Functional genomics analysis reveals the biosynthesis pathways of important cellular components (alginate and fucoidan) of Saccharina

Abstract

Although alginate and fucoidan are unique cellular components and have important biological significance in brown algae, and many possible involved genes are present in brown algal genomes, their functions and regulatory mechanisms have not been fully revealed. Both polysaccharides may play important roles in the evolution of multicellular brown algae, but specific and in-depth studies are still limited. In this study, a functional genomics analysis of alginate and fucoidan biosynthesis routes was conducted in Saccharina, and the key events in these pathways in brown algae were identified. First, genes from different sources, including eukaryotic hosts via endosymbiotic gene transfer and bacteria via horizontal gene transfer, were combined to build a complete pathway framework. Then, a critical event occurred to drive these pathways to have real function: one of the mannose-6-phosphate isomerase homologs that arose by gene duplication subsequently adopted the function of the mannose-1-phosphate guanylyltransferase (MGP) gene, which was absent in algal genomes. Further, downstream pathway genes proceeded with gene expansions and complex transcriptional mechanisms, which may be conducive to the synthesis of alginate and fucoidan with diverse structures and contents depending on the developmental stage, tissue structure, and environmental conditions. This study revealed the alginate and fucoidan synthesis pathways and all included genes from separate phylogenetic sources in brown algae. Enzyme assays confirmed the function of key genes and led to the determination of a substitute for the missing MPG. All gene families had constitutively expressed member(s) to maintain the basic synthesis; and the gene function differentiation, enzyme characterization and gene expression regulation differences separated brown algae from other algae lineages and were considered to be the major driving forces for sophisticated system evolution of brown algae.

Keywords: Alginate; Biosynthesis pathway; Brown algae; Fucoidan; Saccharina.

Fig. 1

Bayesian phylogenetic tree based on the translated amino acids of mannose-6-phosphate isomerase (MPI) with bootstrap values (when >50%) indicated at the nodes. All eukaryotic algal MPIs (including those of brown algae, diatoms, red algae, and green algae) originated from eukaryotic hosts. Brown algae MPIs underwent gene duplication in their common ancestor. Bacterial type II MPIs encode bifunctional enzymes with MPI and MPG activities. All MPI sequences were obtained from GenBank or OneKP databases (Table S1)

Fig. 2

Bayesian phylogenetic tree based on the translated amino acids of phosphomannomutase (PMM) with bootstrap values (when >50%) indicated at the nodes. All eukaryotic algal PMMs (including those of brown algae, diatoms, red algae, and green algae) originated from eukaryotic hosts. All PMM sequences were obtained from GenBank or OneKP databases (Table S1)

Fig. 3

Bayesian phylogenetic tree based on the translated amino acids of GDP-mannose/UDP-glucose 6-dehydrogenases (GMD/UGD) with bootstrap values (when >50%) indicated at the nodes. Brown algal GMD was inherited from bacteria by HGT. UGD may originate from eukaryotic hosts: red algae acquired UGD from primary endosymbiotic hosts, while diatoms and brown algae acquired from secondary endosymbiotic hosts. All GMD/UGD sequences were obtained from the GenBank or OneKP databases (Table S1)

Fig. 4

Summary of a Bayesian tree based on mannuronate C5-epimerases (MC5E). Brown algae acquired MC5E from bacteria via HGT and the gene subsequently duplicated to form five groups (I–V). The detailed phylogenetic tree is shown in Figure S4

Fig. 5

Mannose-1-phosphate guanylyltransferase (MPG) activity of SjaMPI4. Effects of temperature (a), pH (b), and the presence of metal ions (c) on activity. a Enzyme activity at 40 °C was set to 100%. b Enzyme activity at pH 7.0 was set to 100%. c Enzyme activity in the presence of Mn²⁺ was set to 100%. d The double reciprocal plot of enzyme activity for GDP-mannose. Data represent mean ± SD of four independent experiments

Fig. 6

Phosphomannomutase and phosphoglucomutase activity of SjaPMM. Effects of temperature (a), pH (b), and the presence of metal ions (c) on activity. a PMM activity at 25 °C was set to 100%. b PMM activity at pH 7.4 was set to 100%. c PMM activity in the presence of Mg²⁺ was set to 100%. d The double reciprocal plot of enzyme activity for mannose-1-P. e The double reciprocal plot of enzyme activity for glucose-1-P. Data represent mean ± SD of four independent experiments

Fig. 7

Biosynthetic routes of alginate and fucoidan in algae. MPI4 was the actual MPG gene. The GMD/UGD gene family members all encoded enzymes with GMD activity. MC5E catalyzes the isomerization from mannuronic acid (M) to guluronic acid (G) at the alginate polymer level. MPI mannose-6-phosphate isomerase, PMM phosphomannomutase, MPG mannose-1-phosphate guanylyltransferase, GMD/UGD GDP-mannose/UDP-glucose 6-dehydrogenase, MS mannuronan synthase, MC5E mannuronate C5-epimerase, GM46D GDP-mannose 4,6-dehydratase, GFS GDP-fucose synthetase, FK fucokinase, GFPP GDP-fucose pyrophosphorylase, FS fucosyltransferase, ST sulfotransferase

Fig. 8

Summary of gene expression under abiotic stresses (hyperthermia, hyposaline, and continuous darkness). Genes in green exhibited increased expression and genes in red exhibited decreased expression. The circular area represents the relative expression level

Fig. 9

Origins of alginate and fucoidan synthesis genes in algae. The genes in yellow were derived from secondary endosymbiotic eukaryotic hosts and genes in purple were derived from bacteria via horizontal gene transfer