title: One-Pot Production of L-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2).
abstract: We developed a novel process for efficient synthesis of L-threo-3-hydroxyaspartic acid (L-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of L-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize L-THA. By using these recombinant enzymes, L-THA was obtained by L-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. L-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased L-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the L-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, L-THA was successfully obtained from L-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of L-THA.
authors: Research Institute for Science and Engineering, Waseda University, Tokyo, Japan ryo_h@aoni.waseda.jp.
authors: Ryotaro
authors: R
authors: Hara
authors: Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Tokyo, Japan.
authors: Masashi
authors: M
authors: Nakano
authors: Research Institute for Science and Engineering, Waseda University, Tokyo, Japan Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Tokyo, Japan.
authors: Kuniki
authors: K
authors: Kino
mesh_terms: D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D006900:Hydroxylation; D060847:Metabolic Engineering; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; C011274:3-hydroxyaspartic acid; D001224:Aspartic Acid; D006899:Mixed Function Oxygenases