Overexpression of the aminotransferase DsaD and isomerase DsaE as a fusion protein to synthesize L-allo-isoleucine

Abstract:

[Background] L-isoleucine (L-Ile) and L-allo-isoleucine (L-allo-Ile) are two isomers in nature. The anti-infective antibiotic desotamides contain an L-allo-Ile moiety, and the aminotransferase DsaD and isomerase DsaE in their biosynthetic pathway catalyze the interconversion between L-Ile and L-allo-Ile. [Objective] To overexpress the aminotransferase DsaD and isomerase DsaE as a fusion protein DsaDE, and assay its activity to catalyze the interconversion between L-Ile and L-allo-Ile. [Methods] The coding region of dsaE and dsaD-linker (coding region of dasD containing 114 bp linker) were amplified by PCR; the purified DNA fragments were digested and ligated into pET28a(+), and the resulting recombinant plasmid was transformed into Escherichia coli BL21(DE3) to overexpress the fusion protein; the fusion protein DsaDE was purified by Ni-NTA affinity chromatography, then the activity of the fusion protein DsaDE was assayed using L-Ile or L-allo-Ile as substrate, respectively; the reaction mixtures were analyzed by high performance liquid chromatography (HPLC). [Results] PCR analyses, restriction endonuclease digestion and sequencing demonstrated that dasE and dsaD-linker were correctly ligated into pET28a(+); the fusion protein DsaDE with His6 tags at both the N-terminus and C-terminus was solubly produced in E. coli BL21(DE3) and purified to ~95% homogeneity. The purified fusion protein can catalyze the interconversion between L-Ile and L-allo-Ile efficiently in vitro. [Conclusion] The aminotransferase DsaD and isomerase DsaE were successfully overexpressed as a fusion protein; the fusion protein was readily purified by one-step Ni-NTA affinity chromatography; and purified fusion protein can catalyze the interconversion between L-Ile and L-allo-Ile efficiently, laying the foundation for further research on the industrial production of L-allo-Ile.

Key words:Aminotransferase, Isomerase, Fusion protein, Isoleucine