title: Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus.
abstract: Alginate is a marine non-food-competing polysaccharide that has potential applications in biorefinery. Owing to its large size (molecular weight >300,000 Da), alginate cannot pass through the bacterial cell membrane. Therefore, bacteria that utilize alginate are presumed to have an enzyme that degrades extracellular alginate. Recently, Vibrio algivorus sp. SA2Twas identified as a novel alginate-decomposing and alginate-utilizing species. However, little is known about the mechanism of alginate degradation and metabolism in this species. To address this issue, we screened the V. algivorus genomic DNA library for genes encoding polysaccharide-decomposing enzymes using a novel double-layer plate screening method and identified alyB as a candidate. Most identified alginate-decomposing enzymes (i.e., alginate lyases) must be concentrated and purified before extracellular alginate depolymerization. AlyB of V. algivorus heterologously expressed in Escherichia coli depolymerized extracellular alginate without requiring concentration or purification. We found seven homologues in the V. algivorus genome (alyB, alyD, oalA, oalB, oalC, dehR, and toaA) that are thought to encode enzymes responsible for alginate transport and metabolism. Introducing these genes into E. coli enabled the cells to assimilate soluble alginate depolymerized by V. algivorus AlyB as the sole carbon source. The alginate was bioconverted into L-lysine (43.3 mg/l) in E. coli strain AJIK01. These findings demonstrate a simple and novel screening method for identifying polysaccharide-degrading enzymes in bacteria and provide a simple alginate biocatalyst and fermentation system with potential applications in industrial biorefinery.
authors.affiliation: Process Development Laboratories, Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-8681, Japan. hidetaka_doi@ajinomoto.com.|Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. hidetaka_doi@ajinomoto.com.
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Doi
authors.affiliation: Process Development Laboratories, Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-8681, Japan.
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Tokura
authors.affiliation: Process Development Laboratories, Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-8681, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Mori
authors.affiliation: Process Development Laboratories, Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-8681, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Mori
authors.affiliation: Process Development Laboratories, Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-8681, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Asakura
authors.affiliation: Process Development Laboratories, Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-8681, Japan.|Frontier Research Laboratories, Institute for Innovation, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-8681, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Usuda
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Hiroo
authors.initials: H
authors.lastname: Fukuda
authors.affiliation: Process Development Laboratories, Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-8681, Japan.
authors.forename: Akito
authors.initials: A
authors.lastname: Chinen
mesh_terms: D000464:Alginates / Q000378:metabolism*; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000378:metabolism*; D014733:Vibrio / Q000201:enzymology* / Q000378:metabolism*
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase
keywords: Alginate; Alginate lyase; Alginate metabolism; Biorefinery; Polysaccharide-degrading enzyme; Vibrio algivorus