Construction of a targetable cassette for an attempt on aponeocarzinostatin gene replacement in Streptomyces carzinostatics.

• Main Authors: Yu-Wei Chang, 張玉薇
• Other Authors: 金德航
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/08419813198545309995

碩士 === 國立中興大學 === 化學系所 === 94 === Neocarzinostatin, a powerful antitumor antibiotic chromoprotein from Streptomyces carzinostaticus, is composed of a labile enediyne chromophore that is protected by an apoprotein with 113 amino acid residues. Neocarzinostatin complex is secreted as a pro-drug and the biologically active enediyne chromophore must be activated prior to its DNA cleavage action on the target cells. Emerging data on the elucidation of its biosynthetic gene cluster offers a polyketide based framework for the synthesis of notorious enediyne antibiotics. At this juncture, additional functional components of enedyne-defence machinery, including the one that insulates the producer organism from otherwise self-inflictive nature of its own enediyne compound, are also attracting attention. In the present study, we attempted to elucidate the possible role for aponeocarzinostatin in self-resistance function. Since gene knock-out is a proven strategy to identify genes involved in biological processes, we adopted the same in our effort to knock out the gene coding for aponeocarzinostatin. In 1996, Hopwood and coworkers first reported a λ RED system in Streptomyces to develop Streptomyces coelicolor A3(2) library by SuperCos 1 vector. Afterwards, researchers have applied the λ RED system for gene replacements in Streptomyces. Here, we employed the same strategy for creating precise gene replacements in apoprotein gene sequences. We used PCR-targeting to construct streptomycin resistance cassette. We also amplified the apoprotein gene sequences by PCR followed by its ligation with SuperCos 1 vector. A λ Red-mediated recombination with homology extensions guides the replacement of apoprotein in the cosmid clone with streptomycin resistance gene as a selection marker for the purpose of isolating the recombinant Streptomyces. We have successfully transferred streptomycin resistance gene to E. coli to get the target cosmid DNA, and proceeded to replace apoprotein gene by conjugation and protoplast transformation in Streptomyces. However, the apoprotein gene replacement is yet to be confirmed for the putative conjugants/tranformants. Better screening methods from future studies would help to confirm the occurrence of intended gene replacement in S. carzinostaticus.