| The effects of the pathway-specific regulatory genes, the two-component signal conduct genes as well as the genes related to the A-factor regulatory system in the genome DNA of Streptomyces coelicolor on the biosynthesis of undecylprodigiosin (Red) and actinorhodin (Act) were evaluated in this work. The regulation functions of above genes for the secondary metabolism involved in the antibiotics synthesis were analyzed. The temporal and spatial expressions were observed by the fusion between regulative genes and a fluorescent gene. In the mean time, a four-way ligation method was successfully introduced and the main factors affecting ligation were discussed. Also the factors affecting site-mutation method for gene-disruption with homogeneous double-exchange technique were examined and an effective screening method was established.It was reported that the pathway-specific regulatory genes in the genome DNA of Streptomyces coelicolor for the biosynthesis of the antibiotics Red and Act were actII-orf4 and redD, respectively. With an integrative type plasmid, pSET152, as a vector, the additional genes of actII-orf4 and redD were inserted into the genome DNA of S. coelicolor, respectively, and the mutants, lyqAG1521 and lyqRY1522, were obtained. From fermentation dynamics of the mutants, the capabilities for Act and Red biosynthesis with lyqAG1521 and lyqRY1522, respectively, increased about 40% comparing with the wild type strain M145.Two-component genes, ecrAl/ecrA2 and ecrE/ecrF, had been found important in the regulation of antibiotic production in S. coelicolor. By using homogenotisation by reciprocal double-crossover technique, the ecrAl/ecrA2 and ecrE/ecrF gene-disruptive mutants were constructed and four mutants were obtained, namelylyqA2001, lyqA2002, lyqE2003 and lyqF2004. According to the results from batch fermentation, the productivity of antibiotics Red was reduced dramatically, while slightly of antibiotic Act for all four mutants, which explained that both genes ecrAl/ecrA2 and ecrE/ecrF were of strong positive regulation functions for Red biosynthesis, but week for Act.The A-factor, SCB1, is a kind of Y -butyrolactones produced in S. coelicolor. The biosynthesis of SCB1 is related with two genes: scbA is the gene responsible for A-factor synthetase and scbR encodes for Y -butyrolactone binding protein. Again, by using homogenotisation by reciprocal double crossover technique, scbR and scbA gene-disruption mutants, lyqSR2005 (scbR) and lyqSA2006 (scbA), were obtained, respectively. The fermentation experiments showed that the productivities of antibiotics Red and Act with mutant lyqS A2006 were 8 times higher than that of mild strain Ml45, whereas, lower for mutant lyqSR2005. In the mutant lyqSA2006, the biosynthesis gene of A-factor was disrupted, resulted in inability for A-factor synthesis, the great increase in Red and Act productivity indicated that the A-factor was a strong inhibitor for antibiotics biosynthesis. The slight reduction in Act productivity with lyqSR2005 showed the Y -butyrolactone binding protein was a weak positive regulator. From above results, it seems that ScbR/ScbA genes play an important role in the self-regulation system in S. coelicolor for antibiotics synthesis, and they can be regarded as the on/off genes.Confocal laser scanning microscopy was applied to observe the temporal and spatial expressions during cell cultivation for the mutants with the fusion between regulative genes and a fluorescent protein gene.The pathway-specific genes for Red and Act biosynthesis, actll-orf4 and redD, were fused with fluorescent protein genes egfp and eyfp and the fusion genesactll-orf4:: egfp and redD:. eyfp were constructed respectively, and then inserted into the chromosome DNA of S. coelicolor by an integrative plasmid. The temporal expression mode of the fusion protein during submerged cultivation in SMM medium indicated that the expression of redD and actll-orf4 genes was started in the transition phase from exponential growth phase to stationary pha...
|
PDF Full Text Request