Metabolic modeling of Chlamydomonas reinhardtii: energy requirements for photoautotrophic growth and maintenance

In this study, a metabolic network describing the primary metabolism of Chlamydomonas reinhardtii was constructed. By performing chemostat experiments at different growth rates, energy parameters for maintenance and biomass formation were determined. The chemostats were run at low irradiances resulting in a high biomass yield on light of 1.25 g  mol⁻¹. The ATP requirement for biomass formation from biopolymers (K _x ) was determined to be 109 mmol g⁻¹ (18.9 mol mol⁻¹) and the maintenance requirement (m _ATP) was determined to be 2.85 mmol g⁻¹ h⁻¹. With these energy requirements included in the metabolic network, the network accurately describes the primary metabolism of C. reinhardtii and can be used for modeling of C. reinhardtii growth and metabolism. Simulations confirmed that cultivating microalgae at low growth rates is unfavorable because of the high maintenance requirements which result in low biomass yields. At high light supply rates, biomass yields will decrease due to light saturation effects. Thus, to optimize biomass yield on light energy in photobioreactors, an optimum between low and high light supply rates should be found. These simulations show that metabolic flux analysis can be used as a tool to gain insight into the metabolism of algae and ultimately can be used for the maximization of algal biomass and product yield.     

Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.     

An efficient way for obtaining transformants of Chlamydomonas reinhardtii by electroporation

The cells of mutant CW-15, which does not form the cell wall, were transformed by electroporation. It was found that the transformation was optimal at a suspension density of 10(6) cells/ml in the middle of the logarithmic growth phase at a field intensity of 1 kW/cm and pulse duration 2 ms. It was shown that, under these conditions, the efficiency of transformation reached 10(3) HygR-clones per 10(6) recipient cells. Exogenic DNA integrated into the genome of the nucleus of Ch. reinhardtii was inherited constantly for more than 350 generations; however, the tolerance to hygromycin appeared as an unstable feature. The advantages of using mutant CW-15 and the selective system to study the transformation of Ch. reinhardtii by heterologous genes are discussed.     

Photosystem I is indispensable for photoautotrophic growth, CO2 fixation, and H2 photoproduction in Chlamydomonas reinhardtii

Certain Chlamydomonas reinhardtii mutants deficient in photosystem I due to defects in psaA mRNA maturation have been reported to be capable of CO2 fixation, H2 photoevolution, and photoautotrophic growth (Greenbaum, E., Lee, J. W., Tevault, C. V., Blankinship, S. L. , and Mets, L. J. (1995) Nature 376, 438-441 and Lee, J. W., Tevault, C. V., Owens, T. G.; Greenbaum, E. (1996) Science 273, 364-367). We have generated deletions of photosystem I core subunits in both wild type and these mutant strains and have analyzed their abilities to grow photoautotrophically, to fix CO2, and to photoevolve O2 or H2 (using mass spectrometry) as well as their photosystem I content (using immunological and spectroscopic analyses). We find no instance of a strain that can perform photosynthesis in the absence of photosystem I. The F8 strain harbored a small amount of photosystem I, and it could fix CO2 and grow slowly, but it lost these abilities after deletion of either psaA or psaC; these activities could be restored to the F8-psaADelta mutant by reintroduction of psaA. We observed limited O2 photoevolution in mutants lacking photosystem I; use of 18O2 indicated that this O2 evolution is coupled to O2 uptake (i.e. respiration) rather than CO2 fixation or H2 evolution. We conclude that the reported instances of CO2 fixation, H2 photoevolution, and photoautotrophic growth of photosystem I-deficient mutants result from the presence of unrecognized photosystem I.     

Isolation and characterization of xanthine dehydrogenase from Chlamydomonas reinhardtii

Xanthine dehydrogenase (XDH, EC 1.2.1.37) of Chlamydomonas reinhardtii (Sager) 6145c wild strain has been isolated and characterized for the first time in a unicellular green alga. The enzyme has an M_r of 330 kDa, and FAD, molybdenum and iron are cofactors required for its activity as deduced from results obtained using specific inhibitors, ⁵⁹Fe-labelling experiments, activity protection by FAD, physiological responses in vivo to iron and molybdenum deficiencies in the culture medium and work with mutants lacking molybdenum cofactor. Xanthine dehydrogenase exhibited Mi-chaelian kinetics typical for a bisubstrate enzyme with apparent K_m values for NAD ⁺, hypoxanthine and xanthine of 35, 160 and 70 μ M , respectively. Under phototrophic conditions enzyme activity was repressed by ammonium, but xanthine was not required for the enzyme to be induced, since high levels of enzyme activity were found in cells grown on ammonium and transferred to either N-frec media or media containing either of the nitrogen sources adenine, urea, urate, xanthine, hypoxanthine and guanine.     

Acclimation of Chlamydomonas reinhardtii to different growth irradiances

We report on the changes the photosynthetic apparatus of Chlamydomonas reinhardtii undergoes upon acclimation to different light intensity. When grown in high light, cells had a faster growth rate and higher biomass production compared with low and control light conditions. However, cells acclimated to low light intensity are indeed able to produce more biomass per photon available as compared with high light-acclimated cells, which dissipate as heat a large part of light absorbed, thus reducing their photosynthetic efficiency. This dissipative state is strictly dependent on the accumulation of LhcSR3, a protein related to light-harvesting complexes, responsible for nonphotochemical quenching in microalgae. Other changes induced in the composition of the photosynthetic apparatus upon high light acclimation consist of an increase of carotenoid content on a chlorophyll basis, particularly zeaxanthin, and a major down-regulation of light absorption capacity by decreasing the chlorophyll content per cell. Surprisingly, the antenna size of both photosystem I and II is not modulated by acclimation; rather, the regulation affects the PSI/PSII ratio. Major effects of the acclimation to low light consist of increased activity of state 1 and 2 transitions and increased contributions of cyclic electron flow.     

Cloning and characterization of the actin-encoding gene of Chlamydomonas reinhardtii

The genomic and complementary DNA sequences were determined for the unique actin-encoding gene in Chlamydomonas reinhardtii (Cr). The deduced amino acid (aa) sequence of this actin was similar to most known actin sequences, with the highest identity (98.1%) being with that of Volvox carteri actin. The Cr actin-encoding gene has one intron in the 5′-untranslated region and eight introns in the coding region. The latter eight introns occur at the same positions as those in the V. carteri actin-encoding gene. The 5′-upstream region contains four short stretches of sequence similar to the so-called ‘tub box’, a characteristic sequence proposed to be responsible for the regulation of synthesis of various axonemal proteins upon deflagellation and during the cell cycle. Southern blot analysis indicated that the Cr genome has only a single actin-encoding gene. An antibody specific for the 11-aa peptide corresponding to the N-terminal sequence of this actin was found to react with a 43-kDa protein associated with flagellar inner-arm dynein. These findings indicate that a single actin functions in both the cytoplasm and flagella of this organism.     

Molecular characterization of two light-induced, gamete-specific genes from Chlamydomonas reinhardtii that encode hydroxyproline-rich proteins

Gametic differentiation in Chlamydomonas reinhardtii is a two-step process, which is controlled by the sequential action of the two extrinsic signals, nitrogen starvation and blue light. The gamete-specific genes GAS28 and GAS29 are expressed in the late phase of gametogenesis. Their light-induced expression is restricted to cells that have completed the first, nitrogen starvation-activated, phase of differentiation. A comparison of the two genes revealed striking similarities as well as differences. Their most prominent shared feature is an extended sequence homology of over 90% in their 5′-untranslated regions, suggesting a role in translational regulation. GAS28 and GAS29 both encode hydroxyproline-rich proteins (HRGPs) of very similar sizes that exhibit typical features of volvocalean cell wall constituents. GAS28 shows a high degree of homology with the Volvox pherophorin gene family, suggesting a relationship between these genes. Received: 6 August 1998 / Accepted: 16 November 1998     

Plastid DNA content in a cultured soybean line capable of photoautotrophic growth

The levels of chloroplast DNA in a cultured photoautotrophic soybean (Glycine max [L.] Merr. v Corsoy) cell line were determined by molecular hybridization. The cells were also grown photomixotrophically and heterotrophically as suspension cultures and the level of plastid DNA was found to be constant at approximately 26% of the total cellular DNA in all three growth modes. By comparison, total cellular DNA extracted from plants of the same variety used as the explant source for the cultured cells contained 12.3 to 18.9% (leaves and seeds) and 6.1 to 8.9% (roots) plastid DNA.     

Isolation and characterization of arsenate-sensitive and resistant mutants of Chlamydomonas reinhardtii

Arsenate-sensitive and resistant mutants of Chlamydomonas reinhardtii were obtained by screening mutants generated by random insertional mutagenesis for growth in the presence of various concentrations of arsenate. The intracellular concentrations of arsenic in the mutants kept in the arsenate-containing medium were determined with an atomic absorption spectrophotometer. The intracellular levels of arsenic in the arsenate-resistant mutants were all lower than that of the parent strain CC425. Some of the arsenate-sensitive mutants, AS1 and AS3, showed obviously higher levels of arsenic than that of CC425, while other sensitive mutant, AS2, did not accumulate arsenic so much. Analysis of the chemical species of arsenic suggested that inorganic arsenic was converted to dimethylarsinic acid (DMAA) in CC425. However, DMAA was hardly detected in AS2. The mechanisms of the resistance to arsenate are discussed on its uptake and detoxification.     

Purification and characterization of a gamma-like DNA polymerase from Chlamydomonas reinhardtii

A crude in vitro system which initiates chloroplast DNA synthesis near the D-loop site mapped by electron microscopy [Wu, M., Lou, J. K., Chang, D. Y., Chang, C. H., & Nie, Z. Q. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6761-6765] consists of soluble proteins and proteins extracted from purified thylakoid membrane. In this paper, a DNA polymerase activity was purified to near homogeneity from the soluble protein fraction of this in vitro system by sequential chromatographic separations on heparin-agarose, DEAE-cellulose, and single-stranded DNA-agarose columns and sedimentation in a glycerol gradient. In the glycerol gradient, the enzyme activity sedimented at a position corresponding to a 110-kDa protein. Electrophoretic analysis of the highly purified fraction on SDS-polyacrylamide gel revealed a major polypeptide band with an apparent molecular mass of approximately 116 kDa. In situ DNA polymerase activity assay shows that the DNA polymerization function is associated with the 116-kDa band and an 80-kDa band which could be a subunit of the enzyme. Polymerization activity is inhibited by N-ethylmaleimide, ethidium bromide, and dideoxycytosine triphosphate and is relatively resistant to aphidicolin. Poly(dA).(dT)10 and gapped double-stranded DNA are preferred templates. The purified enzyme contains no exonuclease activity and can initiate DNA replication in a supercoiled plasmid DNA template containing the chloroplast DNA replication origin.     

Network reduction in metabolic pathway analysis: elucidation of the key pathways involved in the photoautotrophic growth of the green alga Chlamydomonas reinhardtii

Metabolic pathway analysis aims at discovering and analyzing meaningful routes and their interactions in metabolic networks. A major difficulty in applying this technique lies in the decomposition of metabolic flux distributions into elementary mode or extreme pathway activity patterns, which in general is not unique. We propose a network reduction approach based on the decomposition of a set of flux vectors representing adaptive microbial metabolic behavior in bioreactors into a minimal set of shared pathways. Several optimality criteria from the literature were compared in order to select the most appropriate objective function. We further analyze photoautotrophic metabolism of the green alga Chlamydomonas reinhardtii growing in a photobioreactor under maximal growth rate conditions. Key pathways involved in its adaptive metabolic response to changes in light influx are identified and discussed using an energetic approach.     

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.     

Isolation and characterization of a thioredoxin-dependent peroxidase from Chlamydomonas reinhardtii.

All living organisms contain redox systems involving thioredoxins (Trx), proteins featuring an extremely conserved and reactive active site that perform thiol-disulfide interchanges with disulfide bridges of target proteins. In photosynthetic organisms, numerous isoforms of Trx coexist, as revealed by sequencing of Arabidopsis genome. The specific functions of many of them are still unknown. In an attempt to find new molecular targets of Trx in Chlamydomonas reinhardtii, an affinity column carrying a cytosolic Trx h mutated at the less reactive cysteine of its active site was used to trap Chlamydomonas proteins that form mixed disulfides with Trx. The major protein bound to the column was identified by amino-acid sequencing and mass spectrometry as a thioredoxin-dependent 2Cys peroxidase. Isolation and sequencing of its gene revealed that this peroxidase is most likely a chloroplast protein with a high homology to plant 2Cys peroxiredoxins. It is shown that the Chlamydomonas peroxiredoxin (Ch-Prx1) is active with various thioredoxin isoforms, functions as an antioxidant toward reactive oxygen species (ROS), and protects DNA against ROS-induced degradation. Expression of the peroxidase gene in Chlamydomonas was found to be regulated by light, oxygen concentration, and redox state. The data suggest a role for the Chlamydomonas Prx in ROS detoxification in the chloroplast.     

Isolation and characterization of a new transposable element in Chlamydomonas reinhardtii

A new transposable element, Tcr3, was identified in the unicellular green alga Chlamydomonas reinhardtii. The Tcr3 element contained imperfect terminal inverted repeat sequences of 56 bp and created a 2 bp target site duplication upon insertion. Insertion of Tcr3 into the 3-untranslated region of the NIT8 gene, which is essential for nitrate assimilation, prevented expression of the gene. Excision of the Tcr3 element correlated with reversion of the mutant phenotype and left behind a 3 bp footprint. Tcr3 was found in all Chlamydomonas isolates tested and should prove to be useful for transposon-tagging experiments in Chlamydomonas.     

Chemoresponses of Chlamydomonas reinhardtii

Cells of Chlamydomonas reinhardtii have been found to respond to chemicals in two ways: chemokinesis and chemotaxis. Several amino acids, fatty acids, and inorganic salts can stimulate these responses.     

An endonuclease from Chlamydomonas reinhardtii that cleaves the sequence TATA

An endodeoxyribonuclease, designated CreI, was purified 16,000-fold from zygotes of the eukaryote Chlamydomonas reinhardtii. CreI preferentially attacks the sequence TATA producing double strand breaks with 3'-phosphomonoester and 5'-hydroxyl termini. The endonuclease has an Mr = 27,000 and requires Ca2+ at pH 7.5 for optimal activity.     

Biochemical characterization of the small ubiquitin-like modifiers of Chlamydomonas reinhardtii

Dynamic modification of target proteins by small ubiquitin-like modifier (SUMO) is known to modulate many important cellular processes and is required for cell viability and development in all eukaryotes. However, understanding of SUMO systems in plants, especially in unicellular green algae, remains elusive. In this study, Chlamydomonas reinhardtii CrSUMO96, CrSUMO97 and CrSUMO148 were characterized. We show that the formation of polymeric CrSUMO96 and CrSUMO97 chains can be catalyzed either by the human SAE1/SAE2 and Ubc9 SUMOylation system in vitro or by an Escherichia coli chimeric SUMOylation system in vivo. An exposed C-terminal di-glycine motif of CrSUMO96 or CrSUMO97 is essential for functional SUMOylation. The human SUMO-specific protease, SENP1, demonstrates more processing activity for CrSUMO97 than for CrSUMO96. The CrSUMO148 precursor notably has four repeated di-glycine motifs at the C-terminus. This unique feature is not found in other known SUMO proteins. Interestingly, only 83-residual CrSUMO148_1–83 with the first di-glycine motif can form SAE1/SAE2–SUMO complex and further form polymeric chains with the help of Ubc9. More surprisingly, CrSUMO148 precursor is digested by SENP1, solely at the peptide bond after the first di-glycine motif although there are four theoretically identical processing sites in the primary sequence. This process directly generates 83-residual CrSUMO148_1–83 mature protein, which is exactly the form suitable for activation and conjugation. We also show that SENP1 displays similar isopeptidase activity in the deconjugation of polymeric CrSUMO96, CrSUMO97 or CrSUMO148 chains, revealing that the catalytic mechanisms of processing and deconjugation of CrSUMOs by SENP1 may differ.