Comparative characterization of two GDP-mannose dehydrogenase genes from Saccharina japonica (Laminariales, Phaeophyceae)

SDS-PAGE analysis of recombinant SjGMDs. (PDF 79 kb

Verification of the Saccharina japonica Translocon Tic20 and its Localization in the Chloroplast Membrane in Diatoms

Tic20 is an important translocon protein that plays a role in protein transport in the chloroplast. The sequence of Tic20 was determined in the lower brown alga Saccharina japonica. Structural analysis of SjTic20 revealed a noncanonical structure consisting of an N-terminal non-cyanobacterium-originated EF-hand domain (a helix-loop-helix structural domain) and a C-terminal cyanobacterium-originated Tic20 domain. Subcellular localization and transmembrane analysis indicated that SjTic20 featured an “M”-type Nin-Cin-terminal orientation, with four transmembrane domains in the innermost membrane of the chloroplast in the microalga Phaeodactylum tricornutum, and the EF-hand domain was entirely extruded into the chloroplast stroma. Our study provides information on the structure, localization, and topological features of SjTic20, and further functional analysis of SjTic20 in S. japonica is needed

Glutamate Dehydrogenase Functions in Glutamic Acid Metabolism and Stress Resistance in <i>Pyropia haitanensis</i>

Pyropia haitanensis is an important laver species in China. Its quality traits are closely related to the content of glutamic acid. Glutamate dehydrogenase (GDH) is a crucial enzyme in the glutamic acid metabolism. In this study, two GDH genes from P. haitanensis, PhGDH1 and PhGDH2, were cloned and successfully expressed in Escherichia coli. The in vitro enzyme activity assay demonstrated that the catalytic activity of PhGDHs is mainly in the direction of ammonium assimilation. The measured Km values of PhGDH1 for NADH, (NH4)2SO4, and α-oxoglutarate were 0.12, 4.99, and 0.16 mM, respectively, while the corresponding Km values of PhGDH2 were 0.02, 3.98, and 0.104 mM, respectively. Site-directed mutagenesis results showed that Gly193 and Thr361 were important catalytic residues for PhGDH2. Moreover, expression levels of both PhGDHs were significantly increased under abiotic stresses. These results suggest that PhGDHs can convert α-oxoglutarate to glutamic acid, and enhance the flavor and stress resistance of P. haitanensis

Full-Length Transcriptome of Thalassiosira weissflogii as a Reference Resource and Mining of Chitin-Related Genes

β-Chitin produced by diatoms is expected to have significant economic and ecological value due to its structure, which consists of parallel chains of chitin, its properties and the high abundance of diatoms. Nevertheless, few studies have functionally characterised chitin-related genes in diatoms owing to the lack of omics-based information. In this study, we first compared the chitin content of three representative Thalassiosira species. Cell wall glycosidic linkage analysis and chitin/chitosan staining assays showed that Thalassiosira weissflogii was an appropriate candidate chitin producer. A full-length (FL) transcriptome of T. weissflogii was obtained via PacBio sequencing. In total, the FL transcriptome comprised 23,362 annotated unigenes, 710 long non-coding RNAs (lncRNAs), 363 transcription factors (TFs), 3113 alternative splicing (AS) events and 3295 simple sequence repeats (SSRs). More specifically, 234 genes related to chitin metabolism were identified and the complete biosynthetic pathways of chitin and chitosan were explored. The information presented here will facilitate T. weissflogii molecular research and the exploitation of β-chitin-derived high-value enzymes and products

Glutamate Dehydrogenase Functions in Glutamic Acid Metabolism and Stress Resistance in Pyropia haitanensis

Pyropia haitanensis is an important laver species in China. Its quality traits are closely related to the content of glutamic acid. Glutamate dehydrogenase (GDH) is a crucial enzyme in the glutamic acid metabolism. In this study, two GDH genes from P. haitanensis, PhGDH1 and PhGDH2, were cloned and successfully expressed in Escherichia coli. The in vitro enzyme activity assay demonstrated that the catalytic activity of PhGDHs is mainly in the direction of ammonium assimilation. The measured Km values of PhGDH1 for NADH, (NH4)2SO4, and α-oxoglutarate were 0.12, 4.99, and 0.16 mM, respectively, while the corresponding Km values of PhGDH2 were 0.02, 3.98, and 0.104 mM, respectively. Site-directed mutagenesis results showed that Gly193 and Thr361 were important catalytic residues for PhGDH2. Moreover, expression levels of both PhGDHs were significantly increased under abiotic stresses. These results suggest that PhGDHs can convert α-oxoglutarate to glutamic acid, and enhance the flavor and stress resistance of P. haitanensis

Characterization of Mannitol-2-Dehydrogenase in <i>Saccharina japonica</i>: Evidence for a New Polyol-Specific Long-Chain Dehydrogenases/Reductase

<div><p>Mannitol plays a crucial role in brown algae, acting as carbon storage, organic osmolytes and antioxidant. Transcriptomic analysis of <i>Saccharina japonica</i> revealed that the relative genes involved in the mannitol cycle are existent. Full-length sequence of mannitol-2-dehydrogenase (M2DH) gene was obtained, with one open reading frame of 2,007 bp which encodes 668 amino acids. Cis-regulatory elements for response to methyl jasmonic acid, light and drought existed in the 5′-upstream region. Phylogenetic analysis indicated that SjM2DH has an ancient prokaryotic origin, and is probably acquired by horizontal gene transfer event. Multiple alignment and spatial structure prediction displayed a series of conserved functional residues, motifs and domains, which favored that SjM2DH belongs to the polyol-specific long-chain dehydrogenases/reductase (PSLDR) family. Expressional profiles of <i>SjM2DH</i> in the juvenile sporophytes showed that it was influenced by saline, oxidative and desiccative factors. SjM2DH was over-expressed in <i>Escherichia coli</i>, and the cell-free extracts with recombinant SjM2DH displayed high activity on D-fructose reduction reaction. The analysis on SjM2DH gene structure and biochemical parameters reached a consensus that activity of SjM2DH is NADH-dependent and metal ion-independent. The characterization of SjM2DH showed that M2DH is a new member of PSLDR family and play an important role in mannitol metabolism in <i>S. japonica</i>.</p></div

Unigenes involved in mannitol pathway identified from transcriptome of <i>S. japonica</i> and verified with BLASTX algorithm.

<p>Unigenes involved in mannitol pathway identified from transcriptome of <i>S. japonica</i> and verified with BLASTX algorithm.</p

Structural comparison of the crystal structure of PfM2DH and putative SjM2DH.

<p>A, global tertiary structure of PfM2DH (Kavanagh et al., 2002). B, stereo-ribbon representation of SjM2DH in two domains. The connection of N- and C-terminal domains (VKDV) was indicated in orange; three deletions of β-sheets were shown in magenta; the insertion of an anti-parallel β-sheet was presented in green.</p

Influence of salinities and NaCl concentrations on <i>SjM2DH</i> expression levels in juvenile sporophytes.

<p>A, expression levels of <i>SjM2DH</i> under various NaCl concentrations. B, expression levels of <i>SjM2DH</i> under various salinities. All the data are the mean values of three independent experiments.</p