Draft genome of Streptomyces tsukubaensis NRRL 18488, the producer of the clinically important immunosuppressant tacrolimus (FK506).
Ontology highlight
ABSTRACT:
SUBMITTER: Barreiro C
PROVIDER: S-EPMC3393489 | BioStudies | 2012-01-01
REPOSITORIES: biostudies
Similar Datasets
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.
2012-01-01 | S-EPMC3551636 | BioStudies
Project description:Streptomyces tsukubaensis NRRL 18488 is the preferred strain for the production of immunosuppressant agent tacrolimus (FK506). To take full advantage of its genetic potential, systematic understanding of secondary metabolism and related regulatory mechanisms is highly demanded. Here, to this end, we complete its 7.9 Mbp linear genome sequence followed by integrating with multi-omics measurements. With accurate reannotation of FK506 gene cluster, total 2,389 transcription start sites were determined by using primary transcriptome analysis. Integrated analysis of transcriptome and translatome data revealed that secondary metabolic gene clusters, especially FK506 cluster, undergo translational control with decrease in translational efficiency according to the growth. Furthermore, we demonstrated that SD motif has little correlation with ribosome pausing but AT-rich codons delay the translational elongation. Strong ribosome pausing was observed in the rare TTA codon in FK506 cluster. This comprehensive genome-scale analysis provides insight to the translational regulation of secondary metabolism in S. tsukubaensis. Overall design: Transcription abundance and ribosomal profiling analysis of Streptomyces tsukubaensis according to the growth phase
2019-10-16 | GSE97637 | GEO
Project description:We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (?70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L(-1) to 5.5 mg L(-1)) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.
2013-01-01 | S-EPMC3708917 | BioStudies
Project description:FK506 (tacrolimus) is a secondary metabolite with a potent immunosuppressive activity, currently registered for use as immunosuppressant after organ transplantation. FK506 and FK520 are biogenetically related natural products that are synthesized by combined polyketide synthase/nonribosomal peptide synthetase systems. The entire gene cluster for biosynthesis of FK520 from Streptomyces hygroscopicus var. ascomyceticus has been cloned and sequenced. On the other hand, the FK506 gene cluster from Streptomyces sp. MA6548 (ATCC55098) was sequenced only partially, and it was reasonable to expect that additional genes would be required for the provision of substrate supply. Here we report the identification of a previously unknown region of the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 containing genes encoding the provision of unusual building blocks for FK506 biosynthesis as well as a regulatory gene. Among others, we identified a group of genes encoding biosynthesis of the extender unit that forms the allyl group at carbon 21 of FK506. Interestingly, we have identified a small independent diketide synthase system involved in the biosynthesis of the allyl group. Inactivation of one of these genes, encoding an unusual ketosynthase domain, resulted in an FK506 nonproducing strain, and the production was restored when a synthetic analog of the allylmalonyl-CoA extender unit was added to the cultivation medium. Based on our results, we propose a biosynthetic pathway for the provision of an unusual five-carbon extender unit, which is carried out by a novel diketide synthase complex.
2010-01-01 | S-EPMC2863216 | BioStudies
Project description: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.
2017-01-01 | S-EPMC5785230 | BioStudies
Project description:Chitin is the second most abundant biopolymer present in soils and is utilized by antibiotic-producing Streptomyces species. Its monomer, N-acetylglucosamine (NAG), regulates the developmental program of the model organism Streptomyces coelicolor. NAG blocks differentiation when growing on rich medium whilst it promotes development on poor culture media. We report here the negative effect of NAG on tacrolimus (FK506) production in Streptomyces tsukubaensis NRRL 18488 growing on a defined rich medium. Using microarrays technology, we found that GlcNAc represses the transcription of fkbN, encoding the main transcriptional activator of the tacrolimus biosynthetic cluster, and of ppt1, encoding a phosphopantheteinyltransferase involved in tacrolimus biosynthesis. On the contrary, NAG stimulated transcription of genes related to amino acid and nucleotide biosynthesis, DNA replication, RNA translation, glycolysis, pyruvate metabolism, and key gene members of the PHO regulon. The results obtained support those previously reported for S. coelicolor, but some important differences were observed Overall design: The transcriptomic experiment is composed of 16 samples from two time series named NAG (culture medium containing N-acetylglucosamine) and CTL (control condition: the culture medium without addition). Each time series was replicated once. Streptomyces tsukubaensis spores (10e9) were inoculated into 0.5-L flasks containing 100 mL of MGm-2.5 medium (PMID 22990582) and cultivated at 28 ºC, 220 rpm. Samples for RNA extraction were taken from flask cultures at 70 h, 70.5 h, 71 h and 72 h (4 timepoints × 4 cultures = 16 samples). The final concentration of N-acetylglcuosamine was 22.6 mmol/L (i. e., 0.5 % w/v).
2020-02-08 | GSE110393 | GEO
Project description: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 kilobases. A whole transcriptome differential shotgun sequencing of S. tsukubaensis was performed to analyze transcription at two 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 two consensus sequence motifs in the promoter regions. The transcription start sites of all gene operons within the FK506 cluster were identified, including three examples of leaderless RNA transcripts. These data provide detailed insight into the transcription of the FK506 biosynthetic gene cluster and supports future regulatory studies and genetic manipulations. Overall design: Differential whole transcriptome shotgun sequencing of Streptomyces tsukubaensis NRRL18488 was performed in duplicates at two time points of growth.
2017-07-05 | GSE92480 | GEO
Project description:BACKGROUND: FK506 is an important immunosuppressant, which can be produced by Streptomyces tsukubaensis. However, the production capacity of the strain is very low. Hereby, a computational guided engineering approach was proposed in order to improve the intracellular precursor and cofactor availability of FK506 in S. tsukubaensis. RESULTS: First, a genome-scale metabolic model of S. tsukubaensis was constructed based on its annotated genome and biochemical information. Subsequently, several potential genetic targets (knockout or overexpression) that guaranteed an improved yield of FK506 were identified by the recently developed methodology. To validate the model predictions, each target gene was manipulated in the parent strain D852, respectively. All the engineered strains showed a higher FK506 production, compared with D852. Furthermore, the combined effect of the genetic modifications was evaluated. Results showed that the strain HT-?GDH-DAZ with gdhA-deletion and dahp-, accA2-, zwf2-overexpression enhanced FK506 concentration up to 398.9 mg/L, compared with 143.5 mg/L of the parent strain D852. Finally, fed-batch fermentations of HT-?GDH-DAZ were carried out, which led to the FK506 production of 435.9 mg/L, 1.47-fold higher than the parent strain D852 (158.7 mg/L). CONCLUSIONS: Results confirmed that the promising targets led to an increase in FK506 titer. The present work is the first attempt to engineer the primary precursor pathways to improve FK506 production in S. tsukubaensis with genome-scale metabolic network guided metabolic engineering. The relationship between model prediction and experimental results demonstrates the rationality and validity of this approach for target identification. This strategy can also be applied to the improvement of other important secondary metabolites.
2013-01-01 | S-EPMC3680238 | BioStudies
Project description:Triacsins are a family of natural products having in common an N-hydroxytriazene moiety not found in any other known secondary metabolites. Though many studies have examined the biological activity of triacsins in lipid metabolism, their biosynthesis has remained unknown. Here we report the identification of the triacsin biosynthetic gene cluster in Streptomyces aureofaciens ATCC 31442. Bioinformatic analysis of the gene cluster led to the discovery of the tacrolimus producer Streptomyces tsukubaensis NRRL 18488 as a new triacsin producer. In addition to targeted gene disruption to identify necessary genes for triacsin production, stable isotope feeding was performed in vivo to advance the understanding of N-hydroxytriazene biosynthesis.
2019-01-01 | S-EPMC6590916 | BioStudies
Project description:Phosphopantetheinyl transferases (PPTases) play essential roles in both primary metabolisms and secondary metabolisms via post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). In this study, an industrial FK506 producing strain Streptomyces tsukubaensis L19, together with Streptomyces avermitilis, was identified to contain the highest number (five) of discrete PPTases known among any species thus far examined. Characterization of the five PPTases in S. tsukubaensis L19 unveiled that stw ACP, an ACP in a type II PKS, was phosphopantetheinylated by three PPTases FKPPT1, FKPPT3, and FKACPS; sts FAS ACP, the ACP in fatty acid synthase (FAS), was phosphopantetheinylated by three PPTases FKPPT2, FKPPT3, and FKACPS; TcsA-ACP, an ACP involved in FK506 biosynthesis, was phosphopantetheinylated by two PPTases FKPPT3 and FKACPS; FkbP-PCP, an PCP involved in FK506 biosynthesis, was phosphopantetheinylated by all of these five PPTases FKPPT1-4 and FKACPS. Our results here indicate that the functions of these PPTases complement each other for ACPs/PCPs substrates, suggesting a complicate phosphopantetheinylation network in S. tsukubaensis L19. Engineering of these PPTases in S. tsukubaensis L19 resulted in a mutant strain that can improve FK506 production.
2016-01-01 | S-EPMC4823652 | BioStudies