NADP⁺-Dependent Dehydrogenase SCO3486 and Cycloisomerase SCO3480: Key Enzymes for 3,6-Anhydro-L-Galactose Catabolism inStreptomyces coelicolorA3(2)

Abstract

Marine red algal cells contain high amounts of cellular carbohydrates and low amounts of lignin, and thus have gained attention as a sustainable next-generation biomass resource. Red algae-derived carbohydrates can replace fossil fuels, which are expected to be depleted in the near future, and can be converted into chemical feedstocks and bioenergy [1,2].Consisting mainly of carbohydrates that make up the majority of red algal cells, agar is a copolymer of the agarobiose unit (4-O-β-D-galactopyranosyl-3,6-anhydro-L-galactose) and the porphyrobiose unit (4-O-β-Dgalactopyranosyl-L-galactose-6-sulfate), linked by alpha-1,3 glycosidic bonds. The composition ratio of the two units varies depending on the species of red algae. Agar is called 'agarose' when agarobiose is the main component, and 'porphyran' when porphyrobiose is the main component [1,3].Agar has been widely used as a food, food additive, culturing medium, and support material for techniques like electrophoresis and chromatography [2]. Moreover, as the various biological activities of agar-derived oligosaccharides and agar decomposition products are discovered, the industrial use of agar is expected to expand [4]. Agarose, in nature, is mainly hydrolyzed by microbial beta-agarases into neoagarobiose (3,6-anhydro-Lgalactosyl-α-1,3-D-galactose), which is further hydrolyzed by neoagarooligosaccharide (NAOS) hydrolase into monomers D-galactose (D-Gal) and 3,6-anhydro-L-galactose (L-AHG) [5]. D-Gal is a valuable chemical feedstock that can be converted into bioenergy or other useful chemicals [4]. Additionally, L-AHG is a rare monosaccharide Agarose is a linear polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose (AHG). It is a major component of the red algal cell wall and is gaining attention as an abundant marine biomass.However, the inability to ferment AHG is considered an obstacle in the large-scale use of agarose and could be addressed by understanding AHG catabolism in agarolytic microorganisms. Since AHG catabolism was uniquely confirmed in Vibrio sp. EJY3, a gram-negative marine bacterial species, we investigated AHG metabolism in Streptomyces coelicolor A3(2), an agarolytic gram-positive soil bacterium. Based on genomic data, the SCO3486 protein (492 amino acids) and the SCO3480 protein (361 amino acids) of S. coelicolor A3(2) showed identity with H2IFE7.1 (40% identity) encoding AHG dehydrogenase and H2IFX0.1 (42% identity) encoding 3,6-anhydro-L-galactonate cycloisomerase, respectively, which are involved in the initial catabolism of AHG in Vibrio sp. EJY3. Thin layer chromatography and mass spectrometry of the bioconversion products catalyzed by recombinant SCO3486 and SCO3480 proteins, revealed that SCO3486 is an AHG dehydrogenase that oxidizes AHG to 3,6-anhydro-L-galactonate, and SCO3480 is a 3,6-anhydro-L-galactonate cycloisomerase that converts 3,6-anhydro-L-galactonate to 2-keto-3-deoxygalactonate. SCO3486 showed maximum activity at pH 6.0 at 50°C, increased activity in the presence of iron ions, and activity against various a...

SCO6992, a Protein with β-Glucuronidase Activity, Complements a Mutation at the absR Locus and Promotes Antibiotic Biosynthesis in Streptomyces coelicolor

“…S. coelicolor J1501 was cultured in R2YE medium and used for chromosomal DNA isolation, as described previously [ 16 ]. To clone the abs R - complementing DNA fragment, the chromosomal DNA was partially digested with restriction enzyme Sau 3A1, and separated elctrophoretically on a 1% agarose gel.…”

Utilization of Macroalgae for the Production of Bioactive Compounds and Bioprocesses Using Microbial Biotechnology

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