Glycerol Cycle Research Articles

Genomic adaptations of the green alga Dunaliella salina to life under high salinity

Life in high salinity environments poses challenges to cells in a variety of ways: maintenance of ion homeostasis and nutrient acquisition, often while concomitantly enduring saturating irradiances. Dunaliella salina has an exceptional ability to thrive even in saturated brine solutions. This ability has made it a model organism for studying responses to abiotic stress factors. Here we describe the occurrence of unique gene families, expansion of gene families, or gene losses that might be linked to osmoadaptive strategies. We discovered multiple unique genes coding for several of the homologous superfamily of the Ser-Thr-rich glycosyl-phosphatidyl-inositol-anchored membrane family and of the glycolipid 2-alpha-mannosyltransferase family, suggesting that such components on the cell surface are essential to life in high salt. Gene expansion was found in families that participate in sensing of abiotic stress and signal transduction in plants. One example is the patched family of the Sonic Hedgehog receptor proteins, supporting a previous hypothesis that plasma membrane sterols are important for sensing changes in salinities in D. salina. We also investigated genome-based capabilities regarding glycerol metabolism and present an extensive map for core carbon metabolism. We postulate that a second broader glycerol cycle exists that also connects to photorespiration, thus extending the previously described glycerol cycle. Further genome-based analysis of isoprenoid and carotenoid metabolism revealed duplications of genes for 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and phytoene synthase (PSY), with the second gene copy of each enzyme being clustered together. Moreover, we identified two genes predicted to code for a prokaryotic-type phytoene desaturase (CRTI), indicating that D. salina may have eukaryotic and prokaryotic elements comprising its carotenoid biosynthesis pathways. In brief, our genomic data provide the basis for further gene discoveries regarding sensing abiotic stress, the metabolism of this halophilic alga, and its potential in biotechnological applications.

Metabolic Engineering of Saccharomyces cerevisiae for Redox Balance of Xylose Fermentation

The bioconversion of cellulosic biomass hydrolyzates consisting mainly of glucose and xylose requires the use of engineered Saccharomyces cerevisiae expressing a heterologous xylose pathway. However, there is concern that a fungal xylose pathway consisting of NADPH-specific xylose reductase (XR) and NAD+-specific xylitol dehydrogenase (XDH) may result in a cellular redox imbalance. However, the glycerol biosynthesis and glycerol degradation pathways of S. cerevisiae, termed here as the glycerol cycle, has the potential to balance the cofactor requirements for xylose metabolism, as it produces NADPH by consuming NADH at the expense of one mole of ATP. Therefore, this study tested if the glycerol cycle could improve the xylose metabolism of engineered S. cerevisiae by cofactor balancing, as predicted by an in-silico analysis using elementary flux mode (EFM). When the GPD1 gene, the first step of the glycerol cycle, was overexpressed in the XR/XDH-expressing S. cerevisiae, the glycerol production significantly increased, while the xylitol and ethanol yields became negligible. The reduced xylitol yield suggests that enough NAD+ was supplied for XDH by the glycerol cycle. However, the GPD1 overexpression completely shifted the carbon flux from ethanol to glycerol. Thus, moderate expression of GPD1 may be necessary to achieve improved ethanol production through the cofactor balancing.

Glycerol-3-phosphatase and not lipid recycling is the primary pathway in the accumulation of high concentrations of glycerol in rainbow smelt (Osmerus mordax)

Rainbow smelt is a small fish that accumulates glycerol in winter as a cryoprotectant when the animal is in seawater. Glycerol is synthesized in liver from different substrates that all lead to the formation of glycerol-3-phosphate (G3P). This study assesses whether glycerol is produced by a direct dephosphorylation of G3P by a phosphatase (G3Pase) or by a cycling through the glycerolipid pool followed by lipolysis. Foremost, concentrations of on-board glycerolipids and activity of G3Pase and of enzymes involved in lipid metabolism were measured in smelt liver over the glycerol cycle. Concentrations of on-board glycerolipids did not change over the cycle and were too low to significantly contribute directly to glycerol production but activities of enzymes involved in both potential pathways were up-regulated at the onset of glycerol accumulation. A second experiment conducted with isolated hepatic cells producing glycerol showed 1) that on-board glycerolipids were not sufficient to produce the glycerol released even though phospholipids could account for up to 17% of it, 2) that carbon cycling through the glycerolipid pool was not involved as glycerol was produced at similar rates following inhibition of this pathway, and 3) that G3Pase activity measured was sufficient to allow the synthesis of glycerol at the rate observed. These results are the first to clearly support G3Pase as the metabolic step leading to glycerol production in rainbow smelt and the first to provide strong support for a G3Pase in any animal species.

Correction: Comparative Analysis on the Key Enzymes of the Glycerol Cycle Metabolic Pathway in Dunaliella salina under Osmotic Stresses

Correction: Comparative Analysis on the Key Enzymes of the Glycerol Cycle Metabolic Pathway in Dunaliella salina under Osmotic Stresses

Comparative Analysis on the Key Enzymes of the Glycerol Cycle Metabolic Pathway in Dunaliella salina under Osmotic Stresses

The glycerol metabolic pathway is a special cycle way; glycerol-3-phosphate dehydrogenase (G3pdh), glycerol-3-phosphate phosphatase (G3pp), dihydroxyacetone reductase (Dhar), and dihydroxyacetone kinase (Dhak) are the key enzymes around the pathway. Glycerol is an important osmolyte for Dunaliella salina to resist osmotic stress. In this study, comparative activities of the four enzymes in D. salina and their activity changes under various salt stresses were investigated, from which glycerol metabolic flow direction in the glycerol metabolic pathway was estimated. Results showed that the salinity changes had different effects on the enzymes activities. NaCl could stimulate the activities of all the four enzymes in various degrees when D. salina was grown under continuous salt stress. When treated by hyperosmotic or hypoosmotic shock, only the activity of G3pdh in D. salina was significantly stimulated. It was speculated that, under osmotic stresses, the emergency response of the cycle pathway in D. salina was driven by G3pdh via its response to the osmotic stress. Subsequently, with the changes of salinity, other three enzymes started to respond to osmotic stress. Dhar played a role of balancing the cycle metabolic pathway by its forward and backward reactions. Through synergy, the four enzymes worked together for the effective flow of the cycle metabolic pathways to maintain the glycerol requirements of cells in order to adapt to osmotic stress environments.

Molecular Analysis, Tissue Profiles, and Seasonal Patterns of Cytosolic and Mitochondrial GPDH in Freeze-Resistant Rainbow Smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) is an anadromous teleost that, beginning in late fall, accumulates plasma glycerol in excess of 200 mM, which subsequently decreases in the spring. The activity of cytosolic glycerol-3-phosphate dehydrogenase (cGPDH) is higher (i) in liver of smelt than in that of Atlantic salmon and capelin (nonglycerol accumulators), (ii) in liver of smelt maintained at 1°C than in that of smelt held at 8°-10°C, and (iii) in smelt liver than in smelt muscle, heart, brain, or kidney. In addition, transcript levels of cGPDH in liver peak in December during the onset of glycerol production and then decline over the remainder of the season. There are four cGPDH protein isoforms in smelt liver that are present regardless of glycerol production status. A minimum of four cGPDH gene copies identified by Southern blotting provide adequate genetic potential to yield multiple protein isoforms. A full-length cDNA for smelt mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) was cloned and characterized. The 2,790-bp cDNA contains a 109-bp 5'UTR, a 2,193-bp open reading frame, and a 488-bp 3'UTR; transcripts are ubiquitously expressed in both warm- and cold-acclimated smelt tissues. Smelt mGPDH encodes a 730-aa protein that clusters with that of zebrafish and frog and contains several common structural motifs. mGPDH transcript levels generally increase late in the seasonal glycerol cycle, and mGPDH enzyme activity increases significantly during the glycerol decrease phase. Taken together, these findings suggest that liver cGPDH and mGPDH play a key role in the glycerol accumulation and decrease phases, respectively.

Glycerol loss to water exceeds glycerol catabolism via glycerol kinase in freeze-resistant rainbow smelt (Osmerus mordax)

Rainbow smelt accumulate high amounts of glycerol in winter. In smelt, there is a predictable profile of plasma glycerol levels that starts to increase in November (<5 μmol/ml), peaks in mid-February (>200 μmol/ml), and thereafter decreases to reach the initial levels in the beginning of May. The aim of this study was to investigate the respective role of the two main mechanisms that might be involved in glycerol clearance from mid-February: 1) breakdown of glycerol to glycerol-3-phosphate through the action of the glycerol kinase (GK) and 2) direct loss toward the environment. Over the entire glycerol cycle, loss to water represents a daily loss of ∼10% of the total glycerol content of fish. GK activities were very low in all tissues investigated and likely have a minor quantitative role in the glycerol cycle. These results suggest that glycerol levels are dictated by the rate of glycerol synthesis (accelerated and deactivated during the accumulation and decrease stages, respectively). Although not important in glycerol clearance, GK in liver might have an important metabolic function for other purposes, such as gluconeogenesis, as evidenced by the significant increase of activity at the end of the cycle.

Glycerol cycle enzymes and intermediates during adaption of Dunaliella teriolecta cells to hyperosmotic stress

Abstract Dunaliella tertiolecta cells subjected to a hyperosmotic shock of 0.930 osmol kg−1 start almost immediately to synthesize glycerol at a rate of some 100 nmol min−1 mg protein−1. Glycerol synthesis was equally fast in both light and darkness, and was not affected by the nature but only by the concentration of solutes. During the period of rapid glycerol synthesis, which lasted about 1h, the concentration of glycerol‐3‐phosphate transiently increased. During the same period, ATP, fructose 1‐6‐bisphosphate, and triosephosphate content decreased markedly, especially when 0.1 kmol m−3 NaCl‐grown cells were used. The content of hexose‐6‐phosphates, nicotinamide coenzymes, and phosphate underwent no dramatic changes. Since no in vitro activity changes of the glycerol cycle enzymes could be detected during the adaptation period, the activity of glycerol‐3‐phosphate dehydrogenase in vivo is probably increased by a change in concentration of its effectors such as ATP.

Glycerol cycle enzymes and intermediates during adaption of Dunaliella teriolecta cells to hyperosmotic stress

Glycerol cycle enzymes and intermediates during adaption of Dunaliella teriolecta cells to hyperosmotic stress

Osmoregulation in eukaryotic algae

The cells of marine and halotolerant eukaryotic algae can achieve osmotic balance by ion accumulation mechanisms or by the synthesis and degradation of compatible solutes. The latter mechanism has been extensively studied in Dunaliella tertiolecta, in which the compatible solute glycerol is synthesised and metabolised through the glycerol cycle. Osmoregulation in Poterioochromonas malhamensis by isofloridoside as the compatible solute has a different control mechanism. The results obtained with unicellular algae might lead to strategies for the improvement of salt and water stress resistance in crop plants.

Osmoregulation in eukaryotic algae

The cells of marine and halotolerant eukaryotic algae can achieve osmotic balance by ion accumulation mechanisms or by the synthesis and degradation of compatible solutes. The latter mechanism has been extensively studied in Dunaliella tertiolecta, in which the compatible solute glycerol is synthesised and metabolised through the glycerol cycle. Osmoregulation in Poterioochromonas malhamensis by isofloridoside as the compatible solute has a different control mechanism. The results obtained with unicellular algae might lead to strategies for the improvement of salt and water stress resistance in crop plants.

Glycerol‐3‐phosphate dehydrogenase (EC 1.1.1.8) from Dunaliella tertiolecta. I. Purification and kinetic properties

A rapid purification procedure for glycerol‐3‐phosphate dehydrogenase from Dunaliella tertiolecta (strain 19‐6 of the algal collection of the Univ. of Göttingen), the initial enzyme in the glycerol cycle, has been developed on the basis of affinity chromatography on Blue Sepharose and subsequent desalting by Sephadex G‐50. The achieved purification was 126‐fold. The pH optimum of dihydroxyacetone phosphate reduction is 7, that of glycerol‐3‐phosphate oxidation is about 9. The in vitro enzymatic activity obtained from cell extracts is higher than the required activity for the observed glycerol production rates under osmotic stress in vivo.

Osmoregulation in Dunaliella. Intracellular Distribution of Enzymes of Glycerol Metabolism

Abstract Dunaliella tertiolecta was disrupted mechanically and resolved by centrifugation into chloro­plast- and cytosol-enriched fractions. Intact chloroplasts could not be isolated because peripheral extensions of the single large chloroplast reached almost to the flagellar pole of the cell. The chloroplast envelope was closely appressed to the plasmalemma and, because of this and its dimensions, was vulnerable to mechanical damage to the cell. Distribution of enzymes of the glycerol cycle between the two fractions was compared with that of two marker enzymes, phosphoenolpyruvate carboxylase (cytosol) and ribulose bisphosphate carboxylase (chloroplast). The two reversible steps of the cycle were found to be spatially separated; glycerol-3-phosphate dehydrogenase (NAD-specific) was located in the chloroplast whereas glycerol dehydrogenase (NADP-specific) was located in the cytosol. The distribution of the two irreversible enzymes, glycerol phosphate phosphatase and dihydroxyacetone kinase is uncertain. These enzymes might occur about equally in both major compartments (cytoplasm and chloroplast) or be mitochondrial or they might be loosely associated with a membrane system. Implications of these results for regulation of the glycerol cycle are discussed.

The Intracellular Distribution of Enzymes of the Glycerol Cycle in the Unicellular Alga Dunaliella parva

Abstract The intracellular distribution of the enzymes of the glycerol cycle in the unicellular alga Dunaliella parva was investigated using the technique of differential extraction by the polycation DEAE-dextran. Results suggest a cytoplasmic localization of the DHA reductase, but a chloroplastic localization of the glycerol-3-phosphate dehydrogenase and phosphatase.

DEAE-Dextran Induced Increase of Membrane Permeability and Inhibition of Photosynthesis in Dunaliella parva

Abstract As a prerequisite for studies of the intracellular distribution of enzym es of the glycerol cycle in Dunaliella parva, the effect of the polycation DEAE-dextran on the permeability of membranes for various endogenous compounds and on photosynthesis was investigated. DEAE-dextran induces an increase of the permeability of membranes for low and high molecu­lar weight compounds: Under the influence of DEAE-dextran low molecular com pounds of the cells diffuse more rapidly along the chemical concentration gradient into the medium than macro-molecules. Furthermore enzymes of the cytoplasm do occur more rapidly in the outer medium than enzymes of the chloroplasts. Photosynthetic CO2 fixation is inhibited already at low DEAE-dextran concentrations. This in­ direct inhibition is due to an unspecific efflux of compounds of low molecular weight, such as inorganic cations, metabolites or nucleotides. At high DEAE-dextran concentrations photo­ synthetic electron transport is directly affected at the level of the thylakoids: Electron transport is inhibited between plastoquinone and photosystem I. The efflux characteristics of marker enzymes of DEAE-dextran treated D. parva cells show that under optimal experimental conditions this technique may be a suitable tool to get information about the intracellular distribution of enzym es of unknown localization in the alga.

Effect of 2,3-dinitrilo-1,4-dithia-9,10-anthraquinone on Mycobacterium smegmatis.

2,3-Dinitrilo-1,4-dithia-9,10-anthraquinone (DDA) is an effective inhibitor of respiration of intact cells of Mycobacterium smegmatis in the presence of glucose, glycerol, pyruvate, acetate and other citric acid cycle intermediates or substrates associated with this cyclic (glutamate, asparagine). DDA inhibits the incorporation of both 14C-leucine and 14C-adenine into appropriate macromolecules of M. smegmatis (TCA-precipitable fractions), and causes a drop in the incorporated activity of U-14C-glycine or its degradation products in all the cell fractions studied (lipids, RNA, DNA, proteins). DDA suppresses the growth of M. smegmatis probably through an interference with the cell energy-carbon metabolism.