A Number Of Antibiotic Biosynthesis Genes And Chemical Synthesis Research

Aminoglycoside antibiotics are mostly regarded as compounds mainly based on amino-N-containing-sugars and/or cyclitol derivatives, including streptomycins, kanamycins, apramycins, and so on. Most of them are produced by Streptomyces.Streptomyces tenebrarius H₆ produces a complex of aminoglycoside antibiotics, such as apramycin, tobramycin and kanamycin B etc. To study the possible genes involved in the biosynthesis of aminoglycosides in Streptomyces tenebrarius H6 ,primers were designed according to the highly conserved sequence of the dTDP-glucose-4,6-dehydratse gene , and 0.6kb PCR product was obtained from S. tenebrarius H₆ genome. Using this fragment as probe, an ORF of putative dTDP-glucose-4,6-dehydratase gene consisted of 1,132bp, designated as orfE was obtained.Based on the result, three other ORFs orfG1, orfG2 and orfM encoding the glycosyltransferase and mannosyltransferase linked to orfE were obtained by subcloning and sequencing (Bankit number:476862; accession number: AY131228) .The function of the orfE gene was studied by gene disruption. The orfE gene was disrupted by inserting the erythromycin resistance gene (ermE) into the unique SalI site of orfE. An E.coli/Streptomyces shuttle vector pHZ132 was used in conjugation, which contains a temperature sensitive replicon that functions only at temperatures below 34℃. Gene disruption was done by conjugation between E.coli ET12567 (pUZ8002/pHZ132E0.6) and S.tenebrarius H₆. The conjugation was first carried out on SM medium at 37℃. Potential exconjugants then were picked and inoculated in selected medium containing nalidixic acid and Em and incubated at 42℃.At this temperature, only the strains in which the recombinated plasmid integrated into the chromosome of the wild-type strain by means of homologous recombination could grow. Five potential orfE gene disruptant stains B7-1⁵, which did not show tobramycin and kanamycin B production were obtained. To confirm the result, B7-1⁵ were incubated as liquid culture, the fermentation broth was analyzed by TLC...