title: Three trehalose synthetic pathways in the acarbose-producing Actinoplanes sp. SN223/29 and evidence for the TreY role in biosynthesis of component C.
abstract: In this study, three trehalose gene clusters, treX-Y-Z, tpS1, and treS, of the acarbose-producing strain, Actinoplanes sp. SN223/29, have been identified. In particular, five trehalose synthetic genes were sequenced and characterized in detail. They were cloned and expressed in Escherichia coli BL21(DE3)pLysS using the His-tag vector pET19b. The recombinant proteins were purified by Ni(2+)-nitrilotriacetic acid agarose affinity chromatography, and their functions were characterized biochemically. Both the maltooligosyltrehalose synthase (TreY-TreZ) pathway and the trehalose synthase (TreS) pathway have maximum activity at 40 degrees C and at pH 7.5 and 7.0, respectively, in 100-mM phosphate buffer. Meanwhile, the trehalose-6-phosphate synthase (TpS1) showed maximum activity at 35 degrees C and at pH 7.5 in 100 mM Tris-HCl. As a cofactor candidate, Mg(2+) enhanced the activities of all three trehalose synthetic reactions significantly. TreY produced component C from acarbose by its proposed isomerase activity, but TreS did not. This study suggests that the mutation of treY can improve acarbose production by repressing component C production. Based on the data obtained in this study, a model for component C production in Actinoplanes sp. SN 223/29 is proposed.
authors.affiliation: Institute of Molecular Microbiology and Biotechnology, Westfällische Wilhelms-Universität Münster, Münster, Germany.
authors.forename: Jin-Sook
authors.initials: JS
authors.lastname: Lee
authors.forename: Tran
authors.initials: T
authors.lastname: Hai
authors.forename: Hermann
authors.initials: H
authors.lastname: Pape
authors.forename: Tae-Jong
authors.initials: TJ
authors.lastname: Kim
authors.forename: Joo-Won
authors.initials: JW
authors.lastname: Suh
mesh_terms: D020909:Acarbose / Q000031:analogs & derivatives* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D053898:Biosynthetic Pathways; D003001:Cloning, Molecular; D015870:Gene Expression; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008848:Micromonosporaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D014199:Trehalose / Q000096:biosynthesis*
chemical_list: D001426:Bacterial Proteins; C488330:acarbose byproduct, component C; D014199:Trehalose; D005964:Glucosyltransferases; C099084:maltooligosyl trehalose synthase; C099692:trehalose synthase; D020909:Acarbose