Study On Directed Biosynthesis Of Nebramycins

Tobramycin is an important clinical antibiotic. Streptomyces tenebrarius Tt-49 is an industrial strain, mainly producing apramycin, carbamoyltobramycin, tobramycin and other compounds. In order to obtain a single compound tobramycin producing strain, the biosynthesis of apramycin was firstly blocked by using genetic engineering techniques. And then, the carbamylation was further interdicted. On this basis, to explore the possibility of combinating the exogenous genes with apramycin and tobramycin biosynthesis genes, gene modules, which may be responsible for gentamicin C3’, C4’dehydroxylation, was inserted. The details are as follows:1. Construction of aprH gene deletion strain TH304:The single crossover mutant TH303 was obtained after the recombinant plasmid pTH407 has been introduced to S. tenebrarius Tt49 by conjugation. The aprH gene deletion strain TH304 was selected by replica plating and PCR analysis after TH303 has been continuously cultured in the absence of erythromycin. The result of fermentation showed that the antibiotic production of TH304 was 2250 μ/mL, about 75% of Tt49’s. TLC and MS analysis indicated that TH304 mainly produced carbamoyltobramycin, apramycin was no longer produced. However, in contrast to Tt-49, TH304 had a longer growth cycle and poor spore production ability, it was not conducive to the industrial manufacture.2. Construction of aprJgene deletion strain TJ302:The aprJ gene deletion strain TJ302 was obtained after the recombinant plasmid pTJ401 has been introduced to S. tenebrarius Tt49 by conjugation. The result of fermentation showed that the antibiotic production of TJ302 was 2500 μ/mL, about 85% of Tt49’s. TJ302 mainly produced carbamoyltobramycin, apramycin was no longer produced. The research found that TJ302 had a more stable growth characteristics, it was more suitable for further reform.3. Construction of tobZ gene deletion strain TJZ308:The tobZ gene deletion strain TJZ308 was obtained after the recombinant plasmid pTZ501 has been introduced to S. tenebrarius TJ302 by conjugation. The antimicrobial activity of TJZ308 was as same as TJ302. TLC and MS analysis indicated that, TJZ308 was a single compound tobramycin producing strain. The quality of tobramycin could achieve the standards set by the pharmacopoeia without chromatography separation. It has not noly solved the key technical problems, which have troubled company about for decades, but also filled the blank at domestic and abroad research.4. The combination of gentamicin 3’,4’ dehydroxylation genes and tobramycin biosynthesis genes:The recombinant plasmid pZP606, which has been constructed for ermE* and genB3-P-B4 genes insertion, was introduced to S. tenebrarius TJZ308. The gene insertion strain TZP310 was obtained after ermE* and genB3-P-B4 genes has been integrated to tobramycin biosynthesis gene tobZ. The total titer of antibiotic production of TZP310 has seriously reduced, which was only about 300 μ/mL. MS analysis showed that, tobramycin was no longer produced. TZP310 only produced nebremine as the insertion of genB3-P-B4 genes.5. The combination of gentamicin 3’,4’ dehydroxylation genes and apramycin biosynthesis genes: The recombinant plasmid pDP605 was introduced to S. tenebrarius ST314. The gene insertion strain TDP306 was obtained after ermE* and genB3-P-B4 genes has been integrated to apramycin biosynthesis genes aprD3-D4. The antibiotic production of TDP306 was as same as ST314. MS analysis showed that, TDP306 still accumulated kanamycin B, dibekacin was not biosynthesis as expected as the insertion of genB3-P-B4 gene. However, the insertion loci of exogenous gene has been affirmed, which was essential to further research the expression of gentamicin 3’,4’ dehydroxylation genes in the S. tenebrarius and provide the condition of dibekacin biosynthesis.