 2008). For dynamic systems, kinetic modeling is regarded as the generally most suitable method (McNeil et al., 2000; Poolman et al., 2004; Bruggeman and Westerhoff, 2006; Rios-Estepa and Lange, 2007; Mendes et al., 2009). Building on the rich body of published data on the enzymology and physiology of the peppermint monoterpene pathway (for review, see Croteau et al., 2005), we recently developed a first generation kinetic model to simulate the dynamics of peppermint monoterpene composition (Rios-Estepa et al., 2008). Modeling indicated that the monoterpene profiles observed in leaves of plants grown under low-light conditions could be explained if one assumed that (+)-menthofuran, a dead-end side product, acted as a heretofore unknown competitive inhibitor against (+)-pulegone, the primary substrate of the branch point enzyme (+)-pulegone reductase (PR; Fig. 1). Follow-up biochemical studies established that this prediction was correct (Rios-Estepa et al., 2008), thus illustrating the utility of an approach that integrates mathematical modeling with experimental testing.Open in a separate windowFigure 1.Outline of p-menthane monoterpene biosynthesis in peppermint glandular trichomes. The following enzymes are involved in this pathway: 1, 1-deoxy-d-xylulose 5-phosphate synthase; 2, 1-deoxy-d-xylulose 5-phosphate reductoisomerase; 3, 2C-methyl-d-erythritol 4-phosphate cytidyltransferase; 4, 4-(cytidine 5′-diphospho)-2C-methyl-d-erythritol kinase; 5, 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; 6, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate synthase; 7, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase; 8, isopentenyl diphosphate isomerase; 9, geranyl diphosphate synthase; 10, (−)-limonene synthase; 11, (−)-limonene 3-hydroxylase; 12, (−)-trans-isopiperitenol dehydrogenase; 13, (−)-trans-isopiperitenone reductase; 14, (+)-cis-isopulegone isomerase; 15, (+)-menthofuran synthase; 16a, (+)-pulegone reductase [(−)-menthone-forming activity]; 16b, (+)-pulegone reductase [(+)-isomenthone-forming activity]; 17a, (−)-menthone:(−)-menthol reductase [(−)-menthol-forming activity]; 17b, (−)-menthone:(−)-menthol reductase [(+)-neoisomenthol-forming activity]; 18a, (−)-menthone:(+)-neomenthol reductase [(+)-neomenthol-forming activity]; 18b, (−)-menthone:(+)-neomenthol reductase [(+)-isomenthol-forming activity]. The subcellular compartmentation of p-menthane metabolic enzymes is color coded as follows: Cyt (blue), cytosol; ER (orange), endoplasmic reticulum; Lpl (green), leucoplasts; Mit (red), mitochondria. The inhibitory effects of (+)-menthofuran on (+)-pulegone reductase and geranyl diphosphate on isopentenyl diphosphate isomerase are indicated by red arcs with orthogonal red lines. Names of selected metabolites are shown in the colors that are used to indicate the corresponding profiles in Figures 2 to 5​5​​.As part of this study, a computational perturbation analysis was used to predict factors with the potentially greatest impacts on peppermint essential oil yield and composition (specific biosynthetic enzymes and the density of oil-synthesizing trichomes). To test these modeling predictions experimentally, we first acquired biometric data with peppermint plants grown under several environmental conditions known to adversely affect oil accumulation (Burbott and Loomis, 1967; Clark and Menary, 1980) and the transgenic line MFS7a, for which an altered essential oil profile had been reported earlier (Mahmoud and Croteau, 2001). Building on these experimental data sets, we then developed a second generation model that accounts for biochemical, developmental, environmental, and genotypic factors of essential oil formation. This updated model was then used to simulate monoterpenoid essential oil profiles for the transgenic line MFS7a grown under low-light environmental stress conditions and the transgenic line L3H20, which had previously been shown to have vastly reduced expression levels of the gene encoding L3H. In both cases, simulated and measured monoterpene patterns were very similar, indicating that mathematical modeling has great potential for guiding efforts aimed at developing peppermint lines with high oil yields and favorable composition, even under adverse environmental conditions.     

Electropolymerization molecularly imprinted polymer (E-MIP) SPR sensing of drug molecules: pre-polymerization complexed terthiophene and carbazole electroactive monomers

A novel chemosensitive ultrathin film with high selectivity was developed for the detection of naproxen, paracetamol, and theophylline using non-covalent electropolymerized molecular imprinted polymers (E-MIP). A series of monofunctional and bifunctional H-bonding terthiophene and carbazole monomers were compared for imprinting these drugs without the use of a separate cross-linker. A key step is the fast and efficient potentiostatic method of washing the template, which facilitated enhanced real-time sensing by surface plasmon resonance (SPR) spectroscopy. Various surface characterizations (contact angle, ellipsometry, XPS, AFM) of the E-MIP film verified the templating and release of the drug from the cross-linked conducting polymer film.     

Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity

It is generally accepted that copper toxicity is a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber-Weiss reactions. Copper ions display high affinity for thiol and amino groups occurring in proteins. Thus, specialized proteins containing clusters of these groups transport and store copper ions, hampering their potential toxicity. This mechanism, however, may be overwhelmed under copper overloading conditions, in which copper ions may bind to thiol groups occurring in proteins non-related to copper metabolism. In this study, we propose that indiscriminate copper binding may lead to damaging consequences to protein structure, modifying their biological functions. Therefore, we treated liver subcellular membrane fractions, including microsomes, with Cu²⁺ ions either alone or in the presence of ascorbate (Cu²⁺/ascorbate); we then assayed both copper-binding to membranes, and microsomal cytochrome P450 oxidative system and GSH-transferase activities. All assayed sub-cellular membrane fractions treated with Cu²⁺ alone displayed Cu²⁺-binding, which was significantly increased in the presence of Zn²⁺, Hg²⁺, Cd²⁺, Ag⁺¹ and As³⁺. Treatment of microsomes with Cu²⁺ in the μM range decreased the microsomal thiol content; in the presence of ascorbate, Cu²⁺ added in the nM concentrations range induced a significant microsomal lipoperoxidation; noteworthy, increasing Cu²⁺ concentration to ≥50 μM led to non-detectable lipoperoxidation levels. On the other hand, μM Cu²⁺ led to the inhibition of the enzymatic activities tested to the same extent in either presence or absence of ascorbate. We discuss the possible significance of indiscriminate copper binding to thiol proteins as a possible mechanism underlying copper-induced toxicity.     

Demography of the cycad Ceratozamia mirandae (Zamiaceae) under disturbed and undisturbed conditions in a biosphere reserve of Mexico

The cycad Ceratozamia mirandae is endemic to Chiapas, Mexico. Demographic studies were made in two of its populations in the Sepultura Biosphere Reserve under different conservation conditions; in the nucleus zone “Tres Picos” (conserved) and buffer zone “La Sombra” (disturbed and under management). Spatial distribution of C. mirandae was aggregated, showed a clumped local distribution on shallow soils on steep slopes and male and female cones appear to be synchronous in both populations. The population structure was of type I (Bongers) for both sites. Individuals between the sites showed differences in growth pattern. The oldest plants (80–90 cm tall) were estimated to be about 490 years at “La Sombra”. The finite growth rate () in the buffer zone population showed a tendency for decrease whilst in the nucleus zone this estimate remained stable. The highest elasticity values lied in the transition of the first three classes of the “La Sombra” population, in “Tres Picos” this corresponded to adult plants between 20 and 30 cm tall. Given the above, it is proposed that in the nucleus zone, reproductive adults should be of highest conservation priority, whereas in the buffer zone seedling reintroduction should be carried out regularly until the population increases. We recommend an IUCN Red List category of Vulnerable (VU C, 2a), largely due to difficult-to-control destructive annual forest fires that occur in this Reserve.     

Bioconversion of agrowastes by Lentinula edodes: the high potential of viticulture residues

The production of four strains of edible mushroom Lentinula edodes was evaluated through solid-state fermentation (SSF) of vineyard pruning (VP), barley straw (BS), and wheat straw (WS). Biological efficiency, proximal composition, and energy value of the fruiting bodies, as well as substrate chemical changes after harvest, were determined. The shortest primordium formation time (28 days), highest biological efficiency (93.25%), highest yield (37.46%), and shortest production cycle (6 days) were observed in VP. The fruiting bodies obtained from VP had high energy value (379.09 to 392.95 kcal) and contents of protein (12.37 to 17.19%), but low contents of fat (1.82 to 2.15%). After SSF, phenol concentration decreased on VP (1.2 mmol/L) and BS (0.31 mmol/L), but on WS remained practically the same. Hemicellulose decreased in all substrates; cellulose increased on WS and decreased in the rest of the treatments. Lignin decreased on WS and BS, but its concentration increased on VP. The variability observed in the degradation capacity of lignocellulosic components was influenced by the substrate's nature, environmental factors, and genetic factors among strains. VP has great potential for shiitake production due to its low cost, short production cycles, and high biological efficiency.Research was conducted at Instituto de Ecología, AC, and Centro de Investigación en Alimentación y Desarrollo, AC