Functional heterologous expression and characterization of mannuronan C5-epimerase from the brown alga Saccharina japonica

Abstract

Brown algae produce alginate that has various ratios and diverse sequences of two uronic acids, β-D-mannuronic acid and α-L-guluronic acid, compared with those of alginate produced by bacteria. This diversity of alginate in brown algae is caused by mannuronan C5-epimerases (MC5Es), which catalyze the conversion of β-D-mannuronic acid to α-L-guluronic acid. Although several bacterial MC5E enzymes have been well characterized, to date, there exists no information on the biochemical properties of eukaryotic MC5E. In this study, MC5E expression was detected in a brown alga Saccharina japonica sporophyte by immunoblot analysis. We also searched for MC5E mRNA from S. japonica by RT-PCR and revealed eight partial amino acid sequences, SjC5-I to-VIII. We focused on the highest frequency clone, SjC5-VI, and elucidated its full-length cDNA and putative gene structure. The translated SjC5-VI protein consists of 499 amino acids, with the N-terminal 21 amino acids predicted as a secretion signal sequence. Functional recombinant SjC5-VI (rSjC5-VI) was successfully expressed as a secreted protein using an insect-cell expression system, and we determined the optimal temperature, pH, and NaCl concentrations to be 35°C, 7.0-8.2, and 300 mM, respectively, using the Ca 2+-induced gel-formation assay. In addition, Ca 2+ enhanced gelation by 1.7-fold following rSjC5-VI activity. Furthermore, 1 H-NMR spectroscopy of rSjC5-VI-treated polyM revealed alternate epimerization of β-D-mannuronic acid to 3 α-L-guluronic acid. To the best of our knowledge, this is the first report on the characterization of MC5E activity in eukaryotes.

Single‐base methylome profiling of the giant kelp Saccharina japonica reveals significant differences in DNA methylation to microalgae and plants

“…vIPO) were found in SP (Fig. S21), which suggests that cytosine methylation contributes to regulating fundamental processes underpinning cell differentiation, growth and stress response (Pear et al ., ; Colin et al ., ; Ye et al ., ; Inoue et al ., ). Genes of the sex‐determination system do not show a negative correlation between their level of methylation and gene expression (Fig.…”

Growth of theFucusembryo: insights into wall-mediated cell expansion through mechanics and transcriptomics

“…However, the exact function of almost all of these epimerases remains unknown. Two of the currently known algal MC5Es have been functionally described: in Saccharina japonica, a recombinant epimerase (SjC5-VI) epimerizes M to G (96). In Ectocarpus siliculosus , a mannuronan C5-epimerase MEP13-C5 is thought to epimerize block MM regions, although its exact function is not completely clear (97).…”

Dynamics of cell wall assembly during early embryogenesis in the brown algaFucus

“…alginate lyases (Thomas et al , 2013), fucanase (Colin et al , 2006)] and alginate synthesis [i.e. GDP-mannose dehydrogenase (Tenhaken et al , 2011), ManC5-E (Fischl et al , 2016; Inoue et al , 2016)]. Together these new implementations will be important tools for the study of cell wall biology in brown algae.…”