Engineered ketocarotenoid biosynthesis in the polyextremophilic red microalga Cyanidioschyzon merolae 10D.

The polyextremophilic Cyanidiophyceae are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal ß-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments.

The genomes of polyextremophilic cyanidiales contain 1% horizontally transferred genes with diverse adaptive functions.

The role and extent of horizontal gene transfer (HGT) in eukaryotes are hotly disputed topics that impact our understanding of the origin of metabolic processes and the role of organelles in cellular evolution. We addressed this issue by analyzing 10 novel Cyanidiales genomes and determined that 1% of their gene inventory is HGT-derived. Numerous HGT candidates share a close phylogenetic relationship with prokaryotes that live in similar habitats as the Cyanidiales and encode functions related to polyextremophily. HGT candidates differ from native genes in GC-content, number of splice sites, and gene expression. HGT candidates are more prone to loss, which may explain the absence of a eukaryotic pan-genome. Therefore, the lack of a pan-genome and cumulative effects fail to provide substantive arguments against our hypothesis of recurring HGT followed by differential loss in eukaryotes. The maintenance of 1% HGTs, even under selection for genome reduction, underlines the importance of non-endosymbiosis related foreign gene acquisition.

Phytoene Accumulation in the Novel Microalga Chlorococcum sp. Using the Pigment Synthesis Inhibitor Fluridone.

Carotenoids are lipophilic pigments found in plants and algae, as well as some bacteria, archaea, and fungi that serve two functions-(1) as light harvesting molecules-primary carotenoids, and (2) as antioxidants, acting against reactive oxygen species⁻secondary carotenoids. Because of their strong antioxidant properties, they are also valuable for the development of anti-aging and photo-protective cosmetic applications. Of particular interest is the carotenoid phytoene, for its colorless and UV absorption characteristics. In this study, we targeted a reduction of phytoene desaturase (PDS) activity with the pigment-inhibiting herbicide 1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]pyridin-4-one (fluridone), which leads to the over-accumulation of phytoene in the recently characterized microalgal strain Chlorococcum sp. (UTEX B 3056). After post-incubation with fluridone, phytoene levels were measured at ~33 ug/mg cell tissue, as opposed to non-detectable levels in control cultures. Hence, the novel microalga Chlorococcum sp. is a viable candidate for the production of the high-value carotenoid phytoene and subsequent applications in cosmeceuticals, as well as more obvious nutraceutical and pharmaceutical applications.

Transgenic alfalfa (Medicago sativa) with increased sucrose phosphate synthase activity shows enhanced growth when grown under N2-fixing conditions.

MAIN CONCLUSION: Overexpression of SPS in alfalfa is accompanied by early flowering, increased plant growth and an increase in elemental N and protein content when grown under N2-fixing conditions. Sucrose phosphate synthase (SPS; EC 2.3.1.14) is the key enzyme in the synthesis of sucrose in plants. The outcome of overexpression of SPS in different plants using transgenic approaches has been quite varied, but the general consensus is that increased SPS activity is associated with the production of new sinks and increased sink strength. In legumes, the root nodule is a strong C sink and in this study our objective was to see how increasing SPS activity in a legume would affect nodule number and function. Here we have transformed alfalfa (Medicago sativa, cv. Regen SY), with a maize SPS gene driven by the constitutive CaMV35S promoter. Our results showed that overexpression of SPS in alfalfa, is accompanied by an increase in nodule number and mass and an overall increase in nitrogenase activity at the whole plant level. The nodules exhibited an increase in the level of key enzymes contributing to N assimilation including glutamine synthetase and asparagine synthetase. Moreover, the stems of the transformants showed higher level of the transport amino acids, Asx, indicating increased export of N from the nodules. The transformants exhibited a dramatic increase in growth both of the shoots and roots, and earlier flowering time, leading to increased yields. Moreover, the transformants showed an increase in elemental N and protein content. The overall conclusion is that increased SPS activity improves the N status and plant performance, suggesting that the availability of more C in the form of sucrose enhances N acquisition and assimilation in the nodules.

Hybrid organic/inorganic thin-film multijunction solar cells exceeding 11% power conversion efficiency.

Hybrid multijunction solar cells comprising hydrogenated amorphous silicon and an organic bulk heterojunction are presented, reaching 11.7% power conversion efficiency. The benefits of merging inorganic and organic subcells are pointed out, the optimization of the cells, including optical modeling predictions and tuning of the recombination contact are described, and an outlook of this technique is given.

Impact of concurrent overexpression of cytosolic glutamine synthetase (GS1) and sucrose phosphate synthase (SPS) on growth and development in transgenic tobacco.

MAIN CONCLUSION: The outcome of simultaneously increasing SPS and GS activities in transgenic tobacco, suggests that sucrose is the major determinant of growth and development, and is not affected by changes in N assimilation. Carbon (C) and nitrogen (N) are the major components required for plant growth and the metabolic pathways for C and N assimilation are very closely interlinked. Maintaining an appropriate balance or ratio of sugar to nitrogen metabolites in the cell, is important for the regulation of plant growth and development. To understand how C and N metabolism interact, we manipulated the expression of key genes in C and N metabolism individually and concurrently and checked for the repercussions. Transgenic tobacco plants with a cytosolic soybean glutamine synthetase (GS1) gene and a sucrose phosphate synthase (SPS) gene from maize, both driven by the CaMV 35S promoter were produced. Co-transformants, with both the transgenes were produced by sexual crosses. While GS is the key enzyme in N assimilation, involved in the synthesis of glutamine, SPS plays a key role in C metabolism by catalyzing the synthesis of sucrose. Moreover, to check if nitrate has any role in this interaction, the plants were grown under both low and high nitrogen. The SPS enzyme activity in the SPS and SPS/GS1 co-transformants were the same under both nitrogen regimens. However, the GS activity was lower in the co-transformants compared to the GS1 transformants, specifically under low nitrogen conditions. The GS1/SPS transformants showed a phenotype similar to the SPS transformants, suggesting that sucrose is the major determinant of growth and development in tobacco, and its effect is only marginally affected by increased N assimilation. Sucrose may be functioning in a metabolic capacity or as a signaling molecule.

Nodule-enhanced expression of a sucrose phosphate synthase gene member (MsSPSA) has a role in carbon and nitrogen metabolism in the nodules of alfalfa (Medicago sativa L.).

Sucrose phosphate synthase (SPS) catalyzes the first step in the synthesis of sucrose in photosynthetic tissues. We characterized the expression of three different isoforms of SPS belonging to two different SPS gene families in alfalfa (Medicago sativa L.), a previously identified SPS (MsSPSA) and two novel isoforms belonging to class B (MsSPSB and MsSPSB3). While MsSPSA showed nodule-enhanced expression, both MsSPSB genes exhibited leaf-enhanced expression. Alfalfa leaf and nodule SPS enzymes showed differences in chromatographic and electrophoretic migration and differences in V (max) and allosteric regulation. The root nodules in legume plants are a strong sink for photosynthates with its need for ATP, reducing power and carbon skeletons for dinitrogen fixation and ammonia assimilation. The expression of genes encoding SPS and other key enzymes in sucrose metabolism, sucrose phosphate phosphatase and sucrose synthase, was analyzed in the leaves and nodules of plants inoculated with Sinorhizobium meliloti. Based on the expression pattern of these genes, the properties of the SPS isoforms and the concentration of starch and soluble sugars in nodules induced by a wild type and a nitrogen fixation deficient strain, we propose that SPS has an important role in the control of carbon flux into different metabolic pathways in the symbiotic nodules.

Repercussion of mesophyll-specific overexpression of a soybean cytosolic glutamine synthetase gene in alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.).

Glutamine synthetase (GS) plays a central role in plant nitrogen metabolism. Plant GS occurs as a number of isoenzymes present in either the cytosol (GS1) or chloroplast/plastid (GS2). There are several reports of improved performance in transgenic plants overexpressing GS1 transgenes driven by the constitutive CaMV35S promoter. Improvement has been attributed to the GS1 transgene product functioning to enhance re-assimilation of NH4+ released by photorespiration or protein degradation. In this paper, alfalfa and tobacco transformants expressing a soybean gene driven by a photosynthetic cell-specific promoter have been compared to transformants with the same transgene driven by the stronger CaMV35S promoter. The two classes of alfalfa and tobacco transformants showed differences in the level of GS1 transcript and GS1 protein accumulation, but the difference in the total GS activity was small. The discrepancy in the transgene expression level and GS activity has been attributed to posttranslational regulation at the level of holoprotein stability. Both classes of transformants exhibited similar level of improvement in soluble protein and in the rates of photosynthesis and photorespiration. The data supports the hypothesis that GS1 made in the mesophyll cells is involved in the re-assimilation of NH4+ released via photorespiration and/or protein degradation.

NMR investigation of the behavior of an organothiophosphate pesticide, chlorpyrifos, sorbed on soil components.

Phosphorus-31 nuclear magnetic resonance (31P NMR) was used to follow the decomposition of chlorpyrifos (an organothiophosphate pesticide) adsorbed on soil, humic acid, partially hydrated kaolin clay, and partially hydrated montmorillonite clay at high concentration (typically 2-10 wt %). Solid-state 31P NMR (using magic-angle spinning and cross polarization or direct polarization) and liquid-solution 31P NMR of DMSO and acetone extracts indicate that chlorpyrifos is initially physisorbed, appearing by solid-state 31P NMR to exhibit significant motion on the molecular level, which results in almost liquidlike solid-state spectra. Over periods ranging from hours to years, the signals due to unreacted chlorpyrifos sorbed on the clays diminish and are replaced by new 31P NMR peaks resulting from hydrolysis, isomerization, mineralization, and oxidation reactions. The 31P NMR signal characteristics indicate that these decomposition products are much more tightly bound to the clay than is chlorpyrifos. Solid-state 13C and 27AI NMR spectra were less useful for following the decomposition of chlorpyrifos than those obtained by 31P NMR. Solid-state 31P NMR results indicate that a chlorpyrifos loading level of 10% by weight, used in some of the samples to facilitate 31P NMR detection of less-than-dominant decomposition products, exceeds the adsorption capacity of the soil, humic acid, and kaolinite tested, but not Ca2+ -exchanged montmorillonite. This pattern is consistent with intercalation into the montmorillonite, but only surface adsorption on kaolinite.

NMR investigation of the behavior of an organothiophosphate pesticide, chlorpyrifos, sorbed on montmorillonite clays.

Phosphorus-31 nuclear magnetic resonance (31P NMR) was used to explore the decomposition of chlorpyrifos (an organothiophosphate pesticide) sorbed at high concentration (typically 2-10 wt %) on partially hydrated montmorillonite clays in four different cation-exchanged forms (Ca2+, Cu2+, Zn2+, and Al3+). Solid-state 31P NMR (using magic-angle spinning and cross polarization or direct polarization) and liquid-solution 31P NMR of DMSO and acetone extracts indicate that chlorpyrifos is initially physisorbed, appearing by solid-state 31P NMR to exhibit significant motion on the molecular level, which results in almost liquidlike solid-state spectra. Over periods ranging from hours to years, the signals due to unreacted chlorpyrifos diminish and are replaced by new 31P NMR peaks resulting from hydrolysis, isomerization, mineralization, and oxidation reactions. The 31P NMR signal characteristics indicate that these decomposition products are much more tightly bound to the clay than is chlorpyrifos. Partially hydrated Cu(II)- and Al-montmorillonites most effectively catalyzed chlorpyrifos decomposition (but with different product distributions); Ca-montmorillonites (and, as previously shown, kaolin) were least effective. Solid-state 13C and 27Al NMR spectra were less useful for following the decomposition of chlorpyrifos than those obtained by 31P NMR. Pesticide loading levels (1-10% w/w) that are very much higher than those typically found in the environment were used to facilitate 31P NMR detection of less-than-dominant decomposition species.

NMR investigation of the behavior of an organothiophosphate pesticide, methyl parathion, sorbed on clays.

The decomposition of methyl parathion (an organothiophosphate pesticide) sorbed on partially hydrated kaolin and montmorillonite clays (in Ca2+, Cu2+, Zn2+, and Al3+ forms) at high concentration (typically 1-10 wt %) has been examined by nuclear magnetic resonance (NMR), using solid-state 31P NMR (based on magic-angle spinning and cross polarization or direct polarization) and liquid-state 31P NMR of DMSO and acetone extracts. The results indicate that methyl parathion is initially physisorbed, appearing by solid-state 31P NMR to exhibit substantial molecular-level motion. The signals due to unreacted methyl parathion diminish and are replaced by new 31P NMR peaks resulting from hydrolysis, isomerization, and oxidation reactions over periods ranging from hours to years. 31P NMR characteristics indicate that these decomposition products are much more tightly bound to the clay than is methyl parathion. Methyl parathion decomposition is most effectively catalyzed by partially hydrated Cu(II)- and Al-montmorillonites (but with different product distributions); Ca-montmorillonite and kaolin were least effective.

Quantitative 13C NMR analysis of sequence distributions in poly(ethylene-co-1-hexene).

Different 13C NMR methods of determining triad distributions in two poly(ethylene-co-1-hexene) copolymers are examined using high signal-to-noise ratio 13C NMR spectra of the copolymers dissolved in deuterated 1,2,4-trichlorobenzene at 398 K. This examination includes a comparison of three integration techniques. The experimental impact of decoupler sidebands and significantly nonequal 13C NOE values are examined. A least-squares regression analysis technique for solving for triad mole fractions is tested and appears to be more reliable than two published algebraic expressions (and other expressions examined in the work reported here). The resultant triad mole fractions are compared to sequence distribution parameters expected by Bernoullian and first-order Markovian statistical models. On the basis of 13C NMR-determined average reactivity ratios, the copolymer designated sample B (5.3 mol % 1-hexene) appears to be a Bernoullian copolymer resulting from a single-site catalytic system. The copolymer designated sample S (3.6 mol % 1-hexene overall) is better described as a mixture of polyethylene and a Bernoullian copolymer with 6.4 mol % 1-hexene content, and thus appears to result from a multisite catalytic system.