Study On The Kigamicin Biosynthesis

Background:kigamicins are 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, kigamicins will serve as the drug candidates for developing the specific therapy of treating cancers. Purpose:in light of the practical drugs for the clinical examination as well as production and application, characterized by the small molecules, it has been aimed to explore the pharmaceutical cores for the specific function of anti-cancer. Methods:this paper has aimed at the successful cloning of kigamicins gene cluster to come true that its biosynthetic pathway can be interpreted thoroughly, with the focus that the chemical structures of novel components in the biosynthesis have been illuminated. Significance:this research will make basis on the following speculation and verification of the relationship between structures and functions for kigamicins, which will lay the foundation for developing small-molecule drugs of specific anti-cancer activity in the near future.In order to identify the kigamicin biosynthetic gene cluster, a genomic library for the strain ML630was constructed and subsequently screened by PCR via using the specific primer designed on the basis of genome sequencing on the strain. As a result, three cosmids have been selected from the library, named respectively Fos2, Fos4and Fos7, and furthermore selected for DNA sequencing, giving rise to the putative kigamicin gene cluster of93kb. Sequence analysis of the overall93kb cosmids insert with the Frameplot program identified74open reading frames(ORFs), such as the minimal PKS consisting of orf25,、orf37and、orf38, as well as the post-PKS tailoring enzymes containing orf51and orf56which are responsible for the formation o(?),enous heterocyclic ring. By comparison, only Fos4consists of both genes mentioned above, which, therefore, is most likely used to heterologously express the products of kigamicin or its early intermediates.To verify the kigamicin biosynthetic gene cluster, integrative plasmid pLL59 derived from Fos4were expressed in Streptomyces lividans K4and subsequently its products containing kigamicin derivatives were identified. By protoplast transformation, pLL59was integrated into the genome of K4with the transformation of pLL68subsequently. With the simultaneous fermentation of mutant LQ5968and wild-type ML630, both fermentation products summited to LC-MS and it showed that both strains produced two compounds with the same UV absorption spectrum followed by m/z538and552respectively, of which the component signaled at m/z552has accorded with the aglycon of kigamicin reported in the literature, and the other compound with m/z538might be the early intermediates or derivatives of kigamicin.Following the fermentation of LQ5968with solid culture plate of R5, kigamicin derivatives were isolated and purified through ethyl acetate extraction, reversed phase chromatography on silica gel. With LC-MS analysis for the mixture,6compounds were identified and named LQ-1, LQ1-1, LQ-2, LQ-3, LQ-4and LQ4-1respectively according to their retention time, all of which are characterized by the same UV absorption spectrum and m/z are signaled accordingly at497,512,552,538,494and538. Followed by the purification of HPLC,6compounds were obtained, weighting31.4mg,9.8mg,4.0mg,13.8mg and6.7mg respectively. The second purification with HPLC was imposed on LQ-1and LQ-4, giving the final weight of11mg and13mg respectively.The expression plasmid pLQ10was constructed after the cloning and sequencing for the minimal PKS genes containing orf25, orf37, orf38and orf39were transformed into Streptomyces coceilor, resulting the three mutant strains accordingly. Through the LC-MS analysis for the fermented products, it showed that no putative compounds were identified. In addition, pLQ22containing the negative regulator gene orf29was constructed and transformed into mutant LQ59. Through the LC-MS analysis for its fermented products, it showed that the products profile in the new mutant was not changed but the yield diminished. In addition, pLQ7containing hydroxylase gene orf67was constructed and transformed into LQ59, resulting that there was no change in the products profile.Concludsion:The genomic library for ML630was constructed and furthermore screened by PCR resulting Fos4which contained most of kigamicin gene cluster and was verified through heterologous expression of kigamicin derivatives successfully. Subsequently, kigamicin biosynthetic pathway has been proposed through the bioinformatics analysis of kigamicin gene cluster and corresponding experimental results in this paper, whereas the details about the pathway demands further study.