Cloning and Expression of D-Threonine Aldolase from Ensifer arboris NBRC100383

Authors: Sang-Ho Baik

Abstract:

D-erythro-cyclohexylserine (D chiral unnatural β-hydroxy amino acid expected for the synthesis of drug for AIDS treatment. To develop a continuous bioconversion system with whole cell biocatalyst of D-threonine aldolase (D genes for the D-erythro-CHS production, D-threonine aldolase gene was amplified from Ensifer arboris 100383 by direct PCR amplication using two degenerated oligonucleotide primers designed based on genomic sequence of Shinorhizobium meliloti Sequence analysis of the cloned DNA fragment revealed one open-reading frame of 1059 bp and 386 amino acids. This putative D-TA gene was cloned into NdeI and EcoRI (pEnsi His-tag sequence or BamHI (pEnsi-DTA[2]) sequence of the pET21(a) vector. The expression level of the cloned gene was extremely overexpressed by E. coli BL21(DE3) transformed with pEnsi-DTA[1] compared to E. coli BL21(DE3) transformed with pEnsi-DTA[2]. When the cells expressing the wild used for D-TA enzyme activity, 12 mM glycine was successfully detected in HPLC analysis. Moreover, the whole cells harbouring the recombinant D-TA was able to synthesize D-erythro of 0.6 mg/ml in a batch reaction.

Keywords: About four key words or phrases in alphabetical order, separated by commas.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1330655

References:

[1] J.Q. Liu, M. Odani, T. Yasuoka, Shimizu, H. Yamada (2000) Gene cloning and overexpression of low-specific D-threonine aldolase f application for production of a key intermediate for parkinsonism drug. Appl Microbiol Biotechnol 54: 44
[2] J. Sambrook, E. F. Fritsch, and T. Maniatis. Molecular cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Spring Harbor, New York 1989.