Functional characterization of DOI synthase (kanA) and glycosyltransferase (kanE) involved in kanamycin gene cluster in Streptomyce kanamyceticus
Le Thuy Mai
The Graduate School of Sunmoon University
Department of Chemistry Biochemistry Major
DOI synthase Streptomyce kanamyceticus kanA kanE glycosyltransferase kanamycin gene cluster;
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Kanamycins (Kms) are important antibiotics, which belong to 2-deoxystreptamine (DOS)-containing AmAcs (kanamycin, gentamicin, tobramycin, ribostamycin, neomycin etc.), they cover the majority of clinically important AmAcs. All of the members of these antibiotics are characterized by the presence of an aglycon (DOS). The glycosylations of DOS with a variety of sugars at positions (C-4 and C-6) have brought structural diversities in Km antibiotics (KmA, KmB and KmC). In fact, their identification and isolation from bacteria were several decades ago. The genetic and biochemical investigations on these antibiotics have begun in the past few years. The isolation of a putative Km cluster from Streptomyce kanamyceticus is the major genetic study on this antibiotic hitherto. However, none of these genes has been fully characterized with respect to the biosynthesis of Kms. A cluster for the Km biosynthesis has been isolated from the genomic library of S. kanamyceticus ATCC 12853. Sequencing of 47 kb chromosomal DNA revealed 40 open reading frames (ORFs) including Km biosynthetic, regulatory and resistant genes. The first, the study on Kms started with the feeding experiments. Following, the function of kanA (DOI synthase) was confirmed by protein expression and in vitro enzyme assay. However, high level expressions of many cloned from Streptomyces genes into E. coli are often hindered by the formation of inclusion bodies, very dense aggregates of insoluble proteins. The kanA was expressed in E. coli BL21 (DE3) with the form of His6-fusion protein under the control of T7 promotor. The expression of the target protein was observed absolutely as inclusion bodies at the various conditions of induction temperature (15-37°C) and IPTG concentration (0.2-1.5 mM). For expressing aminoglycoside (Ams) genes in Streptomyces spp. an expression vector (pIBR25) was constructed and used to express a gene encoding 2-deoxy-scyllo-inosose synthase (kanA) in Streptomyce lividans TK24. The activity of KanA was dependent on the culture period of the transformants reaching maximum at 72 hr. The expression of kanA in pIBR25 advantaged over that in pSET152 and improved the formation of insoluble KanA, when it was expressed in Escherichia coli. The results provide a valuable tool for expressing actinomycetes biosynthetic genes in Streptomyces spp. Despite the broad range antibacterial activity of Kms, but their clinical utility is restricted to treat the tuberculosis due to the readily emergence of Km-resistant bacteria. The resistance of Kms follows by various mechanisms. The understanding of the inactivation mechanisms has revived the clinical utilities, from Kms generated their semi-synthetic analogues (dibekacin, amikacin and arbekacin) which are proved to be effective against Km-resistant bacteria. The usage of arbekacin as an anti-MRSA (methicillin-resistant Staphyllococcus areus) orphan drug in Japan since 1990 is an example, it suggested that the characterization and manipulation of glycosyltransferases are valuable for altering the sugar subunit, and ultimately modulating the biological activities of various antibiotics. Originally, based on general method for the deglycosidation of glycosylated antibiotics that has been developed. An improved method was carried out which be enabling for the hydrolysis of aminoglycoside antibiotics. The treatment of Km with H2SO4 results in efficient cleavage of the amino-sugars to produce the corresponding components in high yield. The hydrolytic experiment provides efficient tool not only for the analysis of Km production, but also going to set up in vitro enzyme assay of glycosylation. The substrates were formed from the hydrolysis can be used to produce hybrid antibiotics. Structurally, Kms have three sugar-derived subunits, 2-deoxystreptamine, kanosamine (or its analog) and neosamine (or its analog) linked by esthetic linkages. Sequence analyses of the putative Km biosynthetic gene cluster revealed two putative glycosyltransferases (kanE and kanF). The kanE was independently inactivated by gene deletion. Finally, the success of the deleted mutant generated (W4) abolished Km production was confirmed by the HPLC and LC-Mass analysis of the culture broths. The detection of neosamine-deoxystreptamine in the culture broth of W4 mutant confirmed that the former gene involves in the glycosylation of DOS (deoxystreptamine) with kanosamine during the biosynthesis of Kms. In this study, we provide systematic protocols for cloning, expression, transformation into Streptomyce kanamyceticus and screening strategy of mutants. Practically, we also demonstrated the function of genes and proposed the Km biosynthetic pathway. Finally, We target on engineering the production of Kms and synthesizing of hybrid antibiotics.