Expression of the Chlamydomonas reinhardtii Sedoheptulose‐1,7‐bisphosphatase in Dunaliella bardawil leads to enhanced photosynthesis and increased glycerol production

Bioengineering of photoautotrophic microalgae into CO₂ scrubbers and producers of value‐added metabolites is an appealing approach in low‐carbon economy. A strategy for microalgal bioengineering is to enhance the photosynthetic carbon assimilation through genetically modifying the photosynthetic pathways. The halotolerant microalgae Dunaliella posses an unique osmoregulatory mechanism, which accumulates intracellular glycerol in response to extracellular hyperosmotic stresses. In our study, the Calvin cycle enzyme sedoheptulose 1,7‐bisphosphatase from Chlamydomonas reinhardtii (CrSBPase) was transformed into Dunaliella bardawil, and the transformant CrSBP showed improved photosynthetic performance along with increased total organic carbon content and the osmoticum glycerol production. The results demonstrate that the potential of photosynthetic microalgae as CO₂ removers could be enhanced through modifying the photosynthetic carbon reduction cycle, with glycerol as the carbon sink.     

Isolation and characterisation of components of the <Emphasis Type="Italic">Dunaliella tertiolecta</Emphasis> chloroplast genome

Three chloroplast genes, psbA, psbB and rbcL, of the microalgae Dunaliella tertiolecta were targeted with the view to using these components in the construction of a chloroplast transformation vector. The three genes and surrounding genomic regions were isolated by screening libraries and using degenerate primers to amplify by PCR conserved coding regions and unknown flanking sequences. The putative Dunaliella psbA, psbB and rbcL proteins show high levels of sequence conservation sharing approximately 87, 92 and 97% similarity to the homologues of Chlamydomonas reinhardtii. Interestingly, four of the five introns of the psbA gene contain long open-reading frames which have sequence similarity to the H-N-H and GIY-YIG site-specific homing endonucleases suggesting that, like other microalgae, the Dunaliella gene contains group I introns. Putative promoter regions of the psbB and rbcL genes were isolated and found to contain the required signals necessary for gene expression.     

The unusual di‐domain structure of Dunaliella salina glycerol‐3‐phosphate dehydrogenase enables direct conversion of dihydroxyacetone phosphate to glycerol

Dunaliella has been extensively studied due to its intriguing adaptation to high salinity. Its di‐domain glycerol‐3‐phosphate dehydrogenase (GPDH) isoform is likely to underlie the rapid production of the osmoprotectant glycerol. Here, we report the structure of the chimeric Dunaliella salina GPDH (DsGPDH) protein featuring a phosphoserine phosphatase‐like domain fused to the canonical glycerol‐3‐phosphate (G3P) dehydrogenase domain. Biochemical assays confirm that DsGPDH can convert dihydroxyacetone phosphate (DHAP) directly to glycerol, whereas a separate phosphatase protein is required for this conversion process in most organisms. The structure of DsGPDH in complex with its substrate DHAP and co‐factor nicotinamide adenine dinucleotide (NAD) allows the identification of the residues that form the active sites. Furthermore, the structure reveals an intriguing homotetramer form that likely contributes to the rapid biosynthesis of glycerol.     

The crystal structure of galactitol-1-phosphate 5-dehydrogenase from Escherichia coli K12 provides insights into its anomalous behavior on IMAC processes

Endogenous galactitol-1-phosphate 5-dehydrogenase (GPDH) (EC 1.1.1.251) from Escherichia coli spontaneously interacts with Ni²⁺-NTA matrices becoming a potential contaminant for recombinant, target His-tagged proteins. Purified recombinant, untagged GPDH (rGPDH) converted galactitol into tagatose, and d-tagatose-6-phosphate into galactitol-1-phosphate, in a Zn²⁺- and NAD(H)-dependent manner and readily crystallized what has permitted to solve its crystal structure. In contrast, N-terminally His-tagged GPDH was marginally stable and readily aggregated. The structure of rGPDH revealed metal-binding sites characteristic from the medium-chain dehydrogenase/reductase protein superfamily which may explain its ability to interact with immobilized metals. The structure also provides clues on the harmful effects of the N-terminal His-tag.

Structured summary of protein interactions

GPDH and GPDHbind by molecular sieving (View interaction)GPDH and GPDHbind by x-ray crystallography (View interaction)GPDH and GPDHbind by cosedimentation in solution (View interaction)     

Glycerol‐3‐phosphate metabolism plays a role in stress response in the red alga Pyropia haitanensis

Glycerol‐3‐phosphate (G3P) has been suggested as a novel regulator of plant defense signaling, however, its role in algal resistance remains largely unknown. The glycerol kinase (also designated as NHO1) and NAD‐dependent G3P dehydrogenase (GPDH) are two key enzymes involved in the G3P biosynthesis. In our study, we cloned the full‐length cDNA of NHO1 (NHO1_Ph) and GPDH (GPDH_Ph) from the red alga Pyropia haitanensis (denoted as NHO1_Ph and GPDH_Ph) and examined their expression level under flagellin peptide 22 (flg22) stimulation or heat stress. We also measured the level of G3P and floridoside (a downstream product of G3P in P. haitanensis) under flg22 stimulation or heat stress. Both NHO1_Ph and GPDH_Ph shared high sequence identity and structural conservation with their orthologs from different species, especially from red algae. Phylogenetic analysis showed that NHO1s and GPDHs from red algae were closely related to those from animals. Under flg22 stimulation or heat stress, the expression levels of NHO1_Ph and GPDH_Ph were up‐regulated, G3P levels increased, and the contents of floridoside decreased. But the floridoside level increased in the recovery period after heat stress. Taken together, we found that G3P metabolism was associated with the flg22‐induced defense response and heat stress response in P. haitanensis, indicating the general conservation of defense response in angiosperms and algae. Furthermore, floridoside might also participate in the stress resistance of P. haitanensis.     

Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina

A cDNA encoding a nicotinamide adenine dinucleotide (NAD+) -dependent glycerol 3-phosphate dehydrogenase (GPDH) has been cloned by rapid amplification of cDNA ends from Dunaliella salina. The cDNA is 3032 base pairs long with an open reading frame encoding a polypeptide of 701 amino acids. The polypeptide shows high homology with published NAD+ -dependent GPDHs and has at its N-terminal a chloroplast targeting sequence. RNA gel blot analysis was performed to study GPDH gene expression under different conditions, and changes of the glycerol content were monitored. The results indicate that the cDNA may encode an osmoregulated isoform primarily involved in glycerol synthesis. The 701-amino-acid polypeptide is about 300 amino acids longer than previously reported plant NAD+ -dependent GPDHs. This 300-amino-acid fragment has a phosphoserine phosphatase domain. We suggest that the phosphoserine phosphatase domain functions as glycerol 3-phosphatase and that, consequently, NAD+ -dependent GPDH from D. salina can catalyze the step from dihydroxyacetone phosphate to glycerol directly. This is unique and a possible explanation for the fast glycerol synthesis found in D. salina.     

The <Emphasis Type="Italic">Dunaliella salina</Emphasis> organelle genomes: large sequences,inflated with intronic and intergenic DNA

Background  

Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri.     

Glycerol-3-phosphatase and the control of glycerol metabolism in Dunaliella tertiolecta cells

Glycerol-3-phosphatase (EC 3.1.3.2.1) was studied by following the release of radioactive glycerol from L-(U-¹⁴C)glycerol-3-phosphate in Dunaliella tertiolecta enzyme extracts. The reaction showed a neutral pH optimum and had an absolute requirement for Mg²⁺. The substrate saturation curve was hyperbolic with an apparent K _m value for glycerol-3-phosphate of 0.7 mM in the absence of phosphate. Inorganic orthophosphate was a competitive inhibitor of the enzyme with an estimated K _j of 0.1 mM. The glycerol-3-phosphatase reaction was blocked nearly completely by millimolar Ca²⁺ concentrations. Ca²⁺ inhibition did not depend on the presence of calmodulin in the reaction medium. The characteristics of glycerol-3-phosphatase are discussed in relation to the regulation of the cyclic glycerol metabolism in Dunaliella cells during periods of osmotic stress.     

盐生杜氏藻3-磷酸甘油脱氢酶基因克隆及其蛋白质结构预测

本文通过对盐生杜氏藻(Dunaliella salina) cDNA文库进行大规模EST测序并结合RACE实验克隆了盐生杜氏藻3-磷酸甘油脱氢酶基因, 并且通过Southern blotting实验确定了该基因在这个物种中拷贝数。通过生物信息学的方法, 预测该基因所编码的蛋白质结构, 验证了该蛋白质具有3-磷酸甘油脱氢酶完整的功能性结构域和磷酸水解酶类结构域。本实验为解释盐生杜氏藻在面临高渗胁迫下快速合成甘油的机制提供了帮助。     

盐生杜氏藻3-磷酸甘油脱氢酶基因克隆及其蛋白质结构预测

本文通过对盐生杜氏藻（Dunaliella salina）cDNA文库进行大规模EST测序并结合RACE实验克隆了盐生杜氏藻3-磷酸甘油脱氢酶基因,并且通过Southern blotting实验确定了该基因在这个物种中拷贝数。通过生物信息学的方法,预测该基因所编码的蛋白质结构,验证了该蛋白质具有3-磷酸甘油脱氢酶完整的功能性结构域和磷酸水解酶类结构域。本实验为解释盐生杜氏藻在面临高渗胁迫下快速合成甘油的机制提供了帮助。     

Functional Properties of Lactate Dehydrogenase from Dunaliella salina and Its Role in Glycerol Synthesis

The dependence of the catalytic properties of lactate dehydrogenase (LDH, EC 1.1.1.27) from a halophilic alga Dunaliella salina, a glycophilic alga Chlamydomonas reinhardtii, and from porcine muscle on glycerol concentration, medium pH, and temperature was investigated. Several chemical properties of the enzyme from D. salina differentiated it from the LDH preparation obtained from C. reinhardtii and any homologous enzymes of plant, animal, and bacterial origin. (1) V _max of pyruvate reduction manifested low sensitivity to the major intracellular osmolyte, glycerol. (2) The affinity of LDH for its coenzyme NADH dropped in the physiological pH region of 6–8. Above pH 8, NADH virtually did not bind to LDH, while the enzyme affinity for pyruvate did not change considerably. (3) The enzyme thermostability was extremely low: LDH was completely inactivated at room temperature within 30 min. The optimum temperature for pyruvate reduction (32°C) was considerably lower than with the enzyme preparations from C. reinhardtii (52°C) and porcine muscle (61°C). (4) NADH greatly stabilized LDH: the ratio of LDH inactivation constants in the absence of the coenzyme and after NADH addition at the optimum temperature in the preparation from D. salina exceeded the corresponding indices of LDH preparations from C. reinhardtii twelve times and from porcine muscle eight times. The authors believe that these LDH properties match the specific metabolism of D. salina which is set at rapid glycerol synthesis under hyperosmotic stress conditions. The increase of cytoplasmic pH value produced in D. salina by the hyperosmotic shock can switch off the terminal reaction of the glycolytic pathway and thus provide for the most efficient utilization of NADH in the cycle of glycerol synthesis. As LDH is destabilized in the absence of NADH, this reaction is also switched off. In the course of alga adaptation to the hyperosmotic shock, glycerol accumulation and the neutralization of intracellular pH stabilize LDH, thus creating the conditions for restoring the complete glycolytic cycle.     

不同品系杜氏藻的多相特征研究

杜氏藻(Dunaliella)是一类独特的嗜盐单细胞真核微藻,为对该藻不同地理来源各品系间的特征进行总结及分类鉴定,挖掘特色品系,研究搜集国内外20株不同品系的杜氏藻,利用PCR扩增内部转录间隔区(ITS)和细胞色素C氧化酶(cox2-3)基因,生物信息学软件构建系统发育树对其进行分子鉴定;形态学方法对其显微结构进行观察,利用生理生化研究方法对杜氏藻盐胁迫下的4个代表性指标(最大光合效率、中性脂含量、β-胡萝卜素含量和3-磷酸甘油磷酸酶活性)进行了测定。结果表明, 20株供试藻均属杜氏藻属, ITS和cox2-3的系统发育结果相似,均聚为两大簇,各品系间亲缘关系较为接近;成熟期的D13细胞最大, D14细胞最小并呈长颈形,颜色以绿色或黄绿色为主,鞭毛和眼点各异; D6和D10生长周期短, D18耐盐性最强; D7最大光合效率最高, D6和D18中性脂干重最高, D11的β-胡萝卜含量最高, D7的3-磷酸甘油磷酸酶活性最强。研究结果可为国内外杜氏藻资源的分类鉴定、特色资源的保护、开发与利用奠定基础。     

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.