Alanine racemase from the green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Summary. Chlamydomonas reinhardtii, a unicellular green microalga, could grow to a stationary phase having optical density of 2.0–2.5 at 750 nm in Tris-acetate-phosphate (TAP) medium containing 0.1% D-alanine. D-alanine has no inhibitory effect on growth and induced alanine racemase activity 130-fold more than without D-alanine in the green alga. Although C. reinhardtii cultured in the TAP medium showed alanine racemase activity, the content of free D-alanine was only 0.14%. The enzyme was partially purified by ammonium sulfate fractionation followed by three kinds of liquid chromatography using DEAE Toyopearl, Phenyl Sepharose, and TSK G3000 SWXL columns. The specific activity for L-alanine of the partially purified alanine racemase was 3.8 μmol/min/mg. The molecular weight of the enzyme was determined to be approximately 72,000 by gel filtration. The enzyme showed a maximum activity at 45 °C and pH 8.4 and requires pyridoxal 5′-phosphate as a coenzyme.     

The thioredoxin superfamily in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The thioredoxin (TRX) superfamily includes redox proteins such as thioredoxins, glutaredoxins (GRXs) and protein disulfide isomerases (PDI). These proteins share a common structural motif named the thioredoxin fold. They are involved in disulfide oxido-reduction and/or isomerization. The sequencing of the Arabidopsisgenome revealed an unsuspected multiplicity of TRX and GRX genes compared to other organisms. The availability of full Chlamydomonasgenome sequence offers the opportunity to determine whether this multiplicity is specific to higher plant species or common to all photosynthetic eukaryotes. We have previously shown that the multiplicity is more limited in Chlamydomonas for TRX and GRX families. We extend here our analysis to the PDI family. This paper presents a comparative analysis of the TRX, GRX and PDI families present in Arabidopsis,Chlamydomonas and Synechocystis. The putative subcellular localization of each protein and its relative expression level, based on EST data, have been investigated. This analysis provides a large overview of the redox regulatory systems present in Chlamydomonas. The data are discussed in view of recent results suggesting a complex cross-talk between the TRX, GRX and PDI redox regulatory networks.     

<Emphasis Type="Italic">N</Emphasis>-Acyl derivatives of Asn,new bacterial <Emphasis Type="Italic">N</Emphasis>-acyl <Emphasis Type="Italic">D</Emphasis>-amino acids with surfactant activity

Summary. New N-acyl D-amino acids were isolated from Bacillus pumilus IM 1801. Their structures were determined by chemical analysis and mass spectrometry. The lipid part was identified as a mixture of fatty acids with 11, 12, 13, 15, and 16 carbon atoms in the iso, anteiso or n configuration linked by an amide bond with a D-asparagine. They exhibited surfactant properties.     

An <Emphasis Type="Italic">Escherichia coli</Emphasis> Strain Producing a Leaderless mRNA from the Chromosomal <Emphasis Type="Italic">lac</Emphasis> Promoter

A special Escherichia coli strain capable of producing a leaderless lacZ mRNA from the chromosomal lac promoter was constructed to study the mechanism of the leaderless mRNA translation. The translation efficiency of this noncanonical mRNA is very low in comparison with the canonical cellular templates, but it increases by one order of magnitude in the presence of chromosomal mutations in the genes encoding ribosomal S1 and S2 proteins. The new strain possesses obvious advantages over the commonly used plasmid constructs (first of all, due to the constant dosage of lacZ gene in the cell) and opens the possibilities of investigation of the specific conditions for the leaderless mRNA translation in vivo using the molecular genetic approaches.     

Phosphopantetheinylation in the green microalgae <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Microalgal biofuel is a promising solution to the decline of fossil fuels. However, algal fatty acid metabolism, the machinery producing the raw material for biofuels, remains poorly understood. The central unit of the fatty acid synthase (FAS) is the acyl carrier protein (ACP), which is responsible for holding the product. Fatty acid biosynthesis is initiated through posttranslational modification of the ACP by the phosphopantetheinyl transferase (PPTase). We identified two PPTases, PptC1 and PptC2, in the model alga Chlamydomonas reinhardtii by genome analysis and phylogenetic and structural comparison. Both PPTases are of Sfp-type, the archetypical PPTase type for non-ribosomal peptide and polyketide biosynthetic pathways in bacteria and cyanobacteria. In vitro analysis revealed that PptC2 has a broader substrate range than PptC1. Both PPTases were able to activate the cognate ACP of the type II FAS, while PptC2 also recognized ACP of Escherichia coli type II FAS and actinorhodin type II polyketide synthase. Besides FAS as PPTase target, the C. reinhardtii genome encodes a single type I PKS, and we hypothesize that PptC2 is responsible for its activation. Screening of the currently available microalgal genome data revealed that most green microalgae appear to carry two PPTases forming clusters with each C. reinhardtii PPTase, while microalgae of other divisions carry one or two PPTases and do not cluster in the pattern of the green algal data. This new understanding on the PPTases in microalgae shows that microalgae are already primed for biotechnological applications in contrast to other organisms. Thus, microalgae have great potential for metabolic engineering efforts in the realm of biofuel and high-value products including direct engineering of the fatty acid or secondary metabolism using the natural genomic reservoir and as biotechnological platform for heterologous expression.     

Functional characterization of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> with alterations in the <Emphasis Type="Italic">atpE</Emphasis> gene

The FUD17 strain of Chlamydomonas reinhardtii is a photosynthesis-deficient, acetate-requiring mutant with a defect in the chloroplast atpE gene, which codes for the ε subunit of the chloroplast ATP synthase. In this work, the FUD17 mutant was examined in relation to other known ATP synthase mutants as an initial step toward using this strain to generate altered versions of the atpE gene for site-directed mutagenesis of the ε subunit. The FUD17 strain grows well and is normally pigmented in the dark (heterotrophic conditions), but cannot grow autotrophically in the light, even when media are supplemented with acetate. Under heterotrophic conditions, it shows no accumulation of the ε subunit, and much lower levels of the α and β subunits of the chloroplast ATP synthase. FUD17 shows no light-dependent oxygen evolution and shows a strong, light-dependent alteration in its chlorophyll fluorescence. These results show that FUD17 possesses similar characteristics to other ATP synthase mutants and fails to express an assembled ATP synthase complex on its thylakoid membrane. A preliminary attempt at site-directed mutagenesis is described which produced a slightly truncated form of the ε subunit, which is expressed normally in the cell. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhancement of photorespiration in immobilized <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells

Immobilization of Chlamydomonas reinhardtii in alginate increases its photorespiration rate. In the immobilized cells, the photorespiratory enzyme, phosphoglycolate phosphatase, was 75% higher than in freely suspended cells. Thus, the immobilized cells produced glycolate at twice the rate than in freely suspended cells when treated with aminooxyacetate (a transaminase inhibitor). With immobilized cells in a batch reactor, 270mol glycolatemg^–1 Chl was produced after 12h.Revisions requested 27 October 2004; Revisions received 13 December 2004     

Strategies to facilitate transgene expression in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The unicellular green alga Chlamydomonas reinhardtii has been identified as a promising organism for the production of recombinant proteins. While during the last years important improvements have been developed for the production of proteins within the chloroplast, the expression levels of transgenes from the nuclear genome were too low to be of biotechnological importance. In this study, we integrated endogenous intronic sequences into the expression cassette to enhance the expression of transgenes in the nucleus. The insertion of one or more copies of intron sequences from the Chlamydomonas RBCS2 gene resulted in increased expression levels of a Renilla-luciferase gene used as a reporter. Although any of the three RBCS2 introns alone had a positive effect on expression, their integration in their physiological number and order created an over-proportional stimulating effect observed in all transformants. The secretion of the luciferase protein into the medium was achieved by using the export sequence of the Chlamydomonas ARS2 gene in a cell wall deficient strain and Renilla-luciferase could be successfully concentrated with the help of attached C-terminal protein tags. Similarly, a codon adapted gene variant for human erythropoietin (crEpo) was expressed as a protein of commercial relevance. Extracellular erythropoietin produced in Chlamydomonas showed a molecular mass of 33 kDa probably resulting from post-translational modifications. Both, the increased expression levels of transgenes by integration of introns and the isolation of recombinant proteins from the culture medium are important steps towards an extended biotechnological use of this alga. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CITRIC: cold-inducible translational readthrough in the chloroplast of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> using a novel temperature-sensitive transfer RNA

Background

The chloroplast of eukaryotic microalgae such as Chlamydomonas reinhardtii is a potential platform for metabolic engineering and the production of recombinant proteins. In industrial biotechnology, inducible expression is often used so that the translation or function of the heterologous protein does not interfere with biomass accumulation during the growth stage. However, the existing systems used in bacterial or fungal platforms do not transfer well to the microalgal chloroplast. We sought to develop a simple inducible expression system for the microalgal chloroplast, exploiting an unused stop codon (TGA) in the plastid genome. We have previously shown that this codon can be translated as tryptophan when we introduce into the chloroplast genome a trnW_UCA gene encoding a plastidial transfer RNA with a modified anticodon sequence, UCA.

Results

A mutated version of our trnW_UCA gene was developed that encodes a temperature-sensitive variant of the tRNA. This allows transgenes that have been modified to contain one or more internal TGA codons to be translated differentially according to the culture temperature, with a gradient of recombinant protein accumulation from 35 °C (low/off) to 15 °C (high). We have named this the CITRIC system, an acronym for cold-inducible translational readthrough in chloroplasts. The exact induction behaviour can be tailored by altering the number of TGA codons within the transgene.

Conclusions

CITRIC adds to the suite of genetic engineering tools available for the microalgal chloroplast, allowing a greater degree of control over the timing of heterologous protein expression. It could also be used as a heat-repressible system for studying the function of essential native genes in the chloroplast. The genetic components of CITRIC are entirely plastid-based, so no engineering of the nuclear genome is required.

     

Altered expression of the chloroplast ATP synthase through site-directed mutagenesis in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The chloroplast ATP synthase gates the flow of protons out of the thylakoid lumen. In Chlamydomonas reinhardtii deletion of any of the genes for the ATP synthase subunits, or misfolding of the peptides results in photosynthetic membranes devoid of the enzyme (Lemaire and Wollman, J Biol Chem 264:675–685, 1989). This work examines the physiologic response of an algal strain in which the epsilon subunit of the chloroplast ATP synthase has been truncated. Removal of 10 amino acids from the C-terminus of the peptide results in a sharp decrease in the content of the enzyme, but does not result in its exclusion from the thylakoid membranes. The ATP synthase of this mutant strain has a higher rate of ATP hydrolysis than the wild-type enzyme. This strain of C. reinhardtii exhibits reduced growth in the light, dependence on acetate, and a low threshold for the onset of photoinhibition. The role of the ATP synthase in regulating the proton concentration of the lumen is discussed. This work was supported in part by a grant from the National Science Foundation (MCB0110232).     

Whole cell solid-state NMR study of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> microalgae

In vivo or whole-cell solid-state NMR is an emerging field which faces tremendous challenges. In most cases, cell biochemistry does not allow the labelling of specific molecules and an in vivo study is thus hindered by the inherent difficulty of identifying, among a formidable number of resonances, those arising from a given molecule. In this work we examined the possibility of studying, by solid-state NMR, the model organism Chlamydomonas reinhardtii fully and non-specifically ¹³C labelled. The extension of NMR-based dynamic filtering from one-dimensional to two-dimensional experiments enabled an enhanced selectivity which facilitated the assignment of cell constituents. The number of resonances detected with these robust and broadly applicable experiments appears to be surprisingly sparse. Various constituents, notably galactolipids abundant in organelle membranes, carbohydrates from the cell wall, and starch from storage grains could be unambiguously assigned. Moreover, the dominant crystal form of starch could be determined in situ. This work illustrates the feasibility and caveats of using solid-state NMR to study intact non-specifically ¹³C labelled micro-organisms.     

Chemotactic Behavior of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> Is Altered During Gametogenesis

Chemotactic behavior of Chlamydomonas reinhardtii is altered during the sexual life cycle. Unlike vegetative cells and noncompetent pregametes, mature gametes did not show chemotaxis to ammonium. Loss of chemotaxis to ammonium in mating-competent cells is controlled by gamete-specific genes that are common for both mating-type gametes. Change of chemotaxis mode requires the sequential action of the two environmental signals: removal of ammonium from the medium and light. The mutants lrg1, lrg3, and lrg4 affected in the light-dependent step of sexual differentiation exhibited the loss of chemotaxis to ammonium in the absence of light. These data indicate that there are common components in the signaling pathways that control change of chemotactic behavior and forming of mating competence in gametes. Received: 14 May 2002 / Accepted: 21 June 2002     

Co-cultivation of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> with <Emphasis Type="Italic">Azotobacter chroococcum</Emphasis> improved H<Subscript>2</Subscript> production

Objectives

To improve H₂ production, the green algae Chlamydomonas reinhardtii cc849 was co-cultured with Azotobacter chroococcum.

Results

The maximum H₂ production of the co-culture was 350% greater than that of the pure algal cultures under optimal H₂ production conditions. The maximum growth and the respiratory rate of the co-cultures were about 320 and 300% of the controls, and the dissolved O₂ of co-cultures was decreased 74%. Furthermore, the in vitro maximum hydrogenase activity of the co-culture was 250% greater than that of the control, and the in vivo maximum hydrogenase activity of the co-culture was 1.4-fold greater than that of the control. In addition, the maximum starch content of co-culture was 1400% that of the control.

Conclusions

Azotobacter chroococcum improved the H₂ production of the co-cultures by decreasing the O₂ content and increasing the growth and starch content of the algae and the hydrogenase activity of the co-cultures relative to those of pure algal cultures.

     

<Emphasis Type="Italic">SulP</Emphasis>, a nuclear gene encoding a putative chloroplast-targeted sulfate permease in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Genomic, proteomic, phylogenetic and evolutionary aspects of a novel gene encoding a putative chloroplast-targeted sulfate permease of prokaryotic origin in the green alga Chlamydomonas reinhardtii are described. This nuclear-encoded sulfate permease gene (SulP) contains four introns, whereas all other known chloroplast sulfate permease genes lack introns and are encoded by the chloroplast genome. The deduced amino acid sequence of the protein showed an extended N-terminus, which includes a putative chloroplast transit peptide. The mature protein contains seven transmembrane domains and two large hydrophilic loops. This novel prokaryotic-origin gene probably migrated from the chloroplast to the nuclear genome during evolution of C. reinhardtii. The SulP gene, or any of its homologues, has not been retained in vascular plants, e.g. Arabidopsis thaliana, although it is encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). A comparative structural analysis and phylogenetic origin of chloroplast sulfate permeases in a variety of species is presented.