Characterization Of Aprd3, Aprd4 Gene From Streptomyces Tenebrarius And Construction Of Kanamycin B Overproducing Strain

Streptomyces tenebrarius produce carbamoyltobramycin, carbamoylkanamycin B and apramycin. Carbamoyltobramycin and carbamoylkanamycin B are 4,6 substituted 2-DOS containing aminoglycosides. Whereas apramycin is 4 substituted, and has a unique octose. Carbamoyltobramycin and carbamoylkanamycin B are similar in structure, and they were biosynthesized by the same pathway. Their biosynthetic gene cluster has been cloned and named as tobramycin biosynthetic gene cluster. Apramycin has an independently biosynthetic pathway, whose gene cluster has also been cloned. The C-3'of carbamoyltobramycin and apramycin are hydrogen, but the C-3'of precursors of them are hydroxyl, so genes responsible for turning hydroxyl into hydrogen should exist in their biosynthetic gene clusters. To find those genes, the gene clusters of 2-DOS containing aminoglycosides were analysed by bioinformatics approach. Gene aprD3 and aprD4 of apramyin gene cluster were proposed involved in deoxygenation.Gene aprD3 was disrupted to clarify its function. Construct disruption plasmid pSPU307 with homologous fragments of Streptomyces tenebrarius H6, these fragments amplified by PCR. Introduce disruption plasmid pSPU307 by conjugation. After select transformant, then screen single crossover and double crossover strains sequently. Compared with wild type strain, aprD3 disruption strain produced a new antibiotic. The new antibiotic is oxyapramycin, which was clarified by MS and NMR. This result indicates aprD3 indeed involved in deoxygenation in apramycin biosynthesis. The aprD3 was the first reported gene related with deoxygenation in biosynthesis of 2-DOS containing aminoglycosides.Carbamoyltobramycin was not detectable in the broth of aprD4 disruption strain SPU308, but the production of carbamoylkanamycin B increased, and oxyapramycin was also present in the broth of SPU308. The difference of products between SPU308 and wild type strain proved aprD4 not only participated in dexoygenation in apramycin biosynthesis, but also in tobramycin biosynthesis.Carbamoylkanamycin B can be hydrolysis into kanamycin B, which is raw material for synthesis dibekacin and arbekacin. Therefore, it will be great interesting if we can construct kanamycin B overproducing strain. To block biosynthesis of apramycin and carbamoyltobramycin, a fragment was deleted from genome of S.tenebrarius H6. This fragment contains intact aprQ, partial aprD3 and aprD4. The fermentation products of disruption strain SPU313 were analysed. SPU313 did not produced apramycin and carbamoyltobramycin, it just produced carbamoylkanamycin B. Compared with the production of carbamoylkanamycin B in wild-type strain, the production in SPU313 is increased 5-fold. Nevertheless, carbamoylkanamycin B have to be hydrolysis to produce kanamycin B, so produced kanamycin B directly by fermentation will be more feasible. To construct kanamycin B producing strain, the carbamoyl-transferase gene tacA was disrupted. Finally, kanamycin B high-producing strain was constructed successfully. This study proves that genetic engineering of strains not only can eliminate impurities, but also can produce interesting product did not produce by wild-type strains.