Studies On The Related Biosynthetic Genes Of Apramycin

To study the biosynthetics genes of apramycin in Streptomyces tenebrarius, the apramycin resistant gene was isolated by gunshot cloning firstly. S.tenebrarius chromosomal DNA , partially digested with Sau3Al to yield fragments of 6 ~ 15kb, was ligated with vector pIJ702. The ligation mixture was used to transform S.lividans TK24 protoplasts. Six colonies capable of growing in the presence of apramycin were isolated. Plasmid pSYXl, containing a 12kb insert of S.tenebrarius DNA, was isolated from one of six colonies. Apramycin resistant gene was located in 1.5-kb Sphl-Kpnl fragment of plasmid pSYXl though further studies.The 2.0-kb BamHl-Sphl fragment, upstream fragment of the apramycin resistant gene, was sequenced. Analysis of the nt sequence revealed that it contained an ORF from nt positions 877 to 1995. Composed of 109A, 388C, 489G and 136T, the ORF encoded a protein of 373 amino acids. The G+C percentage of this ORF was 78.2% and that of the third base of the codons was 96.3%. No homologous sequence was found by comparing it with the sequences obtained from the net, so that the cloned gene was supposed to be an unknown new gene, designated as oprA.A fragment, containing 2.0kb cloned 5. tenebrarius DNA and reported genes of ermE and xylE , was inserted in plasmid pHZ132 ( anE.coli-Streptomyces shuttle plasmid incorporating oriT from RK2) to construct disruption plasmid pZXB0l4. The plasmid was transformed into E.coli ET12567(pUZ8002) to construct recombinant E.coli ET12867(pUZ8002, pZXB014 ). Recombinant S.tenebrarius was obtained by conjugation E.coli ET12867(pUZ8002, pZXB014) with S.tenebrarius H6 to scan strains of which plasmids integrated into S.tenebrarius H6 chromosomal DNA. The gene recombinant strain NO.42 can't generate ampramycin, which indicated that the cloned gene is involved in apramycin biosynthesis in S.tenebrarius. It was also proved that the biosynthestic genes of apramycin was linked to the apramycin resistant gene in S.tenebrarius.