Project description:Global transcriptional profile of Streptomyces avermitilis wild-type strain ATCC31267 and avermectin overproducing strain 76-02-e at different time points
Project description:Streptomyces avermitilis is a avermectin producer.Since the avermectin biosynthesis rate has a increased significantly in P3 fermentation stage( P1,24–96 h; P2:96–192 h, P3:192–240 h), but the sugar absorption rate decreased significantly in P3 fermentation stage, in order to improve the titer of avermectins, we conducted transcriptomic analysis of Streptomyces avermitilis S0 in fourth and eighth day, and selected native promoters with appropriate temple using to express sugar transporters.
Project description:The gram-positive bacterium, Streptomyces avermitilis holds industrial importance, which produces widely used anthelmintic agent, avermectin. Furthermore, S. avermitilis is generally considered as a prominent heterologous gene expression host for diverse secondary metabolites biosynthesis. However, despite of its industrial importance, it largely remains unknown how its genome is organized and regulated for timely gene expression. Here, we determined 1,601 transcription units (TU) encoded in its genome using the integrated analysis of high-throughput sequencing data including dRNA-Seq, Term-Seq, RNA-Seq, and Ribo-Seq. In addition to TU cataloguing, these information-rich results also revealed the presence of diverse regulatory elements for the transcriptional and translational control of individual TU, such as promoters, 5¢-UTRs, terminators, 3¢-UTRs, and riboswitches. The conserved promoter sequences for transcription initiation were identified from 2,361 transcription start sites as 5¢-TANNNT and 5¢-TGAC for -10 and -35 elements, respectively. Interestingly, the -35 element and spacer length between them were critical for transcriptional regulation of functionally distinct genes. Total 2,017 transcription termination sites were detected from Term-Seq analysis, revealing that stem structure formation is a prerequisite for transcription termination and that Rho-independent termination prevails in S. avermitilis. Lastly, the TU architecture suggests the presence of novel small RNAs and cis-regulatory elements in the genome. Our findings will serve as invaluable resources for comprehensive understanding on regulatory features of S. avermitilis. Moreover, it is anticipated the elevation of its potential as the heterologous expression host for diverse secondary metabolite biosynthesis.
Project description:Gram-positive bacteria of the genus Streptomyces are industrially important microorganisms, producing >70% of commercially important antibiotics. The production of these compounds is often regulated by low-molecular-weight bacterial hormones called autoregulators. Although 60% of Streptomyces strains may use ?-butyrolactone-type molecules as autoregulators and some use furan-type molecules, little is known about the signaling molecules used to regulate antibiotic production in many other members of this genus. Here, we purified a signaling molecule (avenolide) from Streptomyces avermitilis--the producer of the important anthelmintic agent avermectin with annual world sales of $850 million--and determined its structure, including stereochemistry, by spectroscopic analysis and chemical synthesis as (4S,10R)-10-hydroxy-10-methyl-9-oxo-dodec-2-en-1,4-olide, a class of Streptomyces autoregulator. Avenolide is essential for eliciting avermectin production and is effective at nanomolar concentrations with a minimum effective concentration of 4 nM. The aco gene of S. avermitilis, which encodes an acyl-CoA oxidase, is required for avenolide biosynthesis, and homologs are also present in Streptomyces fradiae, Streptomyces ghanaensis, and Streptomyces griseoauranticus, suggesting that butenolide-type autoregulators may represent a widespread and another class of Streptomyces autoregulator involved in regulating antibiotic production.
Project description:The present work aimed at selecting appropriate native temporal promoters with the identical profile of the cumate-inducible promoter under the optimal induction condition. This strategy enabled us to optimized the expression of biosynthetic gene cluster of desired secondary metabolites in Streptomyces, while avoid the use of inducer during fermentation. By clustering the genes with quite similar transcriptional profile with that of gfp controlled by the cumate-inducible promoter based on time-series microarray data, 50 qualified genes were identified, which were controlled by 24 putative promoters. Then, the cumate-inducible promoter were replaced by the 24 native temporal promoters, and fermentation results showed the good performance of these promoters compared to that of the cumate-inducible promoter. Therefore, our strategy could be used to fine-tune the expression of target BGCs for production improvement.
Project description:DNA/DNA microarray hybridization was used to compare the genome content of Streptomyces avermitilis, Streptomyces cattleya, Streptomyces maritimus and Kitasatospora aureofaciens with that of Streptomyces coelicolor A3(2). The array data showed an about 93% agreement with the genome sequence data available for S. avermitilis and also showed a number of trends in the genome structure for Streptomyces and closely related Kitasatospora. A core central region was well conserved, which might be predicted from previous research and this was linked to a low degree of gene conservation in the terminal regions of the linear chromosome across all four species. Between these regions there are two areas of intermediate gene conservation by microarray analysis where gene synteny is still detectable in S. avermitilis. Nonetheless, a range of conserved genes could be identified within the terminal regions. Variation in the genes involved in differentiation, transcription, DNA replication, etc. provides interesting insights into which genes in these categories are generally conserved and which are not. The results also provide target priorities for possible gene knockouts in a group of bacteria with a very large numbers of genes with unknown functions compared to most bacterial species.