Study On Orf60 In Kigamicin Biosynthesis Gene Cluster

Kigamicin is the natural products isolated from actinomyces with such biological functions as anti-microbial and anti-tumor, for which the distinctive bio-activity is characterized by the specific inhibition of tumor cells in the nutrition starvation while the normal cells can be free from the special inhibition. Therefore, kigamicin can serve as a important source for developing specific anti-cancer drugs. In this paper, we study on the biosynthetic pathways of kigamicin and spend more efforts on new compound in the process. Thus, we can clearly understand the corresponding relationships of kigamicin between its chemical structure and function. In order to study the function of orf60 in Kigamicin biosynthesis, in this paper, our main research works are carried out as follows:First of all, we analysis the orf60 gene by bio informatics analysis method, we suspect that its encoded protein may be a single oxygenase. Then, we use PIJ778 as a template to get a gene fragment with spectinomycin resistant for replacement by using PCR amplification technology, whose two ends are homologous to those of orf60. Then, we transfer the gene fragment and integrated plasmid pLL59 into E. coil BW 25113/pIJ 790. By using PCR-Targeting technology we get a recombinant plasmid and named pLL200. By using PCR checking and resistance screening, we demonstrate that the orf60 gene has been knocked out successfully. Then, we transfer recombinant plasmid pLL200 and plasmid pLL68 (contain activating factor) into Steptomyces coelicolor ML154 together, we get a mutant strain ML20068 by using intergeneric conjugation technology. With the simultaneous fermentation of mutant ML20068 and wide-type ML154, both fermentation products summited to HPLC-MS and we Found that the mutant strains ML20068 produced [M+H]/Z538 and [M+H]/Z 513 two new components. The two new components and kigamicin have same UV absorption spectra. They might be the early intermediates or derivatives of kigamicin during its biosynthesis process. We obtained 12.6 mg 538 compound and 9.0 mg 513 compound by separating and purifying the fermentation products. Then, we get the molecular formula of the two compounds by employing high resolution mass spectrum (HRMS) and inferring the possible atomic composition depending on its molecular weight. We confirm that the molecular formula of 538 compound is C28H27NO10 and the molecular formula of 513 compound is C26H24O11. At last, we conduct nuclear magnetic resonance (NMR) to parse its chemical structure. We get the chemical structure of 538 compound. Thus, we could speculate the function of orf 60 in kigamicin biosynthesis.