| A long term goal of our group is to introduce the very large and high G+C content polyketide synthase(PKS) genes for the biosynthesis of the heptaene macrolide antifungal antibiotic Candicidin from Streptomyces species FR-008 into plant to generate fungal resistance lines.An expression vector carrying a fragment encoding the amino-terminal part of an FR-008 Type I PKS module, containing a keto-synthase(KS) and part of an acetyl-transferase(AT) domain was constructed for trial expression of the extremely high G+C content(76 %) PKS gene in plant. This vector containing the promoter of the Cauliflower mosaic virus 35S RNA for the expression of the 2.7 kb PKS gene. Expression of this truncated gene in plant was expected to give information about expression in plant of high G+C content genes but no antifungal activity was expected in this stage.PKS expression plasmids containing strong, temperature-inducible, bacteriophage lambda promoters with or without the Escherichia coli phage T7 promoter were constructed and some of them lead to high expression of the expected c. 100 kDa PKS protein but some of them did not give reproducible high expression in E. coli. Truncating of the 2.7 kb PKS gene from these expression plasmids resulted in production of an expected 50 kDa protein, suggesting that these over expressed proteins were encoded by the cloned PKS gene. The over expressed PKS proteins in E. coli formed inclusion bodies.Then the FR-008 PKS produced in E. coli was used as antigen to raise polyclonal antibodies against the natural FR-008 PKS. Non-specific anti-E. coli antibodies were removed by precipitation with E. coli extract. Western blot experiments indicated that the partially purified antibody preparations reacted specifically with the PKS proteins expressed in E. coli and, as expected, also with the very large PKS proteins naturally present in S. sp. FR-008. This confirmed that the FR-008 PKS gene in the E. coli expression vectors was cloned in the correct reading frame. The above detection of a very large protein in S. sp. FR-008 indicated that the antiserum had the desired specificity for the FR-008 PKS. The detection limitation of PKS using antibodies was 1000 fold higher than Commassie blue staining, ready for detection of the probably very low level of PKS expression in plant.Two bifunctional Streptomyces-E. coli vectors were constructed that contained the phage lambda promoter(PR) upstream of the His6-tagged recombinant PKS gene. Using these vectors, expression of the PKS gene was achieved both in Streptomyces lividans and in E. coli in a heat-dependent manner, suggesting that the lambda promoter and temperature-sensitive lambda represser functioned in S. lividans as well as in E. coli. Also the CI represser was expressed, presumably from its own phage promoter and prevented transcription from PR at low temperature. Moreover, S. lividans strains expressing the c. 100 kDa PKS were different in sporulation and antibiotic production compared to strains without the 2.7 kb PKS gene, suggesting that the PKS protein was active in an unknown manner in Streptomyces.Integrated plasmids containing phage lambda promoter PR -directed and epitope-tagged 2.7 kb PKS gene were constructed for tagging the natural FR-008 PKS with specific immunodeterminant(epitope). These constructs were transferred into Streptomyces sp. DX600, arestriction deficient strain of the antibiotic producer S. sp. FR-008. Integration through homologous recombination between the 2.7 kb PKS gene and the natural PKS gene was confirmed by Southern hybridization. The resulting derivative strains produced antifungal activity in a manner different from the parental strain, indicating the original promoter was replaced with the engineered promoters and the epitopes.Base on the successful expression of PKS gene by CIts857 controlled lambda promoter, an E. coli expression vector pHZ330, an E. coli and Streptomyces bi-functional vector pHZ1060 were constructed containing the dts857-PR for inducible expression in both hosts. Both vector...
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