Mutational Analysis And Disruption Of The Regions Of The Gene Cluster Governing The Oxazolomycin Biosynthesis

Polyketide and nonribosomal peptide natural products constitute a vast family of compounds mainly synthesized by Streptomyces. They possesed a large range of biological activities. Some of these natural products have proved to be of great therapeutic value. More and more researchers focus on the combinational biosythesis of gene clusters of natural products. Cloning of the gene cluster responsible for natural products, deletion of domains, addition of domains or replacement of domains are essential methods to develop combational biosynthesis. Synthesis of polyketide is just like the way of fatty acid with all genes in clusters of colineary way.Oxazolomycin (OZM), produced by Streptomyces albus JA3453, is a hybrid peptide-polyketide , antibiotic structurally characterized by a unique spiro-linkedβ-lactone/γ-lactam moiety, an oxazole ring, an (E, E)-diene, and a (Z, Z, E)-triene chain. OZM exhibits potent antitumor, antiviral, but limited antibacterial activities. The biosynthesis of OZM has been studied by feeding experiments with isotope-labeled precursors. The carbon backbone of OZM is derived from three molecules of glycine and nine molecules of acetate, with all of the five C-methyl, one O-methyl, and one N-methyl groups originated from methionine, but the origin of the C3-C4 unit and the C16 and C13’carbons of OZM remains unknown.Nontheless, these studies unambiguously established the hybrid nonribosomal peptide-polyketide biosynthetic origin for OZM and implied several features, uncommon or unprecedented to polyketide, nonribosomal peptide, or hybrid nonribosomal peptide-polyketide biosynthesis. Based on the gene cluster of oxazolomycin cloned by Chunhua Zhao in our lab, we analyzed the gene sequence by bioinformation method. The result reveals that there are some differences between PKS of OZM and PKS of traditional polyketide natural products. First, there are only two tandem AT domain on OZM gene cluster while the traditional PKS each set module have one. Second, there are tandem KS domains in module10 of ozmH. While,the traditional way for polyketide synthesis is that each module encodes a functional synthase unit and each synthase unit participates specifically in one of the six FAS-like elongation steps required for formation of the polyketide. In addition, genetic organization and biochemical order of events appear to be colinear. The biosynthetic assembly of polyketide involved acyl carrier protein(ACP), ketosynthase(KS), acyltransferase(AT) . Dehydratase(DH), enoyl reductase(ER) ketoreductase(KR) may also be presented in a specific module and are responsible for modification of the nascentβ-keto group. The colinearity between the modularity of enzyme activities and the structure of the products are corresponding very well. However, we found a special tandem KS domains in the module 10 of ozmH on OZM gene cluster. We tried to do some work to find what role each of them played in oxazolomycin biosynthesis. First, site-directed mutations were carried on respectively to abolish their function by muatation of their active sites. Second, fermentations were carried out for further testing the products of the mutant strains. HPLC analysis of the fermentations suggests that the first KS domain has no effect on the oxazolomycin biosyntheisis. The second KS domain is necessary for oxazomycin biosyntheis.At the same time, we did research on the regulatory genes ozmR and ozmU. Disruptions of ozmR and ozmU were carried out by PCR-targeting method. HPLC analysis of the mutant strains revealed that ozmR is a positive regulatory gene. And ozmU gene has no obivious effect on OZM biosyntheis. These results set the stage for further study on combinational biosyntheis of oxazolomycin.