| Fostriecin and its analogs belong to valuable class of polyketides, which exhibit efficacious in vivo antitumor activity against a wide spectrum of tumor cells. Fostriecin was usually generated by chemical modification. But the procedure of chemosynthesis is tedious and leads to pollute environment. Therefore, exploration of the biosynthetic restructuring pathway engineering of fostriecin is a good choice for novel antibiotic production.In this thesis, the pSET152 vector was uesd to establish the transformation system through investigating genetic engineering operations in FST-producing strain including PEG-mediated protoplast transformation, electroporation and conjugation transfer. At the same time, it was found that the use of MS medium which contains certain concentration of Mg2+, Ca2+ and glycine greatly enhances the conjugation efficiency between FST-producing strain and E. coli and accelerate the growth of transformants.With the development of the combination biosynthesis of erythromycin, Tyr2699 was identified as a key amino acid in the catalytic site of KR6 domain in the polyketide synthase of erythromycin. Comparing the KR4 domains, amino acid sequences with several Glu-tathione reductases in different species, we found that they all have a high conserved NADPH binding region called fingerprint domains (GXGXXGXXXAXXXXXXG). Whether C-11 hydroxyl of Side chain of fostriecin would be reduced depended on the ketoreductase (KR4) domain of the fourth module of polyketide synthase. Therefore the KR4 domain can be completely or partly inactivated and turn C-11 hydroxyl into carbonyl by genetic engineering for the ketolides biosynthesis.Next the Streptomyces pulveraceus subsp. fostreus mutant in which KR4 domain of polyketide synthase lost its function were constructed by mutanting the first Gly condon of the fingerprint region and the same Tyr condon in the KR4 domain corresponding to Tyr2699 in the catalytic site of KR6 domain in the polyketide synthase which produced eyrthromycin. Using genomic DNA of Streptomyces pulveraceus subsp. fostreus as a template, full length of KR4 domain DNA fragment and homologous fragments on the either side of the mutant sites, about 3677bp, was amplified by PCR techniques and cloned into vector pUC19 to build the plasmid pUCYG. Partial-complementary primers used in the site-directed mutagenesis were designed to amplify the whole pUCYG, and then KR4 domain DNA fragment and homologous fragments with the condon TAC of Tyr mutanted to the condon ACC of Thr and the condon GGC of Gly mutanted to the condon GCG of Ala was cloned into homologous recombinant vector pOJ260 to build plasmid pOJTA. Plasmid pOJTA was introduced into S. fostreus through conjugal transfer system and integrated into the gene locus for fostriecin biosynthesis in the chromosome, and the integrants FZ were selected out. After growing the integrant FZ for at least five generations on ISP2 media without apramycin, one mutant, named as FM, which couldnt grow on the ISP2 media with apramycin, and grew without apramycin was picked out. PCR and DNA sequencing proved that the six nucleotides corresponding to YG amino acids in the chromosome of S. fostreus had been mutated. However,11-keto-fostriecin was not detected by HPLC analysis of the shake flask culture. On the other hand, production of fostriecin was dramatically reduced in the mutant. The reason why the 11-keto-fostriecin was not detected was deduced. One reason may be that the production level of the target product was too low to be detected; the second reason may be that FST synthesized by FM has long chain fatty acid and is not optimum substrate for TE, which makes catalytic efficiency of TE decrease; the third possible reason is that space structures of PKS might been changed even though the two amino acids in KR4 had been changed. The results in this study shed light on the enzymatic properties of the biosynthesis of Fostriecin, and also provided basis for the generation of fostriecin derivatives through combinational biosynthesis.
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