Xiangyu Guan

Fatty acid biosynthesis in eukaryotic photosynthetic microalgae: Identification of a microsomal delta 12 desaturase in Chlamydomonas reinhardtii

Polyunsaturated fatty acids (PUFAs) are important components of infant and adult nutrition because they serve as structural elements of cell membranes. Fatty acid desaturases are responsible for the insertion of double bonds into pre-formed fatty acid chains in reactions that require oxygen and reducing equivalents. In this study, the genome-wide characterization of the fatty acid desaturases from seven eukaryotic photosynthetic microalgae was undertaken according to the conserved histidine-rich motifs and phylogenetic profiles. Analysis of these genomes provided insight into the origin and evolution of the pathway of fatty acid biosynthesis in eukaryotic plants. In addition, the candidate enzyme from Chlamydomonas reinhardtii with the highest similarity to the microsomal Δ12 desaturase of Chlorella vulgaris was isolated, and its function was verified by heterologous expression in yeast (Saccharomyces cerevisiae).

Genome-wide survey of putative Serine/Threonine protein kinases in cyanobacteria

BackgroundSerine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution.Results286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains.ConclusionThe number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.

Serine/Threonine Protein Kinase SpkG Is a Candidate for High Salt Resistance in the Unicellular Cyanobacterium Synechocystis sp PCC 6803

Background Seven serine/threonine kinase genes have been predicted in unicellular cyanobacterium Synechocystis sp. PCC6803. SpkA and SpkB were shown to be required for cell motility and SpkE has no kinase activity. There is no report whether the other four STKs are involved in stress-mediated signaling in Synechocystis PCC6803. Methodology/Principal Findings In this paper, we examined differential expression of the other four serine/threonine kinases, SpkC, SpkD, SpkF and SpkG, at seven different stress conditions. The transcriptional level was up-regulated of spkG and down-regulated of spkC under high salt stress condition. Two spk deletion mutants, ΔspkC and ΔspkG, were constructed and their growth characteristic were examined compared to the wild strain. The wild strain and ΔspkC mutant were not affected under high salt stress conditions. In contrast, growth of spkG mutant was completely impaired. To further confirm the function of spkG, we also examined the effect of mutation of spkG on the expression of salt stress-inducible genes. We compared genome-wide patterns of transcription between wild-type Synechocystis sp. PCC6803 and cells with a mutation in the SpkG with DNA microarray analysis. Conclusion In this study, we first study the spkG gene as sensor of high salt signal. We consider that SpkG play essential roles in Synechocystis sp. for sensing the high salt signal directly, rather than mediating signals among other kinases. Our microarray experiment may help select relatively significant genes for further research on mechanisms of signal transduction of Synechocystis sp. PCC6803 under high salt stress.

A potent anti-oxidant property: fluorescent recombinant α-phycocyanin of Spirulina

Aims:  To express and product a fluorescent antioxidant holo‐α‐phycocyanin (PC) of Spirulina platensis (Sp) with His‐tag (rHHPC; recombinant holo‐α‐phycocyaninof Spirulina platensis with His‐tag) in 5‐l bench scale.

Biosynthesis of fluorescent allophycocyanin α-subunits by autocatalysis in Escherichia coli

APC (allophycocyanin) is widely used for fluorescence tagging and may be a promising antioxidant agent for use within the food and pharmaceutical industries. Chromophore attachment to apo‐ApcA (apo‐APC α‐subunit without chromophore) can be autocatalysed both in vitro and in vivo. In the present study, a plasmid containing genes of apo‐ApcA and chromophore synthetases [HO1 (ferredoxin‐dependent haem oxygenase) and PcyA (phycocyanobilin:ferredoxin oxidoreductase)] was constructed and expressed in Escherichia coli. The results show that holo‐ApcA (APC α‐subunit with chromophore) can be synthesized by autocatalysis in E. coli. Recombinant holo‐ApcA showed the same spectral and fluorescent properties as PC (phycocyanin) and could serve as a good substitute for native PC for fluorescent tagging. Moreover, recombinant ApcA can inhibit hydroxyl and peroxyl radicals more strongly than holo‐ApcA and native APC. The EC50 values were 296.4±22.4 μg/ml against hydroxyl radicals and 38.5±2.6 μg/ml against peroxyl radicals.

Isolation and functional analysis of fatty acid desaturase genes from peanut (Arachis hypogaea L.)

Background Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains. Extensive studies of fatty acid desaturases have been done in many plants. However, less is known about the diversity of this gene family in peanut (Arachis hypogaea L.), an important oilseed crop that is cultivated worldwide. Results In this study, twelve novel AhFADs genes were identified and isolated from peanut. Quantitative real-time PCR analysis indicated that the transcript abundances of AhFAB2-2 and AhFAD3-1 were higher in seeds than in other tissues examined, whereas the AhADS and AhFAD7-1 transcripts were more abundant in leaves. AhFAB2-3, AhFAD3-2, AhFAD4, AhSLD-4, and AhDES genes were highly expressed in flowers, whereas AhFAD7-2, AhSLD-2, and AhSLD-3 were expressed most strongly in stems. During seed development, the expressions of AhFAB2-2, AhFAD3-1, AhFAD7-1, and AhSLD-3 gradually increased in abundance, reached a maximum expression level, and then decreased. The AhFAB2-3, AhFAD3-2, AhFAD4, AhADS, and AhDES transcript levels remained relatively high at the initial stage of seed development, but decreased thereafter. The AhSLD-4 transcript level remained relatively low at the initial stage of seed development, but showed a dramatic increase in abundance at the final stage. The AhFAD7-2 and AhSLD-2 transcript levels remained relatively high at the initial stage of seed development, but then decreased, and finally increased again. The AhFAD transcripts were differentially expressed following exposure to abiotic stresses or abscisic acid. Moreover, the functions of one AhFAD6 and four AhSLD genes were confirmed by heterologous expression in Synechococcus elongates or Saccharomyces cerevisiae. Conclusions The present study provides valuable information that improves understanding of the biological roles of FAD genes in fatty acid synthesis, and will help peanut breeders improve the quality of peanut oil via molecular design breeding.

Comparative Analysis of Two-Component Signal Transduction Systems in Six Synechococcus Strains - Two-Component Signal Transduction Systems in Synechococcus

Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments and their signal transduction systems play important roles for environmental adaptation. Two-component signal transduction (TCST) systems show important roles in prokaryotes. Sequencing information of cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this signal transduction system. In this study, we extracted information from six species of sequenced Synechococcus and investigated the diversity, gene structure and conservation and phylogeny of the TCST components. A total of 184 predicted proteins were identified in the putative histidine kinases and response regulators. Twelve types additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. The number and structure character of TCST components in different Synechococcus strains are the results of the ecophysiology and physiological properties of the organism. Histidine kinase (HK) and Response Regulators (RR) have different evolutionary history and gene gain-and-loss play important roles during the evolution along with domain shuffling and insertion.