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.