<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.     

The biosynthetic pathway of carotenoids in the astaxanthin-producing green alga <Emphasis Type="Italic">Chlorella zofingiensis</Emphasis>

The carotenoid composition of the astaxanthin-producing green alga Chlorella zofingiensis was investigated using high-performance liquid chromatography. Astaxanthin, adonixanthin, and zeaxanthin are the major carotenoids in this alga. The pigment pattern was characterized during the accumulation period, and in response to diphenylamine (DPA), an inhibitor of carotenoid biosynthesis. An increase in zeaxanthin followed by a decrease in xanthophyll was seen after the induction of astaxanthin biosynthesis by glucose. This biphasic kinetics of zeaxanthin was parallel to the marked increase in adonixanthin (from 0 mg g⁻¹ to 0.21 mg g⁻¹) and astaxanthin (from 0.05 mg g⁻¹ to 0.35 mg g⁻¹) and decrease of β-carotene (from 0.30 mg g⁻¹ to 0.03 mg g⁻¹). More importantly, unlike the Haematococcus alga, in which there was a high β-carotene accumulation after DPA treatment, C. zofingiensis showed an accumulation of extra zeaxanthin instead of β-carotene after treatment of the cells with DPA. All these results observed in vivo studies corroborate the observations in vitro studies at the enzyme level that zeaxanthin is a substrate for the carotenoid oxygenase in C. zofingiensis. It is suggested that zeaxanthin might be an important intermediate and not an end product of the biosynthetic pathway of astaxanthin. Therefore, a new pathway for astaxanthin formation by C. zofingiensis, which is different from that of the other astaxanthin-producing microorganisms, is proposed. An understanding of the astaxanthin biosynthetic pathway may yield important information toward the optimization of astaxanthin production, especially for the improvement of astaxanthin through genetic manipulations.     

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.     

Recombinant production of <Emphasis Type="Italic">Streptococcus equisimilis</Emphasis> streptokinase by <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Background  

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway.     

Shared molecular characteristics of successfully transformed mitochondrial genomes in <Emphasis Type="Italic">Chlamydomonas</Emphasis><Emphasis Type="Italic">reinhardtii</Emphasis>

Three types of respiratory deficient mitochondrial strains have been reported in Chlamydomonas reinhardtii: a deficiency due to (i) two base substitutions causing an amino acid change in the apocytochrome b (COB) gene (i.e., strain named dum-15), (ii) one base deletion in the COXI gene (dum-19), or (iii) a large deletion extending from the left terminus of the genome to somewhere in the COB gene (dum-1, -14, and -16). We found that these respiratory deficient strains of C. reinhardtii can be divided into two groups: strains that are constantly transformable and those could not be transformed in our experiments. All transformable mitochondrial strains were limited to the type that has a large deletion in the left arm of the genome. For these mitochondria, transformation was successful not only with purified intact mitochondrial genomes but also with DNA-constructs containing the compensating regions. In comparison, mitochondria of all the non-transformable strains have both of their genome termini intact, leading us to speculate that mitochondria lacking their left genome terminus have unstable genomes and might have a higher potential for recombination. Analysis of mitochondrial gene organization in the resulting respiratory active transformants was performed by DNA sequencing and restriction enzyme digestion. Such analysis showed that homologous recombination occurred at various regions between the mitochondrial genome and the artificial DNA-constructs. Further analysis by Southern hybridization showed that the wild-type genome rapidly replaces the respiratory deficient monomer and dimer mitochondrial genomes, while the E. coli vector region of the artificial DNA-construct likely does not remain in the mitochondria.     

Effect of <Emphasis Type="Italic">cra</Emphasis> gene knockout together with <Emphasis Type="Italic">edd</Emphasis> and <Emphasis Type="Italic">iclR</Emphasis> genes knockout on the metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

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.     

Molecular analysis of the genus <Emphasis Type="Italic">Anoxybacillus</Emphasis> based on sequence similarity of the genes <Emphasis Type="Italic">recN</Emphasis>, <Emphasis Type="Italic">flaA</Emphasis>, and <Emphasis Type="Italic">ftsY</Emphasis>

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies. We analyzed three genes, recN, flaA, and ftsY, for determining if these genes are useful tools for systematic analyses in the genus Anoxybacillus. The genes encoding a DNA repair and genetic recombination protein (recN), the flagellin protein (flaA), and GTPase signal docking protein (ftsY) were sequenced for ten Anoxybacillus species. The sequence comparisons revealed that recN sequence similarities range between 61% and 99% in the genus Anoxybacillus. Comparisons to other bacterial recN genes indicated that levels of similarity did not differ from the levels within genus Anoxybacillus. These data showed that recN is not a useful marker for the genus Anoxybacillus. A 550–600-bp region of the flagellin gene was amplified for all Anoxybacillus strains except for Anoxybacillus contaminans. The sequence similarity of flaA gene varies between 61% and 76%. Comparisons to other bacterial flagellin genes obtained from GenBank (Bacillus, Pectinatus, Proteus, and Vibrio) indicated that the levels of similarity were lower (3–42%). Based on these data, we concluded that the variability in this single gene makes it a particularly useful marker. Another housekeeping gene ftsY suggested to reflect the G+C (mol/mol) content of whole genome was analyzed for Anoxybacillus strains. A mean difference of 1.4% was observed between the G+C content of the gene ftsY and the G+C content of the whole genome. These results showed that the gene ftsY can be used to represent whole G+C content of the Anoxybacillus species.     

<Emphasis Type="Italic">PGM2</Emphasis> overexpression improves anaerobic galactose fermentation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium.     

Cloning and knockout of phytoene desaturase gene in <Emphasis Type="Italic">Sphingomonas elodea</Emphasis> ATCC 31461 for economic recovery of gellan gum

A gene encoding phytoene desaturase (crtI) in the carotenoid biosynthetic pathway of Sphingomonas elodea ATCC 31461, an industrial gellan gum-producing strain, was cloned and identified. This gene is predicted to encode a 492-amino acid protein with significant homology to the phytoene desaturase of other carotenogenic organisms. Knockout of crtI gene blocked yellow carotenoid pigment synthesis and resulted in the accumulation of colorless phytoene, confirming that it encodes phytoene desaturase. Further research indicates that the yield of gellan gum production by crtI gene knockout mutants is almost the same as that by the wild-type strain. In addition, a recovery method based on the colorless fermentation broth of the crtI gene knockout mutant was investigated. Compared to the volume of alcohol for the parent strain, much less alcohol (30%) is required in this recovery process; thus, the costs of downstream purification of gellan gum can be substantially reduced.