| Streptomyces spp. are the most versatile and commercially important producers of antibiotics. Genes specifically involved in the production of a particular antibiotic are invariably found clustered together. By now, at least 50 antibiotic biosynthetic gene clusters have been cloned or identified, including those larger than 40kb. For gene manipulation such as heterologous expression of these huge gene clusters, new vectors and strategies are required. The chief aim of the present study is trying to extend the applications of YAC and SCP2*, which have tremendous cloning capacity, for the above mentioned purpose. Streptomyces sp. FR-008 PKS gene cluster(c. 150kb ) was chosen as the target for such an application.Prior to the vector construction, the availability of gene replacement was extensively tested in Streptomyces. sp. FR-008. Most of genes responsible for the biosynthesis of antibiotic FR-008 had been cloned ( Hu et al , 1995 ), but that for the sugar moiety and potential regulator(s) are still unknown. Loss of the 18kb fragment containing pabAB gene and its upstream region abolished or changed the antibiotic FR-008 productivity. To explore the potential regulatory elements or genes for the sugar moiety biosyntheis in this region, a series of gene replacement constructs had been obtained for the replacements of the 5.2kb + 7.2kb + 4.5kb , 5.2kb + 7.2kb , 5.2kb and 7.2kb BamHI or BamHI+ Bglll fragments in this region . All of the six final constructs had been introduced into Streptomyces. sp. FR-008 and corresponding derivatives with gene interuptions , namely BL10 ( FR-008:: PHZ670 ), BL19 ( FR-008:: pHZ679 ), BL14 (FR-008:: pHZ675 ), BL15 ( FR-008:: pHZ674), BL16 ( FR-008:: pHZ676 ) and BL17 ( FR-008:: pHZ677 ), had been obtained through a modified procedure for bi-parental conjugation from E. coli to Streptomyces sp. FR-008 as they carry oriT fragment. Furthermore, two kinds of derivatives losing 5. 2kb + 7.2kb and 7.2kb fragments respectively were available. In the bioassay using Saccharomyces cerevisiae as indicator, BL10 and BL19 were found unable to produce antibiotic FR-008 while the other four had no detectable changes in productivity. Obviously, the gene replacements happened in the 5.2kb + 7. 2kb or 7. 2kb regions abolished the productivity of antibiotic FR-008. The expected strain after genereplacement in the region of 5.2kb have not been obtained for unknown reason.Streptomyces low copy number plasmid SCP2* is also employed for the cloning and recovery of large DNA fragment from Streptomyces. As SCP2* can accommodate large DNA insertions and is efficiently transmissible among its permissive hosts such as S. lividansbut seemed to be unable to replicates in Streptomyces sp. FR-008, it might be a suitable vector for the cloning of the entire FR-008 PKS gene cluster through gene replacement followed by conjugation with 5. lividans. Using pIJ903 bearing tsr gene as a vector, the two furthermost fragments of the cluster, 4. Okb and 1. 5kb in size respectively, were inserted into it. To facilitate the detection of gene replacement, apr gene was placed in the middle of the two inserts. OriT from E. coli plasmid RK2 was also incoporated into the vector, therefore, pIJ903 derivatives can be mobilized from E. coli into Streptomyces sp. FR-008 by either bi-parental conjugation or by conjugation between Streptomyces.The application of SCP2* derivied vectors requires that the physical map of a specific gene cluster is known and SCP2* derivatives are suicidal in the host earring the gene cluster. To clone large and random DNA fragments, the second generation of YAC, pJS97-pJS98 was modified as gene replacement vector in Streptomyces. pHZ621-pHZ622, the new gene replacement vector system, had been constructed through the insertion of the 1.0kb and 1.4kb flanking sequences of HAU3R gene from wild-type S. lividans in the same natural orientation. For desired gene replacement, hyg/cml and str/spc gene cassettes were also incorporated into the new vector system. It is expected that hygwould be integ...
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