The first elucidation of the novel metabolic pathway of 3,6-anhydro-L-galactose, the main component of red macroalgae, by integrating the metabolome and transcriptome analyses

Despite an increasing demand for utilizing marine macroalgae as sustainable resource for bio-based products, a bioconversion scheme of red macroalgae has not yet been developed owing to the non-fermentable rare sugar composed of red macroalgae. The most abundant carbohydrate polymer of red macroalgae is agarose, which is a heteropolysaccharide composed of D-galactose and 3,6-anhydro-L-galactose (AHG). Although AHG is the major sugar in red macroalgae, AHG is known to be not fermentable by any terrestrial microorganisms. For the first time, we have discovered the novel metabolic pathway of AHG in a marine microorganism by integrating metabolome and transcriptome analyses. The analyses of metabolome and transcriptome of EJY3 revealed two key metabolic intermediates of AHG, 3,6-anhydrogalactonate (AHGA) and 2-keto-3-deoxy-galactonate. Also, their corresponding genes were verified both in vitro and in vivo. The oxidation by an NA(D)P⁺‒dependent AHG dehydrogenase and isomerization by an AHGA cycloisomerase are the key metabolic steps for AHG metabolism. This newly discovered metabolic route was verified by demonstrating the growth of E. coli cells harboring the genes of these two enzymes on AHG as a sole carbon source. This is the first report on the discovery of the novel metabolic pathway of AHG in a living organism by systematic analyses of transcriptome and metabolome. These findings provide not only insights for the evolutionary adaptation of a central metabolic pathway to utilize uncommon substrates in organisms, but also a metabolic design principle for bioconversion of red macroalgal biomass into biofuels or industrial chemicals.