Dataset Information

DRNA-seq transcriptional profiling of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis NRRL18488 and general analysis of the transcriptome.

ABSTRACT: FK506 (tacrolimus) is a valuable immunosuppressant produced by several Streptomyces strains. In the genome of the wild type producer Streptomyces tsukubaensis NRRL18488, FK506 biosynthesis is encoded by a gene cluster that spans 83.5 (kb). A whole transcriptome differential shotgun sequencing (dRNA-seq) of S. tsukubaensis was performed to analyze transcription at 2 different time points; before and during active FK506 production. In total, 8,914 transcription start sites were identified in either condition, which enabled precise determination of the 5'-UTR length of the corresponding transcripts as well as the identification of 2 consensus sequence motifs in the promoter regions. The transcription start sites of all gene operons within the FK506 cluster were identified, including 3 examples of leaderless RNA transcripts. These data provide detailed insight into the transcription of the FK506 biosynthetic gene cluster to support future regulatory studies, genetic manipulation, and industrial production.

SUBMITTER: Bauer JS

PROVIDER: S-EPMC5785230 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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dRNA-seq transcriptional profiling of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis NRRL18488 and general analysis of the transcriptome.

Bauer Judith S JS Fillinger Sven S Förstner Konrad K Herbig Alexander A Jones Adam C AC Flinspach Katrin K Sharma Cynthia C Gross Harald H Nieselt Kay K Apel Alexander K AK

RNA biology 20170731 11

FK506 (tacrolimus) is a valuable immunosuppressant produced by several Streptomyces strains. In the genome of the wild type producer Streptomyces tsukubaensis NRRL18488, FK506 biosynthesis is encoded by a gene cluster that spans 83.5 (kb). A whole transcriptome differential shotgun sequencing (dRNA-seq) of S. tsukubaensis was performed to analyze transcription at 2 different time points; before and during active FK506 production. In total, 8,914 transcription start sites were identified in eithe ...[more]

PMID: 28665778

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Project description:BACKGROUND: FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influence on the final yield of target product and can play an important role in development of industrial bioprocesses. RESULTS: Three putative regulatory elements, namely fkbR, belonging to the LysR-type family, fkbN, a large ATP-binding regulator of the LuxR family (LAL-type) and allN, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of fkbN caused a complete disruption of FK506 biosynthesis, while inactivation of fkbR resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the allN gene. Using RT-PCR and a reporter system based on a chalcone synthase rppA, we demonstrated, that in the wild type as well as in fkbN- and fkbR-inactivated strains, fkbR is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas fkbN expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of fkbN (or fkbR) does not abolish the transcription of the genes in the FK506 gene cluster in general, but may reduce expression of some of the tested biosynthetic genes. Finally, introduction of a second copy of the fkbR or fkbN genes under the control of the strong ermE* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively. CONCLUSIONS: Our results clearly demonstrate the positive regulatory role of fkbR and fkbN genes in FK506 biosynthesis in S. tsukubaensis NRRL 18488. We have shown that regulatory mechanisms can differ substantially from other, even apparently closely similar FK506-producing strains, reported in literature. Finally, we have demonstrated the potential of these genetically modified strains of S. tsukubaensis for improving the yield of fermentative processes for production of FK506.

Project description:BACKGROUND: Differential RNA sequencing (dRNA-seq) is a high-throughput screening technique designed to examine the architecture of bacterial operons in general and the precise position of transcription start sites (TSS) in particular. Hitherto, dRNA-seq data were analyzed by visualizing the sequencing reads mapped to the reference genome and manually annotating reliable positions. This is very labor intensive and, due to the subjectivity, biased. RESULTS: Here, we present TSSAR, a tool for automated de novo TSS annotation from dRNA-seq data that respects the statistics of dRNA-seq libraries. TSSAR uses the premise that the number of sequencing reads starting at a certain genomic position within a transcriptional active region follows a Poisson distribution with a parameter that depends on the local strength of expression. The differences of two dRNA-seq library counts thus follow a Skellam distribution. This provides a statistical basis to identify significantly enriched primary transcripts.We assessed the performance by analyzing a publicly available dRNA-seq data set using TSSAR and two simple approaches that utilize user-defined score cutoffs. We evaluated the power of reproducing the manual TSS annotation. Furthermore, the same data set was used to reproduce 74 experimentally validated TSS in H. pylori from reliable techniques such as RACE or primer extension. Both analyses showed that TSSAR outperforms the static cutoff-dependent approaches. CONCLUSIONS: Having an automated and efficient tool for analyzing dRNA-seq data facilitates the use of the dRNA-seq technique and promotes its application to more sophisticated analysis. For instance, monitoring the plasticity and dynamics of the transcriptomal architecture triggered by different stimuli and growth conditions becomes possible.The main asset of a novel tool for dRNA-seq analysis that reaches out to a broad user community is usability. As such, we provide TSSAR both as intuitive RESTful Web service ( http://rna.tbi.univie.ac.at/TSSAR) together with a set of post-processing and analysis tools, as well as a stand-alone version for use in high-throughput dRNA-seq data analysis pipelines.

Dataset Information

DRNA-seq transcriptional profiling of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis NRRL18488 and general analysis of the transcriptome.

Publications

dRNA-seq transcriptional profiling of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis NRRL18488 and general analysis of the transcriptome.

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