Directed evolution and site-specific mutagenesis of l-isoleucine dioxygenase derived from Bacillus weihenstephanensis
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l-isoleucine dioxygenase (IDO) specifically transforms l-isoleucine (Ile) to 4-hydroxyisoleucine (4-HIL), and 4-HIL is a promising drug for diabetes. To enhance the activity and catalytic efficiency of IDO, we used directed evolution and site-specific mutagenesis.
The IDO gene (ido) derived from Bacillus weihenstephanensis was cloned and expressed in Escherichia coli. Directed evolution using error prone (EP)-PCR and site-specific mutagenesis were conducted. Two improved mutants were obtained after one round of EP-PCR, with IdoN126H exhibiting a 2.8-fold increase in activity. Two improved mutants were obtained through site-specific mutagenesis, with IdoT130K showing a 170% increase in activity. Although the activity of the combined mutant IdoN126H/T130K (0.95 ± 0.08 U/mg) was slightly higher than that of the wild-type Ido, its catalytic efficiency was 2.4-fold and 3.0-fold higher than Ido with Ile and α-ketoglutaric acid as substrates. After biotransformation of Ile by E. coli BL21(DE3) expressing IdoN126H/T130K and Ido, 66.50 ± 0.99 mM and 26.09 ± 1.85 mM 4-HIL was synthesized, respectively, in 24 h.
IdoN126H/T130K had a higher enzyme activity and catalytic efficiency and can therefore be used as a more suitable candidate for 4-HIL production.
KeywordsBiotransformation Catalytic efficiency Error prone-PCR 4-Hydroxyisoleucine l-isoleucine dioxygenase Site-specific mutagenesis
The authors thank the “national first-class discipline program of Light Industry Technology and Engineering” (contract no. LITE2018-10) for financial support.
Supplementary Table 1–Primers for amplifying the ido gene and the mutant ido genes.
- Maurya CK, Singh R, Jaiswal N, Venkateswarlu K, Narender T, Tamrakar AK (2014) 4-Hydroxyisoleucine ameliorates fatty acid-induced insulin resistance and inflammatory response in skeletal muscle cells. Mol Cell Endocrinol 39:551–560Google Scholar
- Ogawa J, Kodera T, Smirnov SV, Hibi M, Samsonova NN, Koyama R, Yamanaka H, Mano J, Kawashima T, Yokozeki K, Shimizu S (2011) A novel l-isoleucine metabolism in Bacillus thuringiensis generating (2S, 3R, 4S)-4-hydroxyisoleucine, a potential insulinotropic and anti-obesity amino acid. Appl Microbiol Biotechnol 89:1929–1938CrossRefPubMedGoogle Scholar