Study On Relationship Between Thioesterase Domain And Ketolide Biosynthesis

Since the formation of C-3 ketone group of macrolactone ring depended on the ketoreductase (KR) domain of the sixth module of polyketide synthase that catalyzed the synthesis of 6-deoxy-erythronolide B, we could make KR6 domain inactivated or deleted by genetic engineering for ketolide biosynthesis. Since 2002, several Saccharopolyspora erythraea KR6 mutants have been constructed through chromosomal homologous recombination technique, while 3-deoxy-3-oxo-6-deoxy-erythronolide B (DODEB) and its derivatives were synthesized at a poor production level in these mutants.In polyketide synthase, thioesterase (TE) domain was responsible for release and cyclization of polyketide chain, so it was important to polyketide biosynthesis. Because the precusor of DODEB, p-keto long-chain fatty acid, was not the natural substrate of erythromycin thioesterase (EryTE), it's possible that the efficency of EryTE catalyzing the precusor is very low. Therefore, in this study, to probe into the relationship between TE and ketolide biosynthesis, the gene of EryTE was amplified firstly, then cloned into vector pZM and pZMW to construct expression plasmids respectively. They were expressed in S. erythraea KR6 mutants. The products from the fermentation broth were extracted with ethyl acetate, however, the expected extract wasn't detected by thin layer chromatography and their activities of restraining the growth of Bacillus subtilis PUB 110 wasn't as well, so EryTE could not improve the production level of ketolide antibiotics markedly. The linker between...