One-Pot Production of l - threo -3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

Abstract

bWe 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.H ydroxylated amino acids occurring in nature possess important physiological functions as bioactive substances. For example, 5-hydroxy-L-tryptophan, which can be extracted from the African plant Griffonia simplicifolia, is clinically used for the treatment of depression, insomnia, headache, and obesity, owing to its ability to stimulate serotonin biosynthesis in the human brain (1). cis-4-Hydroxy-L-proline, which occurs in leaves of sandalwood, is a potent antitumor drug that prevents the folding of collagen triple helices in cancer cells (2). In addition, (4S)-4-hydroxy-L-isoleucine, which is contained in the seeds of fenugreek, is effective for the treatment of type II diabetes, since it promotes insulin secretion in a blood glucose-dependent manner (3). Moreover, derivatives of these hydroxylated amino acids can also be used in the synthesis of pharmaceuticals.3-Hydroxyaspartic acid is one such compound, and much attention has been paid to its synthesis and biological properties over the years. 3-Hydroxyaspartic acid contains two asymmetric centers at C-2 and C-3; thus, four stereoisomers are expected:, and D-erythro-3-hydroxyaspartic acid [(2R,3S)-form] (D-EHA) (see Fig. S1 in the supplemental material). L-THA is especially attractive for chemists and biologists owing to its broad clinical and material utility, including as an antimicrobial agent against various microorganisms (4), as an inhibitor of ...

Isolation and amino acid sequence of a dehydratase acting on d-erythro-3-hydroxyaspartate from Pseudomonas sp. N99, and its application in the production of optically active 3-hydroxyaspartate

“…To the best of our knowledge, this is the first example of the enzymatic production or resolution of L-EHA. In addition, the concentration of L-EHA in the reaction mixture achieved in this study (100 mM) was much higher than previously reported values achieved using enzymatic L-THA production (about 10 mM using AsnO-D241N system, 14) and about 48 mM using wild-type AsnO-expressing E. coli system 15) ). We also analyzed the final products using TLC, HPLC, and NMR spectroscopy and confirmed them to be chemically and optically pure L-EHA/D-THA.…”

“…Hara et al also reported whole cell catalysis from asparagine to L-THA using asparaginase-deficientand wild-type AsnO-expressing E. coli cells. 15) However, the enzymatic production of L-EHA has not yet been reported. In this study, we successfully achieved the enzymatic resolution of DL-EHA and DL-THA to obtain optically pure L-EHA and D-THA, respectively, using the recombinant D-EHA DH produced by E. coli cells.…”

“…12) In addition, the enzymatic syntheses of L-THA from L-aspartate or L-asparagine have also been reported. 14,15) However, no dehydratase acting on the D-erythro form of 3-hydroxyaspartate has been reported so far. Therefore, the enzymatic resolution of DL-EHA to produce L-EHA has not been achieved to date.…”