Cloning and expression of neoQ and neoB genes coded for bifunctional 6’- (and 6’’’)-dehydrogenase and 6’- (and 6’’’)-aminotransferase involved in the biosynthesis of neomycin in Streptomyces fradiae BUWKH 66278

Abstract

Neomycin represents an example of 2-deoxystreptamine containing aminocyclitol aminoglycoside antibiotics (2DOS-ACAGAs) which are mainly composed of amino sugars and a unique amincyclitol aglycone (2DOS). Previous analysis of the neomycin biosynthetic gene cluster has revealed two genes namely neoQ (1.626 kb) and neoB (1.25 kb) whose gene products were anticipated to encode bifunctional 6’- (and 6’’’)-dehydrogenase and 6’- (and 6’’’)-aminotransferase during neomycin biosynthesis, respectively. Homologous to NeoQ and NeoB proteins were also found to be conserved in the gene clusters of various ACAGAs, such as paromamycin, lividomycin, butirosin, ribostamycin, kanamycin, gentamicin and tobramycin. In order to investigate and prove the biochemistry of regarded gene products, cloning and expression of the two genes were carried out. Accordingly, the two genes were amplified via PCR from chromosomal DNA of Streptomyces fradiae BUWKH 66278 using the appropriate primers. Both PCR products were cloned into cloning pUCPU21 and expression pET16b vectors. Heterologous expressions of both neoQ and neoB under the control of the T7 promoter in Escherichia coli BL21 (DE3) and E. coli JM 109 (DE3) have been undertaken. Both NeoQ (about 58.0 kDa) and NeoB (44.6 kDa) proteins were obtained in soluble His-tagged form as determined by SDSPAGE and Western blot assay.