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Functional identification of alginate lyase from the brown alga Saccharina japonica.

ABSTRACT: Despite the progress in massive gene analysis of brown algal species, no alginate-degrading enzyme from brown alga has been identified, impeding the understanding of alginate metabolism in brown alga. In the current study, we identified and characterized alginate lyase from Saccharina japonica using a protein-based approach. First, cDNA library was prepared from the S. japonica sporophyte. Expression screening was then performed; the encoding gene was identified and cloned; and the recombinant enzyme was purified and characterized. Alginate lyase production in algal tissues was evaluated by western blotting. The identified alginate lyase, SjAly (359 amino acids, with a predicted N-terminal secretion signal of 27 residues), is encoded by an open reading frame comprising seven exons. Recombinant SjAly exhibited endolytic alginate lyase activity, specifically toward stretches of consecutive ?-D-mannuronic acid units. The optimum temperature, pH, and NaCl concentration were 30?°C, pH 8.0, and 100?mM, respectively. SjAly exhibited pronounced activity below 20?°C, the S. japonica growth temperature. SjAly was highly expressed in the blade but not the stipe and rhizoid. The data indicate that S. japonica possesses at least one active alginate lyase. This is the first report of a functional alginate lyase from brown alga, the major natural alginate producer.

SUBMITTER: Inoue A

PROVIDER: S-EPMC6426991 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Functional identification of alginate lyase from the brown alga Saccharina japonica.

Inoue Akira A Ojima Takao T

Scientific reports 20190320 1

Despite the progress in massive gene analysis of brown algal species, no alginate-degrading enzyme from brown alga has been identified, impeding the understanding of alginate metabolism in brown alga. In the current study, we identified and characterized alginate lyase from Saccharina japonica using a protein-based approach. First, cDNA library was prepared from the S. japonica sporophyte. Expression screening was then performed; the encoding gene was identified and cloned; and the recombinant e ...[more]

PMID: 30894645

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Project description:BackgroundSaccharina japonica (Areschoug) Lane, Mayes, Druehl et Saunders is an economically important and highly morphologically variable brown alga inhabiting the northwest Pacific marine waters. On the basis of nuclear (ITS), plastid (rbcLS) and mitochondrial (COI) DNA sequence data, we have analyzed the genetic composition of typical Saccharina japonica (TYP) and its two common morphological varieties, known as the "longipes" (LON) and "shallow-water" (SHA) forms seeking to clarify their taxonomical status and to evaluate the possibility of cryptic species within S. japonica.ResultsThe data show that the TYP and LON forms are very similar genetically in spite of drastic differences in morphology, life history traits, and ecological preferences. Both, however, are genetically quite different from the SHA form. The two Saccharina lineages are distinguished by 109 fixed single nucleotide differences as well as by seven fixed length polymorphisms (based on a 4,286 bp concatenated dataset that includes three gene regions). The GenBank database reveals a close affinity of the TYP and LON forms to S. japonica and the SHA form to S. cichorioides. The three gene markers used in the present work have different sensitivity for the algal species identification. COI gene was the most discriminant gene marker. However, we have detected instances of interspecific COI recombination reflecting putative historical hybridization events between distantly related algal lineages. The recombinant sequences show highly contrasted level of divergence in the 5'- and 3'- regions of the gene, leading to significantly different tree topologies depending on the gene segment (5'- or 3'-) used for tree reconstruction. Consequently, the 5'-COI "barcoding" region (~ 650 bp) can be misleading for identification purposes, at least in the case of algal species that might have experienced historical hybridization events.ConclusionTaking into account the potential roles of phenotypic plasticity in evolution, we conclude that the TYP and LON forms represent examples of algae phenotypic diversification that enables successful adaptation to contrasting shallow- and deep-water marine environments, while the SHA form is very similar to S. cichorioides and should be considered a different species. Practical applications for algal management and conservation are briefly considered.

Project description:Lipoxygenase (LOX) plays important roles in fatty acid oxidation and lipid mediator biosynthesis. In this study, we give first insights into brown algal LOX evolution. Whole genome searches revealed four, three, and eleven LOXs in Ectocarpus siliculosus, Cladosiphon okamuranus, and Saccharina japonica, respectively. In phylogenetic analyses, LOXs from brown algae form a robust clade with those from prokaryotes, suggesting an ancestral origin and slow evolution. Brown algal LOXs were divided into two clades, C1 and C2 in a phylogenetic tree. Compared to the two species of Ectocarpales, LOX gene expansion occurred in the kelp S. japonica through tandem duplication and segmental duplication. Selection pressure analysis showed that LOX genes in brown algae have undergone strong purifying selection, while the selective constraint in the C2 clade was more relaxed than that in the C1 clade. Furthermore, within each clade, LOXs of S. japonica evolved under more relaxed selection constraints than E. siliculosus and C. okamuranus. Structural modeling showed that unlike LOXs of plants and animals, which contain a β barrel in the N-terminal part of the protein, LOXs in brown algae fold into a single domain. Analysis of previously published transcriptomic data showed that LOXs in E. siliculosus are responsive to hyposaline, hypersaline, oxidative, and copper stresses. Moreover, clear divergence of expression patterns was observed among different life stages, as well as between duplicate gene pairs. In E. siliculosus, all four LOXs are male-biased in immature gametophytes, and mature gametophytes showed significantly higher LOX mRNA levels than immature gametophytes and sporophytes. In S. japonica, however, our RNA-Seq data showed that most LOXs are highly expressed in sporophytes. Even the most recently duplicated gene pairs showed divergent expression patterns, suggesting that functional divergence has likely occurred since LOX genes duplicated, which potentially contributes to the production of various oxylipins in brown algae.

Dataset Information

Functional identification of alginate lyase from the brown alga Saccharina japonica.

Publications

Functional identification of alginate lyase from the brown alga Saccharina japonica.

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