Membrane lipid biosynthesis in Chlamydomonas reinhardtii. In vitro biosynthesis of diacylglyceryltrimethylhomoserine

Diacylglyceryltrimethylhomo-Ser (DGTS) is an abundant lipid in the membranes of many algae, lower plants, and fungi. It commonly has an inverse concentration relationship with phosphatidylcholine, thus seemingly capable of replacing this phospholipid in these organisms. In some places this replacement is complete; Chlamydomonas reinhardtii is such an organism, and was used for these investigations. We have assayed headgroup incorporation to form DGTS in vitro. The precursor for both the homo-Ser moiety and the methyl groups was found to be S-adenosyl-L-Met. DGTS formation was associated with microsomal fractions and is not in plastids. By analogy with phosphatidylcholine and phosphatidylethanolamine biosynthesis in higher plants, the microsomal activity probably is associated with the endoplasmic reticulum. The pH optimum for the total reaction was between 7.5 and 8.0, and the best temperature was 30 degrees C. The apparent K(m) and V(max) for S-adenosyl-L-Met in the overall reaction were 74 and 250 microM, respectively.     

Effect of green and red light in lipid accumulation and transcriptional profile of genes implicated in lipid biosynthesis in Chlamydomonas reinhardtii

Microalgae have the potential to accumulate triacylglycerols under different light spectra. In this work, Chlamydomonas reinhardtii was grown under white (400–700 nm), red (650 nm), and green (550 nm) lights. According to our results, red light (650 nm) has a positive effect in the microalgae growth and chlorophyll concentration. About the lipid content, the control culture (white light‐illuminated) reached a 4.4% of dry cell weight (dcw), whereas the culture grown at 550 nm showed an increase of 1.35‐fold in the lipids accumulation (5.96% dcw). Interestingly, the most significant accumulation was found in the culture grown at 650 nm (14.78% dcw) which means 3.36‐fold higher with respect to the white light‐illuminated culture. The most abundant fatty acids found in lipid extracts obtained from the cultures under different light wavelength were palmitic (C16: 0), oleic (C18: 1n9), stearidonic (C18: 4), and linoleic (C18: 2), which are useful in the biodiesel production. Changes in gene expression in response to different wavelength illuminations were assessed; however, an in‐depth analysis of a larger number of genes involved in lipid biosynthesis is necessary to fully explain the highest accumulation of lipids in the culture grown under red light. This approach will be useful to find a sustainable source of lipids for biodiesel production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1404–1411, 2016     

Regulation of carotenoid biosynthesis genes in response to light in Chlamydomonas reinhardtii

As we had found previously that thapsigargin, an endomembrane Ca2+-ATPase inhibitor, induces production of intracellular platelet-activating factor (PAF) [Br. J. Pharmacol. 116 (1995) 2141], we decided to investigate the possible roles of intracellular PAF in nuclear factor (NF)-kappaB activation of thapsigargin-stimulated rat peritoneal macrophages. When rat peritoneal macrophages were stimulated with thapsigargin, the level of inhibitory protein of NF-kappaB-alpha (IkappaB-alpha) was decreased and the nuclear translocation of NF-kappaB was increased. The thapsigargin-induced activation of NF-kappaB was inhibited by the PAF synthesis inhibitor SK&F 98625 and the PAF antagonist E6123. Structurally unrelated PAF antagonists such as E5880 and L-652,731 also inhibited the thapsigargin-induced activation of NF-kappaB. Lipopolysaccharide (LPS)-induced activation of NF-kappaB was also suppressed by these drugs. In a culture of rat peritoneal macrophages, exogenously added PAF did not induce degradation of IkappaB-alpha. These findings suggest that the intracellular PAF produced by the stimulation with thapsigargin or LPS is involved in activation of the NF-kappaB pathway.     

Membrane lipid biosynthesis in Chlamydomonas reinhardtii. Partial characterization of CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase

Myo-inositol may be incorporated in the formation of phosphatidylinositol by two mechanisms. One reaction utilizes CDP-diacylglycerol and is catalyzed by phosphatidylinositol (PtdIns) synthase (CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11). The second reaction is the phosphatidylinositol: myo-inositol exchange reaction, in which a free inositol is exchanged for an existing inositol headgroup. This characterization of inositol incorporation into phosphatidylinositol in the green alga Chlamydomonas reinhardtii provides evidence for the presence of both reactions. The transferase reaction required a divalent cation and exhibited its maximum activity at 2.0 mM Mn²⁺. The optimal pH for this reaction was 8.5–9.0. The best substrate concentrations were 0.5 mM CDP-diacylglycerol and 1.2 mM myo-inositol, with an estimated K_m for myo-inositol of 0.2 mM. The exchange reaction also required Mn²⁺ for activity, but became saturated at 0.5 mM Mn²⁺. The optimal pH of the exchange reaction was 8.0, the optimal myo-inositol concentration was 0.3 mM, and the estimated K_m for myo-inositol in this reaction was 0.015 mM. Measurement of the transferase reaction in cell fractions of C. reinhardtii indicated that the activity occurred primarily in the microsomal fraction, with little or no activity in the plastids.     

Membrane lipid biosynthesis in Chlamydomonas reinhardtii: ethanolaminephosphotransferase is capable of synthesizing both phosphatidylcholine and phosphatidylethanolamine

Phosphatidylethanolamine, but not phosphatidylcholine, is found in Chlamydomonas reinhardtii. A cDNA coding for diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase (EPT) was cloned from C. reinhardtii. The C. reinhardtii EPT appears phylogenetically more similar to mammalian aminoalcoholphosphotransferases than to those of yeast and the least close to those of plants. Similar membrane topography was found between the C. reinhardtii EPT and the aminoalcoholphosphotransferases from mammals, yeast, and plants. A yeast mutant deficient in both cholinephosphotransferase and ethanolaminephosphotransferase was complemented by the C. reinhardtii EPT gene. Enzymatic assays of C. reinhardtii EPT from the complemented yeast microsomes demonstrated that the C. reinhardtii EPT synthesized both PC and PE in the transformed yeast. The addition of either unlabeled CDP-ethanolamine or CDP-choline to reactions reduced incorporation of radiolabeled CDP-choline and radiolabeled CDP-ethanolamine into phosphatidylcholine and phosphatidylethanolamine. EPT activity from the transformed yeast or C. reinhardtii cells was inhibited nearly identically by unlabeled CDP-choline, CDP-ethanolamine, and CMP when [14C]CDP-choline was used as the primary substrate, but differentially by unlabeled CDP-choline and CDP-ethanolamine when [14C]CDP-ethanolamine was the primary substrate. The Km value of the enzyme for CDP-choline was smaller than that for CDP-ethanolamine. This provides evidence that C. reinhardtii EPT, similar to plant aminoalcoholphosphotransferase, is capable of catalyzing the final step of phosphatidylcholine biosynthesis, as well as that of phosphatidylethanolamine in the Kennedy pathway.     

Purification of the glutamyl-tRNA reductase from Chlamydomonas reinhardtii involved in delta-aminolevulinic acid formation during chlorophyll biosynthesis

The formation of delta-aminolevulinic acid, the first committed precursor in porphyrin biosynthesis, occurs in certain bacteria and in the chloroplasts of plants and algae in a three-step, tRNA-dependent transformation of glutamate. Glutamyl-tRNA reductase, the second enzyme of this pathway, reduces the activated carboxyl group of glutamyl-tRNA (Glu-tRNA) in the presence of NADPH and releases glutamate 1-semialdehyde (GSA). We have purified Glu-tRNA reductase from Chlamydomonas reinhardtii by employing six different chromatographic separations. The apparent molecular mass of the protein when analyzed under both denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 130,000 Da; this indicates that the active enzyme is a monomer. In the presence of NADPH Glu-tRNA reductase catalyzed the reduction to GSA of glutamate acylated to the homologous tRNA. Thus, the reductase alone is sufficient for conversion of Glu-tRNA to GSA. In the absence of NADPH, a stable complex of Glu-tRNA reductase with Glu-tRNA can be isolated.     

Metabolism controls dimerization of the chloroplast FoF1 ATP synthase in Chlamydomonas reinhardtii

Dimers and oligomers of F-type ATP synthases have been observed previously in mitochondria of various organisms and for the CF(o)F(1) ATP synthase of chloroplasts of Chlamydomonas reinhardtii. In contrast to mitochondria, however, dimers of chloroplast ATP synthases dissociate at elevated phosphate concentration. This suggests a regulation by cell physiological processes. Stable isotope labeling of living cells and blue-native PAGE have been employed to quantitate changes in the ratio of monomeric to dimeric CF(o)F(1) ATP synthase. Chlamydomonas reinhardtii cells were cultivated photoautotrophically in the presence of (15)N and photomixotrophically at natural (14)N abundance, respectively. As compared to photoautotrophic growth, an increased assembly of ATP synthase dimers on the expense of preexisting monomers during photomixotrophic growth was observed, demonstrating a metabolic control of the dimerization process.     

Metabolic engineering of ketocarotenoids biosynthesis in the unicelullar microalga Chlamydomonas reinhardtii

Most higher plants and microalgae are not able to synthesize ketocarotenoids. In this study the unicellular chlorophyte Chlamydomonas reinhardtii has been genetically engineered with the beta-carotene ketolase cDNA from Haematococcus pluvialis, bkt1 (GeneBank accession no. X86782), involved in the synthesis of astaxanthin, to obtain a transgenic microalga able to synthesize ketocarotenoids. The expression of bkt1 was driven by the Chlamydomonas constitutive promoter of the rubisco small subunit (RbcS2) and the resulting protein was directed to the chloroplast by the Chlamydomonas transit peptide sequences of Rubisco small subunit (RbcS2) or Ferredoxin (Fd). In all transformants containing the bkt1 gene fused to the RbcS2 or the Fd transit peptides a new pigment with the typical ketocarotenoid spectrum was detected. Surprisingly this ketocarotenoid was not astaxanthin nor canthaxanthin. The ketocarotenoid was identified on the basis of its mass spectrum as 3,3'-dihydroxy-beta,varepsilon-carotene-4-one (4-keto-lutein) or its isomer ketozeaxanthin.     

Chlamydomonas reinhardtii CNX1E reconstitutes molybdenum cofactor biosynthesis in Escherichia coli mutants

We have isolated and characterized the Chlamydomonas reinhardtii genes for molybdenum cofactor biosynthesis, namely, CNX1G and CNX1E, and expressed them and their chimeric fusions in Chlamydomonas and Escherichia coli. In all cases, the wild-type phenotype was restored in individual mutants as well as in a CNX1G CNX1E double mutant. Therefore, CrCNX1E is the first eukaryotic protein able to complement an E. coli moeA mutant.     

Genome-based examination of chlorophyll and carotenoid biosynthesis in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a particularly important model organism for the study of photosynthesis since this alga can grow heterotrophically, and mutants in photosynthesis are therefore conditional rather than lethal. The recently developed tools for genomic analyses of this organism have allowed us to identify most of the genes required for chlorophyll and carotenoid biosynthesis and to examine their phylogenetic relationships with homologous genes from vascular plants, other algae, and cyanobacteria. Comparative genome analyses revealed some intriguing features associated with pigment biosynthesis in C. reinhardtii; in some cases, there are additional conserved domains in the algal and plant but not the cyanobacterial proteins that may directly influence their activity, assembly, or regulation. For some steps in the chlorophyll biosynthetic pathway, we found multiple gene copies encoding putative isozymes. Phylogenetic studies, theoretical evaluation of gene expression through analysis of expressed sequence tag data and codon bias of each gene, enabled us to generate hypotheses concerning the function and regulation of the individual genes, and to propose targets for future research. We have also used quantitative polymerase chain reaction to examine the effect of low fluence light on the level of mRNA accumulation encoding key proteins of the biosynthetic pathways and examined differential expression of those genes encoding isozymes that function in the pathways. This work is directing us toward the exploration of the role of specific photoreceptors in the biosynthesis of pigments and the coordination of pigment biosynthesis with the synthesis of proteins of the photosynthetic apparatus.     

Characterization of a light-dependent glutamate synthase activity in Chlamydomonas reinhardtii

Photosynthetically active vesicles prepared from Chlamydomonas reinhardtii retained a light-dependent glutamate synthase activity which was highly specific for 2-oxoglutarate (K_m=2.1 mM) and L-glutamine (K_m=0.9 mM) as amido group acceptor and donor respectively. This activity was inhibited by azaserine, p-hydroxymercuribenzoate and 3-(p-chlorophenyl)-1,1-dimethyl urea.Light-dependent synthesis of glutamate was also obtained by coupling Chlamydomonas photosynthetic particles to purified ferredoxin-glutamate synthase, using ascorbate and 2,6-dichlorophenol-indophenol as electron donor. This system was also specific for 2-oxoglutarate (K_m=1 mM) and L-glutamine (K_m=0.8 mM) as substrates, and was stimulated by dithioerythritol. Azaserine and p-hydroxymercuribenzoate, but not 3-(p-chlorophenyl)-1,1-dimethyl urea, inhibited the reconstituted activity; high concentrations of 2-oxoglutarate were inhibitory.Abbreviations A Absorbance - CCP p-Trichlorometoxi-carbonylcyanide-phenylhydrazone - Chl Chlorophyll - CMU 3-(p-Chlorophenyl)-1,1-dimethyl urea - DPIP 2,6-Dichlorophenol-indophenol - DTE Dithioerythritol - MSX L-Methionine, D-L, sulfoximine - MV Methyl viologen     

White mutants of Chlamydomonas reinhardtii are defective in phytoene synthase

Carotenoids play an integral and essential role in photosynthesis and photoprotection in plants and algae. A collection of Chlamydomonas reinhardtii mutants lacking carotenoids was characterized for pigment and tocopherol (vitamin E) composition, growth phenotypes under different light conditions, and the molecular basis of their mutant phenotype. The carotenoid-less mutants, or "white" mutants, were also deficient in chlorophylls but had approximately twice the tocopherol content of the wild type. White mutants grew in the dark but were unable to survive in the light, even under very low light conditions on acetate-containing medium. Genetic crosses and recombination tests revealed that all individual white mutants in the collection are alleles of a single gene, lts1, and the white phenotype was closely linked to a marker located in the phytoene synthase gene. DNA sequencing of the phytoene synthase gene from each of the mutants revealed nonsense, missense, frameshift, and splice site mutations. Transformation with a wild-type copy of the phytoene synthase gene was able to complement the lts1-210 mutation. Together, these results show that all the white mutants examined in this work are affected in the phytoene synthase gene.     

Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii.

Summary We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of nonphotosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.     

Fluorescent measurement of lipid content in the model organism Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is one of the most studied microalgae, which has the potential to be used as a model system to study lipid metabolism. Establishment of a method in this organism for rapid and simple measurement of neutral lipids is desirable. Fluorescent measurement of neural lipids by Nile Red staining has been widely used in various cell types including microalgae. However, a systematic study of Nile Red staining to measure neutral lipids in Chlamydomonas has not been reported. Here, we show that Nile Red staining is suitable for relative and absolute quantification of neutral lipids as well as for possible large-scale screening for mutants defective in lipid accumulation. We have compared and optimized the factors involved Nile Red staining including solvents, cell concentration, staining time, and Nile Red concentration. We determined that 5 % DMSO with 1 μg mL^?1 Nile Red and 5–15-min time window after staining was optimal for measuring lipid content of cells within the range of 1 to 8?×?10⁶ cells mL^?1. The absolute quantification of neutral lipids could be achieved by standard addition method. In addition, we developed a protocol that could be potentially used for large-scale screening for cells with different lipid content. Thus, the work reported here provides timely needed techniques to facilitate Chlamydomonas to be used as a model organism for studying lipid metabolism for biodiesel production.     

Thylakoid membrane biogenesis in Chlamydomonas reinhardtii 137+: cell cycle variations in the synthesis and assembly of polar glycerolipid

The synthesis and assembly of thylakoid membrane polar glycerolipid (glycolipid, phospholipid, and ether lipid) have been monitored in synchronous cultures of the green alga Chlamydomonas reinhardtii 137+. A "pulse" protocol using radioactive acetate as the lipogenic precursor was devised to allow assessment of both processes during the 24-h (12-h light/12-h dark) vegetative cell cycle. Under these conditions, acetate incorporation into each chromatographically resolved lipid at the cellular level reliably reflects lipid synthesis, and the appearance of radiolabeled lipid in purified photosynthetic membrane is indicative of the lipid assembly attendant to thylakoid biogenesis. Our results demonstrate that polar glycerolipid is synthesized by the alga and is assembled into its thylakoid membrane continuously, but differentially, with respect to cell cycle time. Synthesis and assembly are most rapid during the photoperiod (mid-to-late G1), reach maximum rates at mid- photoperiod, and are comparatively negligible in the dark (S, M, and early-to-mid G1). The extent to which synthesis and assembly vary within this general kinetic pattern, though, is characteristic of each thylakoid lipid, suggesting that the processes take place in a multistep manner with some temporal coordination among the different lipid types. Parallelism between the cyclic patterns of polar lipid synthesis at the cellular level and of polar lipid assembly into photosynthetic membrane at the subcellular level indicates that lipid production is not only essential to continuing thylakoid biogenesis but is also the critical determinant of the kinetics of thylakoid lipid assembly.