Study On Disruption And Replacement Of DnmV Gene In Daunorubicin Biosynthesis

Epi-daunorubicin, which mainly produced by chemical synthesis, is an important precursor of semi-synthetic epirubicin, a first line anti-tumor chemotherapeutic agent. Thus, direct fermentation of epi-daunorubicin has the advantages in eliminating several synthetic steps, cost reduction and more friendly environment in epirubicin production. According to daunorubicin biosynthesis pathway, it is possible to obtain an epi-daunorubicin-producer by modifying the function of DnmV in TDP-L-daunosamine biosynthesis way. Homologous recombination was first used for dnmV gene disruption in daunorubicin-producing Streptomyces coeruleorubidus SIPI-1482 and a dnmV block mutant was screened and identified. Three heterologous C4 ketoreductase genes were amplified by PCR and the function of these gene products were studied when introduced into dnmV block mutant either via plasmid expression or chromosome integration. The details are described as follows:dnmV, along with its upstream dnmU gene, was PCR amplified from SIPI-1482 genomic DNA. Sequence alignment indicated that the cloned fragment had a 93.8% DNA sequence homology and a 92.5% deduced amino acid sequence homology with the published dnmV gene from S. peucetius ATCC 29050. Three C4 ketoreductase with a stereoselectivity opposite to that of DnmV was selected and their genes were also amplified. aveBIV, from 5. avermitilis, had a 100% homology with the published sequence, while oleU, from S. antibioticus ATCC 11891 and novS, from S. niveus, had a 99.3% DNA sequence homology, 97.3% amino acid sequence and 99% DNA sequence homology, 98.6% amino acid sequence with the published sequence, respectively.Recombinant plasmids pYG807 and pYG809 for dnmV disruption were constructed by inserting 470bp and 716bp fragments from cloned dnmV into pKC1139, an E. coli-Streptomyces shuttle vector, respectively. S. lividans TK24 was first transformed with recombinant plasmid originated from E. coli, followed by transforming SIPI-1482 protoplast with plasmid from TK24 to avoid restricted modification of the host. After high temperature screening, PCR validation and Southern Blotting, a dnmV mutant named BF3 was obtained. 470bp is identified as the minimal length for recombination occurring in S. coeruleorubidus. However, daunorubicin still could be detected in BF3 metabolites. This phenomenon indicates that the mutant allele of dnmV in BF3 remains at least a part of the function of wild type gene.Recombinant plasmid pYG817 was then constructed by inserting apramycin resistant gene into cloned dnmUV fragment and transformed SIPI-1482 protoplasts with single-strand DNA generated by alkaline denaturation. A double crossover mutant with dnmV insertional inactivation was acquired and named strain 12#. Daunorubicin absence from metabolites of 12# indicates that dnmV function was completely interupted.