論文 - 詳細
| RRC ID | 53627 |
|---|---|
| 著者 | Hara R, Nakano M, Kino K. |
| タイトル | One-Pot Production of L-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2). |
| ジャーナル | Appl Environ Microbiol |
| 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. |
| 巻・号 | 81(11) |
| ページ | 3648-54 |
| 公開日 | 2015-6-1 |
| DOI | 10.1128/AEM.03963-14 |
| PII | AEM.03963-14 |
| PMID | 25795668 |
| PMC | PMC4421054 |
| MeSH | Aspartic Acid / analogs & derivatives* Aspartic Acid / metabolism Escherichia coli / enzymology* Escherichia coli / genetics Escherichia coli / metabolism* Hydrolysis Hydroxylation Metabolic Engineering* Mixed Function Oxygenases / genetics Mixed Function Oxygenases / metabolism* Recombinant Proteins / genetics Recombinant Proteins / metabolism* Streptomyces coelicolor / enzymology* Streptomyces coelicolor / genetics |
| IF | 4.016 |
| 引用数 | 3 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
|
| 最多言及媒体 | X(Twitter) |
| 各媒体での言及数の合計 | 1 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 原核生物(大腸菌) | |