Mining Of Polyether-specific Gene Resources With Degenerate Pcr Primers

1. Mining of polyether-specific epoxidase genes with Degenerate PCR primers. Polyether antibiotics are an important kind of polyketide which are widely used in production. Polyether-specfic epoxidase is a key enzyme in the biosynthesis process of the polyether antibiotics. For quickly identifing the polyether biosynthesis gene cluster, pairs of degenerate primers were designed based on the known epoxidase gene sequences to locate the epoxidase genes. Based on different primer design methods Consensus-Degenerate Hybrid Oligonucleotide (CODEHOP) and PaBaLiS (after the authors'name) two types degenerate primers were designed. All the known epoxidase genes and several novel epoxidase-like genes were identified when we used the degenerate primers to screen the polyether producer strains. The novel epoxidase protein sequences show high similarities to the known epoxidase sequences. The PaBaLiS primers work slightly better than the CODEHOP primers. At the same time, we also found that the ratio of forward primer to the reverse primer is also quite important to the effective amplification of the target products. This method has been proven to be very effective as it allow us to identify some fragment encoding epoxidase-like genes in the polyether producer strains.2. Cloning of the salinomycin biosynthetic gene cluster. Salinomycin is a widerly used polyether antibiotics. In order to understanding the mechanism of the biosynthesis process of polyether antibiotics, we designed several primers based on the published sequence to clone the whole gene cluster, and then we got a cosmid of 38kb long with the published sequence among it. But when we were analyzing the sequences of this 38kb cosmid may belong to, we can not find a reasonable relationship between the salinomycin structure and the arrangement of the module in the 38kb cosmid. So we used the method we developed above to screen the gene cluster alternatively, and we got another gene cluster which may responsible for the biosynthesis of salinomycin, and we also confirm that this gene cluster is different from the one published before.3. Cloning of the lasalocid biosynthetic gene cluster. Lasalocid is a polyether antibiotics, its structure is quite different from the other polyether antibiotics which biosynthetic gene cluster have been cloned. The lasalocid biosynthetic gene cluster was cloned successfully by the method developed in our lab and was confirmed by the disruption experiment. This method is based on the conservative distance between the AT domain and KR domain. It is useful for the understanding of the biosynethic mechanism of polyether antibiotics by the cloning of the lasalocid biosynethic gene cluster.