| Erythromycins (Er) were macrolide antibiotics, produced by Saccharopolyspora erythraea. In recent years, the third-generation erythromycin, which with greatly enhanced antibacterial activity and significant inhibition of activity of respiratory drug-resistant strains, had broad development prospect. To explore the reasons for the decline in production of new ketolide compound DOEB which was synthesized by directly transformation of Saccharopolyspora erythraea chromosome structure by way of genetic engineering, the rate-limiting factors of biosynthesis of DOEB in M mutant were studied from the level of gene transcription, translation and ester precursor compound, using Saccharopolyspora erythraea M mutant as the research object, the results were displayed as follows:In the study of mRNA stability, the total RNAs were extracted and purified firstly, then mRNA reverse transcription was carried out to obtain the cDNAs. By taking cDNAs as the templates, then the changes of amplified products were monitored instantaneously by using ABI real-time quantitive 7500 equipment, and the relative mRNA expression level was calculated (RQ= 2-△△Ct), PKS-TE gene expression of M mutant was 0.65 fold of PKS-TE gene expression in A226 strain, with an average decrease by 35%; PKS-ACP gene expression of M mutant was 0.69-fold of PKS-ACP gene expression in A226 strain, with an average decrease by 31%. The result showed that mRNA expression was reduced slightly when reconstituted Saccharopolyspora erythraea by genetic engineering, while its stability had not been significantly affected. Because the DOEB output of M mutant reduced about thousands of times, so the mRNA transcription was not the rate-limiting factor of DOEB biosynthesis. In the study of protein stability in the PKS, the TE domain of PKS complex was studied, the TE protein amino acid sequence was analyzed by bioinformatics tools before the antigen was synthesized chemically, then the rabbits were immuned by synthesized antigen, antiserum was isolated afer two months, and antibody titer was identified by ELISA method. Since then Saccharopolyspora erythraea A226 and M total proteins of different phases were extracted, and its concentration were determinated by use of Bradford method. The reaction between antibody and the same amount of protein in different periods were carried out by ELISA, and the result showed that the TE protein expression of M mutant was slightly reduced, while its stability had not been significantly affected according to A226 strain too. At the same time, PKS expression of protein complexes of different phases were detected by Coomassie brilliant blue-stained by silver staining method, the result showed that there were no obvious differences between M mutant and A226 strain.In the stabilities study of DOEB and its precursors, LC-MS method with high resolution was used to detect the expression level of DOEB and their precursors after the ethylacetate extracts of different phases in M mutant and A226 strain were prepared. The result showed that, in the synthesis of DOEB precursors, compared to A226 strain, the initial synthesis of module 5 product had reduced, and there was a increasing of module five product with the increasing of fermentation time, the product was cumulated; At the same time, due to the pool recognition of EryF for new compound, the transformation process from DODEB to DOEB had also been blocked by a certain degree of obstruction so that transformation rate of DODEB declined greatly and it was not completely transformed, and eventually made the DOEB precursor conversion capability reduced significantly. Therefore, the reduction of DOEB synthase activity was a key factor which caused the significantly decrease in the production of DOEB. (The DOEB amount declined 200 folds according to A226 strain), and the biosynthesis route from DOEB to eryA was obstructed powerfully.
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