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Juan F. Martín - One of the best experts on this subject based on the ideXlab platform.

  • Genome-wide transcriptome response of Streptomyces Tsukubaensis to N-acetylglucosamine: effect on tacrolimus biosynthesis.
    Microbiological research, 2018
    Co-Authors: María Ordóñez-robles, Antonio Rodríguez-garcía, Juan F. Martín
    Abstract:

    Chitin is the second most abundant carbohydrate biopolymer present in soils and is utilized by antibiotic-producing Streptomyces species. Its monomer, N-acetylglucosamine (GlcNAc), regulates the developmental program of the model organism Streptomyces coelicolor. GlcNAc blocks differentiation when growing on rich medium whilst it promotes development on poor culture media. However, it is unclear if the same GlcNAc regulatory profile observed in S. coelicolor applies also to other industrially important Streptomyces species. We report here the negative effect of GlcNAc on differentiation and tacrolimus (FK506) production by Streptomyces Tsukubaensis NRRL 18488. 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, GlcNAc stimulated transcription of genes related to amino acid and nucleotide biosynthesis, DNA replication, RNA translation, glycolysis and pyruvate metabolism. The results obtained support those previously reported for S. coelicolor, but some important differences were observed; for example genes involved in GlcNAc transport and metabolism and genes encoding transcriptional regulators such as crr, ptsI, nagE1, nagE2, nagB, chiA, chiJ, ngcE, dasR or atrA are not significantly induced in S. Tsukubaensis by GlcNAc addition. Differences in the GlcNAc transport systems, in the physiology of S. Tsukubaensis and S. coelicolor and/or the different composition of the culture media used are likely to be responsible for the discrepancies observed between these species.

  • Analysis and validation of the pho regulon in the tacrolimus-producer strain Streptomyces Tsukubaensis: differences with the model organism Streptomyces coelicolor.
    Applied microbiology and biotechnology, 2018
    Co-Authors: Miriam Martínez-castro, Carlos Barreiro, Juan F. Martín
    Abstract:

    Inorganic and organic phosphate controls both primary and secondary metabolism in Streptomyces genus. Metabolism regulation by phosphate in Streptomyces species is mediated by the PhoR-PhoP two-component system. Response regulator PhoP binds to conserved sequences of 11 nucleotides called direct repeat units (DRus), whose organization and conservation determine the binding of PhoP to distinct promoters. Streptomyces Tsukubaensis is the industrial producer of the clinical immunosuppressant tacrolimus (FK506). A bioinformatic genome analysis detected several genes with conserved PHO boxes involved in phosphate scavenging and transport, nitrogen regulation, and secondary metabolite production. In this article, the PhoP regulation has been confirmed by electrophoretic mobility shift assays (EMSA) of the most relevant members of the traditional pho regulon such as the two-component system PhoR-P or genes involved in high-affinity phosphate transport (pstSCAB) and low-affinity phosphate transport (pit). However, the PhoP control over phosphatase genes in S. Tsukubaensis is significantly different from the pattern reported in the model bacteria Streptomyces coelicolor. Thus, neither the alkaline phosphatase PhoA nor PhoD is regulated by PhoP. On the contrary, the binding of PhoP to the promoter of a novel putative phosphatase PhoX was confirmed. A crosstalk of the PhoP and GlnR regulators, which balances phosphate and nitrogen utilization, also occurs in S. Tsukubaensis but slightly modified. Finally, PhoP regulates genes, like afsS, that link phosphate control and secondary metabolite production in S. Tsukubaensis. In summary, there are notable differences between the regulation of specific genes of the pho regulon in S. Tsukubaensis and the model organism S. coelicolor.

  • Unraveling Nutritional Regulation of Tacrolimus Biosynthesis in Streptomyces Tsukubaensis through omic Approaches
    Antibiotics (Basel Switzerland), 2018
    Co-Authors: María Ordóñez-robles, Fernando Santos-beneit, Juan F. Martín
    Abstract:

    Streptomyces Tsukubaensis stands out among actinomycetes by its ability to produce the immunosuppressant tacrolimus. Discovered about 30 years ago, this macrolide is widely used as immunosuppressant in current clinics. Other potential applications for the treatment of cancer and as neuroprotective agent have been proposed in the last years. In this review we introduce the discovery of S. Tsukubaensis and tacrolimus, its biosynthetic pathway and gene cluster (fkb) regulation. We have focused this work on the omic studies performed in this species in order to understand tacrolimus production. Transcriptomics, proteomics and metabolomics have improved our knowledge about the fkb transcriptional regulation and have given important clues about nutritional regulation of tacrolimus production that can be applied to improve production yields. Finally, we address some points of S. Tsukubaensis biology that deserve more attention.

  • Streptomyces Tsukubaensis as a new model for carbon repression: transcriptomic response to tacrolimus repressing carbon sources
    Applied Microbiology and Biotechnology, 2017
    Co-Authors: María Ordóñez-robles, Fernando Santos-beneit, Juan F. Martín, Silvia M. Albillos, Paloma Liras, Antonio Rodríguez-garcía
    Abstract:

    In this work, we identified glucose and glycerol as tacrolimus repressing carbon sources in the important species Streptomyces Tsukubaensis . A genome-wide analysis of the transcriptomic response to glucose and glycerol additions was performed using microarray technology. The transcriptional time series obtained allowed us to compare the transcriptomic profiling of S. Tsukubaensis growing under tacrolimus producing and non-producing conditions. The analysis revealed important and different metabolic changes after the additions and a lack of transcriptional activation of the fkb cluster. In addition, we detected important differences in the transcriptional response to glucose between S. Tsukubaensis and the model species Streptomyces coelicolor . A number of genes encoding key players of morphological and biochemical differentiation were strongly and permanently downregulated by the carbon sources. Finally, we identified several genes showing transcriptional profiles highly correlated to that of the tacrolimus biosynthetic pathway regulator FkbN that might be potential candidates for the improvement of tacrolimus production.

  • Analysis of the Pho regulon in Streptomyces Tsukubaensis
    Microbiological research, 2017
    Co-Authors: María Ordóñez-robles, Fernando Santos-beneit, Antonio Rodríguez-garcía, Juan F. Martín
    Abstract:

    Phosphate regulation of antibiotic biosynthesis in Streptomyces has been studied due to the importance of this genus as a source of secondary metabolites with biological activity. Streptomyces Tsukubaensis is the main producer of tacrolimus (or FK506), an immunosuppressant macrolide that generates important benefits for the pharmaceutical market. However, the production of tacrolimus is under a negative control by phosphate and, therefore, is important to know the molecular mechanism of this regulation. Despite its important role, there are no reports about the Pho regulon in S. Tsukubaensis. In this work we combined transcriptional studies on the response to phosphate starvation with the search for PHO boxes in the whole genome sequence of S. Tsukubaensis. As a result, we identified a set of genes responding to phosphate starvation and containing PHO boxes that include common Pho regulon members but also new species-specific candidates. In addition, we demonstrate for the first time the functional activity of PhoP from S. Tsukubaensis through complementation studies in a Streptomyces coelicolor ΔphoP strain. For this purpose, we developed an anhydrotetracycline inducible system that can be applied to the controlled expression of target genes.

Carlos Barreiro - One of the best experts on this subject based on the ideXlab platform.

  • Regulation of the phosphate metabolism in Streptomyces genus: impact on the secondary metabolites
    Applied Microbiology and Biotechnology, 2019
    Co-Authors: Carlos Barreiro, Miriam Martínez-castro
    Abstract:

    The analysis of the inorganic phosphate effect over the antibiotics production is a long-distance history in the Streptomyces genus, which began almost at the same time that Michael Ende published his book entitled The Neverending Story. In some way, the unveiling of the pho regulon and its influence over the secondary metabolites production is an unfinished story, which keeps this subject as a trending topic, nowadays. Up to date, different studies have been releasing knowledge about particular areas of the pho regulon of different Streptomyces species. Nevertheless, for the first time, these knowledge drops are grouped in a review presenting a broad overview of the phosphate regulation and its impact over the secondary metabolites production in industrially relevant species. Even though the genetic response against phosphate scarcity is similar, as a whole, in different Streptomyces species, the fine-tuning is species-specific. Thus, the response regulator PhoP directly controls the secondary metabolites production in some species, whereas it regulates them in an indirect manner in other species. This information, unraveled in this review, is the result of the intensive analysis along last decade in several species of the genus that is allowing to distinguish how the phosphate response is unleashed in Streptomyces coelicolor , Streptomyces lividans , Streptomyces natalensis , Streptomyces lydicus , Streptomyces avermitilis , and Streptomyces Tsukubaensis .

  • Analysis and validation of the pho regulon in the tacrolimus-producer strain Streptomyces Tsukubaensis: differences with the model organism Streptomyces coelicolor.
    Applied microbiology and biotechnology, 2018
    Co-Authors: Miriam Martínez-castro, Carlos Barreiro, Juan F. Martín
    Abstract:

    Inorganic and organic phosphate controls both primary and secondary metabolism in Streptomyces genus. Metabolism regulation by phosphate in Streptomyces species is mediated by the PhoR-PhoP two-component system. Response regulator PhoP binds to conserved sequences of 11 nucleotides called direct repeat units (DRus), whose organization and conservation determine the binding of PhoP to distinct promoters. Streptomyces Tsukubaensis is the industrial producer of the clinical immunosuppressant tacrolimus (FK506). A bioinformatic genome analysis detected several genes with conserved PHO boxes involved in phosphate scavenging and transport, nitrogen regulation, and secondary metabolite production. In this article, the PhoP regulation has been confirmed by electrophoretic mobility shift assays (EMSA) of the most relevant members of the traditional pho regulon such as the two-component system PhoR-P or genes involved in high-affinity phosphate transport (pstSCAB) and low-affinity phosphate transport (pit). However, the PhoP control over phosphatase genes in S. Tsukubaensis is significantly different from the pattern reported in the model bacteria Streptomyces coelicolor. Thus, neither the alkaline phosphatase PhoA nor PhoD is regulated by PhoP. On the contrary, the binding of PhoP to the promoter of a novel putative phosphatase PhoX was confirmed. A crosstalk of the PhoP and GlnR regulators, which balances phosphate and nitrogen utilization, also occurs in S. Tsukubaensis but slightly modified. Finally, PhoP regulates genes, like afsS, that link phosphate control and secondary metabolite production in S. Tsukubaensis. In summary, there are notable differences between the regulation of specific genes of the pho regulon in S. Tsukubaensis and the model organism S. coelicolor.

  • The gamma-butyrolactone receptors BulR1 and BulR2 of Streptomyces Tsukubaensis: tacrolimus (FK506) and butyrolactone synthetases production control.
    Applied microbiology and biotechnology, 2014
    Co-Authors: Zahra Salehi-najafabadi, Carlos Barreiro, Antonio Rodríguez-garcía, Anthony Cruz, Gustavo E. Lopez, Juan F. Martín
    Abstract:

    Streptomyces Tsukubaensis is a well-established industrial tacrolimus producer strain, but its molecular genetics is very poorly known. This information shortage prevents the development of tailored mutants in the regulatory pathways. A region (named bul) contains several genes involved in the synthesis and control of the gamma-butyrolactone autoregulator molecules. This region contains ten genes (bulA, bulZ, bulY, bulR2, bulS2, bulR1, bulW, bluB, bulS1, bulC) including two γ-butyrolactone receptor homologues (bulR1, bulR2), two putative gamma-butyrolactone synthetase homologues (bulS1, bulS2) and two SARP regulatory genes (bulY, bulZ). Analysis of the autoregulatory element (ARE)-like sequences by electrophoretic mobility shift assays and footprinting using the purified BulR1 and BulR2 recombinant proteins revealed six ARE regulatory sequences distributed along the bul cluster. These sequences showed specific binding of both BulR1 (the gamma-butyrolactone receptor) and BulR2, a possible pseudo γ-butyrolactone receptor. The protected region in all cases covered a 28-nt sequence with a palindromic structure. Optimal docking area analysis of BulR1 proved that this protein can be presented as either monomer or dimer but not oligomers and that it binds to the conserved ARE sequence in both strands. The effect on tacrolimus production was analysed by deletion of the bulR1 gene, which resulted in a strong decrease of tacrolimus production. Meanwhile, the ΔbulR2 mutation did not affect the biosynthesis of this immunosuppressant.

  • FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces Tsukubaensis
    BMC microbiology, 2012
    Co-Authors: Dušan Goranovič, Carlos Barreiro, Miriam Martínez-castro, Javier Santos-aberturas, Marko Blažič, Enej Kuščer, Jaka Horvat, Vasilka Magdevska, Tomaž Polak, Peter Mrak
    Abstract:

    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.

  • Taxonomy and chemically semi-defined media for the analysis of the tacrolimus producer ‘Streptomyces Tsukubaensis’
    Applied microbiology and biotechnology, 2012
    Co-Authors: Miriam Martínez-castro, Juan F. Martín, Zahra Salehi-najafabadi, Francisco Romero, Rodrigo Pérez-sanchiz, Rosa Isabel Fernández-chimeno, Carlos Barreiro
    Abstract:

    ‘Streptomyces Tsukubaensis’ was the first tacrolimus producer strain identified. Although it has been included in the Streptomyces genus, its taxonomic position has not been rigorously determined. By using a polyphasic approach, we have established that the tacrolimus producer strain ‘S. Tsukubaensis’ NRRL 18488 represents a unique species in the Streptomyces genus, which is phylogenetically distant from other subsequently described producers. This fact means a horizontal transference of the tacrolimus-producing gene cluster. Physiology, nutrient requirement, and molecular genetics analyses of tacrolimus biosynthesis in ‘S. Tsukubaensis’ necessitate chemically defined or semi-defined media, which work as a jigsaw puzzle and allow for pieces (nutrients) exchange. To date, studies related to ‘S. Tsukubaensis’ have been mainly focused in the improvement of tacrolimus production using complex industrial fermentation media, which difficulty allows testing of tacrolimus overproduction enhancers or inhibitors because of the presence of non‐defined substances. In the present work, two semi-defined media were developed in order to study the main factors involved in tacrolimus production in ‘S. Tsukubaensis’.

Jianping Wen - One of the best experts on this subject based on the ideXlab platform.

  • Combining metabolomics and network analysis to improve tacrolimus production in Streptomyces Tsukubaensis using different exogenous feedings.
    Journal of industrial microbiology & biotechnology, 2017
    Co-Authors: Cheng Wang, Jiao Liu, Huanhuan Liu, Shaoxiong Liang, Jianping Wen
    Abstract:

    Tacrolimus is widely used as an immunosuppressant in the treatment of various autoimmune diseases. However, the low fermentation yield of tacrolimus has thus far restricted its industrial applications. To solve this problem, the time-series response mechanisms of the intracellular metabolism that were highly correlated with tacrolimus biosynthesis were investigated using different exogenous feeding strategies in S. Tsukubaensis. The metabolomic datasets, which contained 93 metabolites, were subjected to weighted correlation network analysis (WGCNA), and eight distinct metabolic modules and seven hub metabolites were identified to be specifically associated with tacrolimus biosynthesis. The analysis of metabolites within each metabolic module suggested that the pentose phosphate pathway (PPP), shikimate and aspartate pathway might be the main limiting factors in the rapid synthesis phase of tacrolimus accumulation. Subsequently, all possible key-limiting steps in the above metabolic pathways were further screened using a genome-scale metabolic network model (GSMM) of S. Tsukubaensis. Based on the prediction results, two newly identified targets (aroC and dapA) were overexpressed experimentally, and both of the engineered strains showed higher tacrolimus production. Moreover, the best strain, HT-aroC/dapA, that was engineered to simultaneously enhanced chorismate and lysine biosynthesis was able to produce 128.19 mg/L tacrolimus, 1.64-fold higher than control (78.26 mg/L). These findings represent a valuable addition to our understanding of tacrolimus accumulation in S. Tsukubaensis, and pave the way to further production improvements.

  • Comparative proteomic and metabolomic analysis of Streptomyces Tsukubaensis reveals the metabolic mechanism of FK506 overproduction by feeding soybean oil
    Applied microbiology and biotechnology, 2017
    Co-Authors: Jun Wang, Cheng Wang, Huanhuan Liu, Di Huang, Lina Jin, Jianping Wen
    Abstract:

    FK506 (tacrolimus) is a 23-membered polyketide macrolide that possesses powerful immunosuppressant activity. In this study, feeding soybean oil into the fermentation culture of Streptomyces Tsukubaensis improved FK506 production by 88.8%. To decipher the overproduction mechanism, comparative proteomic and metabolomic analysis was carried out. A total of 72 protein spots with differential expression in the two-dimensional gel electrophoresis (2-DE) were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS), and 66 intracellular metabolites were measured by gas chromatography-mass spectrometer (GC-MS). The analysis of proteome and metabolome indicated that feeding soybean oil as a supplementary carbon source could not only strengthen the FK506 precursor metabolism and energy metabolism but also tune the pathways related to transcriptional regulation, translation, and stress response, suggesting a better intracellular metabolic environment for the synthesis of FK506. Based on these analyses, 20 key metabolites and precursors of FK506 were supplemented into the soybean oil medium. Among them, lysine, citric acid, shikimic acid, and malonic acid performed excellently for promoting the FK506 production and biomass. Especially, the addition of malonic acid achieved the highest FK506 production, which was 1.56-fold of that in soybean oil medium and 3.05-fold of that in initial medium. This report represented the first comprehensive study on the comparative proteomics and metabolomics applied in S. Tsukubaensis, and it would be a rational guidance to further strengthen the FK506 production.

  • A genome-scale dynamic flux balance analysis model of Streptomyces Tsukubaensis NRRL18488 to predict the targets for increasing FK506 production
    Biochemical Engineering Journal, 2017
    Co-Authors: Cheng Wang, Jiao Liu, Huanhuan Liu, Junhua Wang, Jianping Wen
    Abstract:

    Abstract FK506 is a 23-membered polyketide macrolide with a good immunosuppressant activity for the treatment of autoimmune diseases. However, the low fermentation yield is a bottleneck to limit its further industrialization and clinical applications. To address this issue, a genome-scale dynamic flux balance analysis (GS-DFBA) model was developed to seek for the bottlenecks of FK506 biosynthesis. After validating under different cultural conditions, lots of targets were predicted successfully with help of Minimization of Metabolic Adjustment (MOMA). Specifically, many uncover targets that heretofore had remained hidden in the complex interconnectedness of central metabolism had also shown significant effects on FK506 accumulation. To validate the model predictions, four identified targets (gcdh, tktB, msdh and ask gene) were engineered in Streptomyces Tsukubaensis NRRL18488. All of the engineered strains showed a higher FK506 production, compared with the parent strain. Finally, the best strain HT-△gcdh-tktB/msdh/ask with gcdh-deletion and tktB-, msdh- and ask-overexpression could produce 126.61 ± 4.66 mg/L FK506, 129.8% higher than that of the wild-type strain (55.1 ± 3.83 mg/L). Results demonstrated that the GS-DFBA model could provide an effective approach in engineering S. Tsukubaensis NRRL18488 for FK506 overproduction. This strategy developed here could also be extended to titer improvement of other products.

  • Improved FK506 production by the precursors and product-tolerant mutant of Streptomyces Tsukubaensis based on genome shuffling and dynamic fed-batch strategies
    Journal of industrial microbiology & biotechnology, 2014
    Co-Authors: Di Huang, Jianping Wen, Menglei Xia, Ming Huang
    Abstract:

    FK506, a secondary metabolite produced by Streptomyces Tsukubaensis, is well known for its immunosuppressant properties to prevent rejection of transplanted organs and treat autoimmune diseases. However, the low titer of FK506 in the original producer strain limits the further industrialization efforts and restricts its clinical applications. To address this issue, a highly efficient method combined genome shuffling and dynamic fed-batch strategies was systematically performed in this work. Firstly, after five rounds of genome shuffling based on precursors and product resistances, a higher yielding strain TJ-P325 was successfully acquired, whose production reached 365.6 mg/L, 11-fold increase compared with the original strain. Then, the possible mechanism of different production capabilities between TJ-P325 and the wild type was explored through comparative gene expression analysis of key genes. Results showed that the transcription level of key genes was altered significantly in the mutant. Moreover, precursors addition enhanced the FK506 production by 28 %, as well as reduced the by-products biosynthesis. Finally, the disodium malonate and disodium methylmalonate dynamic fed-batch strategies dramatically led to the production of 514.5 mg/L in a 7.5-L bioreactor. These results demonstrated that genome shuffling and dynamic fed-batch strategies could be successfully applied to generate high-yield strains with value-added natural products during industrial microbial fermentation.

  • Enhancement of FK506 production by engineering secondary pathways of Streptomyces Tsukubaensis and exogenous feeding strategies
    Journal of industrial microbiology & biotechnology, 2013
    Co-Authors: Di Huang, Jianping Wen, Menglei Xia, Xiaoqiang Jia
    Abstract:

    FK506 is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces Tsukubaensis. However, the low titer at which it is produced is a bottleneck to its application and use in industrial processes. We have overexpressed five potential targets associated with FK506 production (fkbO, fkbL, fkbP, fkbM, fkbD) which were identified in our previous study, with the aim to improve FK506 production. The results of the analysis showed that the constructed strains with an additional copy of each gene increased FK506 production by approximately 10-40 % compared with the wild-type strain D852. The results of the gene expression analysis indicated that each gene was upregulated. Combinatorial overexpression of the five genes resulted in a 146 % increase in the FK506 titer to 353.2 mg/L, in comparison with the titer produced by D852. To further improve the production of FK506 by the engineered strain HT-FKBOPLMD, we supplemented the medium with various nutrients, including soybean oil, lactate, succinate, shikimate, chorismate, lysine, pipecolate, isoleucine and valine. Optimization of feeding concentrations and times resulted in HT-FKBOPLMD being able to produce approximately 70 % more FK506, thereby reaching the maximal titer of 457.5 mg/L, with lower amounts of by-products (FK520 and 37,38-dihydro-FK506). These results demonstrate that the combination of the metabolically engineered secondary pathways and the exogenous feeding strategies developed here was able to be successfully applied to improve the production of industrially and clinically important compounds.

María Ordóñez-robles - One of the best experts on this subject based on the ideXlab platform.

  • Genome-wide transcriptome response of Streptomyces Tsukubaensis to N-acetylglucosamine: effect on tacrolimus biosynthesis.
    Microbiological research, 2018
    Co-Authors: María Ordóñez-robles, Antonio Rodríguez-garcía, Juan F. Martín
    Abstract:

    Chitin is the second most abundant carbohydrate biopolymer present in soils and is utilized by antibiotic-producing Streptomyces species. Its monomer, N-acetylglucosamine (GlcNAc), regulates the developmental program of the model organism Streptomyces coelicolor. GlcNAc blocks differentiation when growing on rich medium whilst it promotes development on poor culture media. However, it is unclear if the same GlcNAc regulatory profile observed in S. coelicolor applies also to other industrially important Streptomyces species. We report here the negative effect of GlcNAc on differentiation and tacrolimus (FK506) production by Streptomyces Tsukubaensis NRRL 18488. 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, GlcNAc stimulated transcription of genes related to amino acid and nucleotide biosynthesis, DNA replication, RNA translation, glycolysis and pyruvate metabolism. The results obtained support those previously reported for S. coelicolor, but some important differences were observed; for example genes involved in GlcNAc transport and metabolism and genes encoding transcriptional regulators such as crr, ptsI, nagE1, nagE2, nagB, chiA, chiJ, ngcE, dasR or atrA are not significantly induced in S. Tsukubaensis by GlcNAc addition. Differences in the GlcNAc transport systems, in the physiology of S. Tsukubaensis and S. coelicolor and/or the different composition of the culture media used are likely to be responsible for the discrepancies observed between these species.

  • Unraveling Nutritional Regulation of Tacrolimus Biosynthesis in Streptomyces Tsukubaensis through omic Approaches
    Antibiotics (Basel Switzerland), 2018
    Co-Authors: María Ordóñez-robles, Fernando Santos-beneit, Juan F. Martín
    Abstract:

    Streptomyces Tsukubaensis stands out among actinomycetes by its ability to produce the immunosuppressant tacrolimus. Discovered about 30 years ago, this macrolide is widely used as immunosuppressant in current clinics. Other potential applications for the treatment of cancer and as neuroprotective agent have been proposed in the last years. In this review we introduce the discovery of S. Tsukubaensis and tacrolimus, its biosynthetic pathway and gene cluster (fkb) regulation. We have focused this work on the omic studies performed in this species in order to understand tacrolimus production. Transcriptomics, proteomics and metabolomics have improved our knowledge about the fkb transcriptional regulation and have given important clues about nutritional regulation of tacrolimus production that can be applied to improve production yields. Finally, we address some points of S. Tsukubaensis biology that deserve more attention.

  • Streptomyces Tsukubaensis as a new model for carbon repression: transcriptomic response to tacrolimus repressing carbon sources
    Applied Microbiology and Biotechnology, 2017
    Co-Authors: María Ordóñez-robles, Fernando Santos-beneit, Juan F. Martín, Silvia M. Albillos, Paloma Liras, Antonio Rodríguez-garcía
    Abstract:

    In this work, we identified glucose and glycerol as tacrolimus repressing carbon sources in the important species Streptomyces Tsukubaensis . A genome-wide analysis of the transcriptomic response to glucose and glycerol additions was performed using microarray technology. The transcriptional time series obtained allowed us to compare the transcriptomic profiling of S. Tsukubaensis growing under tacrolimus producing and non-producing conditions. The analysis revealed important and different metabolic changes after the additions and a lack of transcriptional activation of the fkb cluster. In addition, we detected important differences in the transcriptional response to glucose between S. Tsukubaensis and the model species Streptomyces coelicolor . A number of genes encoding key players of morphological and biochemical differentiation were strongly and permanently downregulated by the carbon sources. Finally, we identified several genes showing transcriptional profiles highly correlated to that of the tacrolimus biosynthetic pathway regulator FkbN that might be potential candidates for the improvement of tacrolimus production.

  • Analysis of the Pho regulon in Streptomyces Tsukubaensis
    Microbiological research, 2017
    Co-Authors: María Ordóñez-robles, Fernando Santos-beneit, Antonio Rodríguez-garcía, Juan F. Martín
    Abstract:

    Phosphate regulation of antibiotic biosynthesis in Streptomyces has been studied due to the importance of this genus as a source of secondary metabolites with biological activity. Streptomyces Tsukubaensis is the main producer of tacrolimus (or FK506), an immunosuppressant macrolide that generates important benefits for the pharmaceutical market. However, the production of tacrolimus is under a negative control by phosphate and, therefore, is important to know the molecular mechanism of this regulation. Despite its important role, there are no reports about the Pho regulon in S. Tsukubaensis. In this work we combined transcriptional studies on the response to phosphate starvation with the search for PHO boxes in the whole genome sequence of S. Tsukubaensis. As a result, we identified a set of genes responding to phosphate starvation and containing PHO boxes that include common Pho regulon members but also new species-specific candidates. In addition, we demonstrate for the first time the functional activity of PhoP from S. Tsukubaensis through complementation studies in a Streptomyces coelicolor ΔphoP strain. For this purpose, we developed an anhydrotetracycline inducible system that can be applied to the controlled expression of target genes.

  • Target genes of the Streptomyces Tsukubaensis FkbN regulator include most of the tacrolimus biosynthesis genes, a phosphopantetheinyl transferase and other PKS genes.
    Applied microbiology and biotechnology, 2016
    Co-Authors: María Ordóñez-robles, Antonio Rodríguez-garcía, Juan F. Martín
    Abstract:

    Tacrolimus (FK506) is a 23-membered macrolide immunosuppressant used in current clinics. Understanding how the tacrolimus biosynthetic gene cluster is regulated is important to increase its industrial production. Here, we analysed the effect of the disruption of fkbN (encoding a LAL-type positive transcriptional regulator) on the whole transcriptome of the tacrolimus producer Streptomyces Tsukubaensis using microarray technology. Transcription of fkbN in the wild type strain increases from 70 h of cultivation reaching a maximum at 89 h, prior to the onset of tacrolimus biosynthesis. Disruption of fkbN in S. Tsukubaensis does not affect growth but prevents tacrolimus biosynthesis. Inactivation of fkbN reduces the transcription of most of the fkb cluster genes, including some all (for allylmalonyl-CoA biosynthesis) genes but does not affect expression of allMNPOS or fkbR (encoding a LysR-type regulator). Disruption of fkbN does not suppress transcription of the cistron tcs6-fkbQ-fkbN; thus, FkbN self-regulates only weakly its own expression. Interestingly, inactivation of FkbN downregulates the transcription of a 4′-phosphopantetheinyl transferase coding gene, which product is involved in tacrolimus biosynthesis, and upregulates the transcription of a gene cluster containing a cpkA orthologous gene, which encodes a PKS involved in coelimycin P1 biosynthesis in Streptomyces coelicolor. We propose an information theory-based model for FkbN binding sequences. The consensus FkbN binding sequence consists of 14 nucleotides with dyad symmetry containing two conserved inverted repeats of 7 nt each. This FkbN target sequence is present in the promoters of FkbN-regulated genes.

Miriam Martínez-castro - One of the best experts on this subject based on the ideXlab platform.

  • Regulation of the phosphate metabolism in Streptomyces genus: impact on the secondary metabolites
    Applied Microbiology and Biotechnology, 2019
    Co-Authors: Carlos Barreiro, Miriam Martínez-castro
    Abstract:

    The analysis of the inorganic phosphate effect over the antibiotics production is a long-distance history in the Streptomyces genus, which began almost at the same time that Michael Ende published his book entitled The Neverending Story. In some way, the unveiling of the pho regulon and its influence over the secondary metabolites production is an unfinished story, which keeps this subject as a trending topic, nowadays. Up to date, different studies have been releasing knowledge about particular areas of the pho regulon of different Streptomyces species. Nevertheless, for the first time, these knowledge drops are grouped in a review presenting a broad overview of the phosphate regulation and its impact over the secondary metabolites production in industrially relevant species. Even though the genetic response against phosphate scarcity is similar, as a whole, in different Streptomyces species, the fine-tuning is species-specific. Thus, the response regulator PhoP directly controls the secondary metabolites production in some species, whereas it regulates them in an indirect manner in other species. This information, unraveled in this review, is the result of the intensive analysis along last decade in several species of the genus that is allowing to distinguish how the phosphate response is unleashed in Streptomyces coelicolor , Streptomyces lividans , Streptomyces natalensis , Streptomyces lydicus , Streptomyces avermitilis , and Streptomyces Tsukubaensis .

  • Analysis and validation of the pho regulon in the tacrolimus-producer strain Streptomyces Tsukubaensis: differences with the model organism Streptomyces coelicolor.
    Applied microbiology and biotechnology, 2018
    Co-Authors: Miriam Martínez-castro, Carlos Barreiro, Juan F. Martín
    Abstract:

    Inorganic and organic phosphate controls both primary and secondary metabolism in Streptomyces genus. Metabolism regulation by phosphate in Streptomyces species is mediated by the PhoR-PhoP two-component system. Response regulator PhoP binds to conserved sequences of 11 nucleotides called direct repeat units (DRus), whose organization and conservation determine the binding of PhoP to distinct promoters. Streptomyces Tsukubaensis is the industrial producer of the clinical immunosuppressant tacrolimus (FK506). A bioinformatic genome analysis detected several genes with conserved PHO boxes involved in phosphate scavenging and transport, nitrogen regulation, and secondary metabolite production. In this article, the PhoP regulation has been confirmed by electrophoretic mobility shift assays (EMSA) of the most relevant members of the traditional pho regulon such as the two-component system PhoR-P or genes involved in high-affinity phosphate transport (pstSCAB) and low-affinity phosphate transport (pit). However, the PhoP control over phosphatase genes in S. Tsukubaensis is significantly different from the pattern reported in the model bacteria Streptomyces coelicolor. Thus, neither the alkaline phosphatase PhoA nor PhoD is regulated by PhoP. On the contrary, the binding of PhoP to the promoter of a novel putative phosphatase PhoX was confirmed. A crosstalk of the PhoP and GlnR regulators, which balances phosphate and nitrogen utilization, also occurs in S. Tsukubaensis but slightly modified. Finally, PhoP regulates genes, like afsS, that link phosphate control and secondary metabolite production in S. Tsukubaensis. In summary, there are notable differences between the regulation of specific genes of the pho regulon in S. Tsukubaensis and the model organism S. coelicolor.

  • FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces Tsukubaensis
    BMC microbiology, 2012
    Co-Authors: Dušan Goranovič, Carlos Barreiro, Miriam Martínez-castro, Javier Santos-aberturas, Marko Blažič, Enej Kuščer, Jaka Horvat, Vasilka Magdevska, Tomaž Polak, Peter Mrak
    Abstract:

    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.

  • Taxonomy and chemically semi-defined media for the analysis of the tacrolimus producer ‘Streptomyces Tsukubaensis’
    Applied microbiology and biotechnology, 2012
    Co-Authors: Miriam Martínez-castro, Juan F. Martín, Zahra Salehi-najafabadi, Francisco Romero, Rodrigo Pérez-sanchiz, Rosa Isabel Fernández-chimeno, Carlos Barreiro
    Abstract:

    ‘Streptomyces Tsukubaensis’ was the first tacrolimus producer strain identified. Although it has been included in the Streptomyces genus, its taxonomic position has not been rigorously determined. By using a polyphasic approach, we have established that the tacrolimus producer strain ‘S. Tsukubaensis’ NRRL 18488 represents a unique species in the Streptomyces genus, which is phylogenetically distant from other subsequently described producers. This fact means a horizontal transference of the tacrolimus-producing gene cluster. Physiology, nutrient requirement, and molecular genetics analyses of tacrolimus biosynthesis in ‘S. Tsukubaensis’ necessitate chemically defined or semi-defined media, which work as a jigsaw puzzle and allow for pieces (nutrients) exchange. To date, studies related to ‘S. Tsukubaensis’ have been mainly focused in the improvement of tacrolimus production using complex industrial fermentation media, which difficulty allows testing of tacrolimus overproduction enhancers or inhibitors because of the presence of non‐defined substances. In the present work, two semi-defined media were developed in order to study the main factors involved in tacrolimus production in ‘S. Tsukubaensis’.

  • Draft genome of Streptomyces Tsukubaensis NRRL 18488, the producer of the clinically important immunosuppressant tacrolimus (FK506).
    Journal of bacteriology, 2012
    Co-Authors: Carlos Barreiro, Carlos Prieto, Alberto Sola-landa, Elena Solera, Miriam Martínez-castro, Rosario Pérez-redondo, Carlos García-estrada, Jesús F. Aparicio, Lorena T. Fernández-martínez, Javier Santos-aberturas
    Abstract:

    The macrocyclic polyketide tacrolimus (FK506) is a potent immunosuppressant that prevents T-cell proliferation produced solely by Streptomyces species. We report here the first draft genome sequence of a true FK506 producer, Streptomyces Tsukubaensis NRRL 18488, the first tacrolimus-producing strain that was isolated and that contains the full tacrolimus biosynthesis gene cluster.