Identification of Lipstatin-Producing Ability in Streptomyces virginiae CBS 314.55 Using Dereplication Approach

Streptomicete su poznate kao bogat izvor bioaktivnih metabolita, poput sekundarnog metabolita lipstatina, koji ima vrlo karakterističnu strukturu β-laktona, a upotrebljava se kao intermedijar u proizvodnji lijeka za mršavljenje, komercijalnog naziva orlistat. Dublje razumijevanje distribucije identičnih ili strukturno sličnih spojeva, izoliranih iz taksonomski sličnih skupina mikroorganizama, bitno je za otkrivanje novih biološki aktivnih spojeva ili kvalitetnijih sojeva što proizvode poznate metabolite od velikog značaja za medicinu. Samo su dva soja, neovisni izolati vrste S. toxytricini, poznati kao proizvođači lipstatina. Prema današnjim taksonomskim spoznajama, vrsta S. toxytricini pripada fenotipskoj skupini S. lavendulae. Taksonomskom dereplikacijom i ispitivanjem in vitro inhibicije lipaze pomoću p-nitrofenolnih derivata C4 i C16 masnih kiselina istraženi su odabrani sojevi fenotipske skupine S. lavendulae iz javno dostupnih kolekcija mikroorganizama. Utvrđeno je da bakterijska kultura soja Streptomyces virginiae CBS 314.55 izrazito inhibira lipazu. Pomoću metoda HPLC i LC-MS/MS po prvi je put otkriveno da ovaj novi soj proizvodi metabolit strukture identične lipstatinu. Dokazano je da se novi soj S. virginiae morfološki i fiziološki bitno razlikuje od sojeva S. toxytricini, ali da im je proizvodni potencijal sličan. Time je potvrđena mogućnost učinkovite primjene metode dereplikacije za brzu identifikaciju novih industrijskih sojeva iz javno dostupnih kolekcija mikroorganizama.Streptomyces species are prolific producers of bioactive metabolites, such as β-lactone-containing lipstatin produced by Streptomyces toxytricini, an intermediate used in semi-synthetic process for production of anti-obesity drug orlistat. Understanding the distribution of identical or structurally similar molecules produced by a taxonomic group is of particular importance when trying to isolate novel biologically active compounds or strains producing known metabolites of medical importance with potentially improved properties. Until now, only two independent isolates of S. toxytricini species have been known to be producers of lipstatin. According to the current taxonomic criteria, S. toxytricini belongs to Streptomyces lavendulae phenotypic cluster. Taxonomy-based dereplication approach coupled with in vitro assay was applied to screen the S. lavendulae phenotypic cluster for production of lipstatin-like lipase inhibitors using synthetic p-nitrophenol derivatives of C4 and C16 lipids. Screening the available strains from public collections belonging to S. lavendulae phenotypic cluster, high lipase inhibitory activity was identified in the Streptomyces virginiae CBS 314.55 culture supernatants. HPLC and LC-MS/MS confirmed lipstatin production by a new Streptomyces species for the first time. We have demonstrated that the new lipstatin-producing strain S. virginiae morphologically and physiologically differs from S. toxytricini substantially; however, the production capacity of the newly identified lipstatin-producing species S. virginiae is comparable to S. toxytricini. We have thus demonstrated the effectiveness of a simple and affordable dereplication approach for identification of potentially novel and useful industrial strains available in public culture collections

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Additional file 14. A table with description of strains and plasmids generated and used in this study and graphical representation of over-expressed operons/genes

Production of extracellular heterologous proteins in Streptomyces rimosus, producer of the antibiotic oxytetracycline

Among the Streptomyces species, Streptomyces lividans has often been used for the production of heterologous proteins as it can secrete target proteins directly into the culture medium. Streptomyces rimosus, on the other hand, has for long been used at an industrial scale for oxytetracycline production, and it holds ‘Generally Recognised As Safe’ status. There are a number of properties of S. rimosus that make this industrial strain an attractive candidate as a host for heterologous protein production, including (1) rapid growth rate(2) growth as short fragments, as for Escherichia coli(3) high efficiency of transformation by electroporationand (4) secretion of proteins into the culture medium. In this study, we specifically focused our efforts on an exploration of the use of the Sec secretory pathway to export heterologous proteins in a S. rimosus host. We aimed to develop a genetic tool kit for S. rimosus and to evaluate the extracellular production of target heterologous proteins of this industrial host. This study demonstrates that S. rimosus can produce the industrially important enzyme phytase AppA extracellularly, and analogous to E. coli as a host, application of His-Tag/Ni-affinity chromatography provides a simple and rapid approach to purify active phytase AppA in S. rimosus. We thus demonstrate that S. rimosus can be used as a potential alternative protein expression system

Elementi koji reguliraju biosintezu tetraciklinskih antibiotika u genskim nakupinama: otcG gen pozitivno regulira proizvodnju oksitetraciklina u vrste Streptomyces rimosus

The expression of bacterial polyketide synthase gene clusters is often controlled by a number of different families of regulatory proteins that can have either a pathway-specific or a pleiotropic mode of action, e.g. the SARP family (Streptomyces antibiotic regulatory proteins), ribosome-associated ppGpp synthetase, γ-butyrolactone-binding regulatory proteins, and two-component regulatory proteins. The molecular genetics of such regulatory mechanisms that govern the biosynthesis of tetracyclines is poorly understood. In this work, a comparative bioinformatic analysis of regulatory genes present in three tetracycline antibiotic gene clusters, namely oxytetracycline (OTC), chlortetracycline and recently cloned chelocardin gene clusters of S. rimosus, S. aureofaciens and Amycolatopsis sulphurea has been performed. A SARP family regulatory protein is located in the chlortetracycline gene cluster, but is not detected in the gene cluster encoding OTC biosynthesis. Interestingly, the only regulatory element identified in chelocardin gene cluster was chdA, an otrR and ctcR homologue from the TetR family of regulators that regulates the expression of the otrB and ctc05 exporter genes in the oxytetracycline and chlortetracycline gene clusters. In the oxytetracycline gene cluster, a new LAL (LuxR) family regulatory gene homologue, otcG, was identified. This homologue is also present in the ctc gene cluster. By gene disruption and overexpression experiments, a \u27conditionally positive\u27 role of otcG in OTC biosynthesis has been demonstrated. The observation, the bioinformatics data and the previous work on phosphate regulation suggest the presence of a more complex, fine tuning role of the otcG gene product in overall expression of genes for OTC biosynthesis.Ekspresiju bakterijskih genskih nakupina poliketidnih sintaza obično reguliraju različiti vrlo specifični proteini koji su uglavnom usmjereni samo na ciljane gene u nakupini gena ili je riječ o regulatornim genima što djeluju šire na stanične procese, takozvani pleiotropni regulatori. Prema načinu djelovanja ti se regulatorni proteini mogu podijeliti u više skupina, kao što su SARP (engl. Streptomyces Antibiotic Regulatory Proteins), skupina ribosomski povezanih regulatornih proteina nazvanih ppGpp sintaze, γ-butirolaktonskih veznih i dvokomponentnih regulatornih proteina. Molekularna genetika takvih regulatornih mehanizama, koji utječu na biosintezu tetraciklina, još je i danas slabo istražena. U ovom je radu prikazana komparativna bioinformatička studija regulatornih proteina triju genskih nakupina koje kodiraju biosintezu oksitetraciklina (OTC) u vrste Streptomyces rimosus, klortetraciklina (u vrste Streptomyces aureofaciens), a i nedavno klonirane genske nakupine za biosintezu kelokardina (CHD) u vrste Amycolatopsis sulphurea. U genskoj nakupini koja kodira biosintezu klortetraciklina nalazi se takozvani SARP regulatorni gen. Međutim, homolog toga gena ne postoji u nakupini gena za biosintezu oksitetraciklina. U nakupini gena za biosintezu kelokardina pronađen je samo jedan regulatorni gen nazvan chdA, homolog gena otrR i ctcR iz skupine TetR regulatornih proteina, koji regulira ekspresiju otrB i ctc05 gena u nakupinama gena za biosintezu oksitetraciklina i klortetraciklina. U genskoj nakupini za biosintezu oksitetraciklina identificiran je novi regulatorni protein iz skupine LAL (LuxR), nazvan OtcG. Taj se genski homolog nalazi i u genskoj nakupini za biosintezu klortetraciklina. Inaktivacijom i povećanom ekspresijom gena otcG eksperimentalno je utvrđeno njegovo djelovanje kao uvjetno pozitivnog regulatora u biosintezi oksitetraciklina. Takvi eksperimentalni rezultati bioinformatičke studije i rezultati koji se odnose na prijašnje istraživanje utjecaja fosfata na reguliranje biosinteze oksitetraciklina upućuju na vrlo kompleksnu organizaciju regulatornih elemenata i njihova djelovanja u procesu biosinteze oksitetraciklina, u kojima sudjeluje i novi regulatorni element, produkt gena otcG

Production of extracellular heterologous proteins in Streptomyces rimosus, producer of the antibiotic oxytetracycline

Among the Streptomyces species, Streptomyces lividans has often been used for the production of heterologous proteins as it can secrete target proteins directly into the culture medium. Streptomyces rimosus, on the other hand, has for long been used at an industrial scale for oxytetracycline production, and it holds ‘Generally Recognised As Safe’ status. There are a number of properties of S. rimosus that make this industrial strain an attractive candidate as a host for heterologous protein production, including (1) rapid growth rate; (2) growth as short fragments, as for Escherichia coli; (3) high efficiency of transformation by electroporation; and (4) secretion of proteins into the culture medium. In this study, we specifically focused our efforts on an exploration of the use of the Sec secretory pathway to export heterologous proteins in a S. rimosus host. We aimed to develop a genetic tool kit for S. rimosus and to evaluate the extracellular production of target heterologous proteins of this industrial host. This study demonstrates that S. rimosus can produce the industrially important enzyme phytase AppA extracellularly, and analogous to E. coli as a host, application of His-Tag/Ni-affinity chromatography provides a simple and rapid approach to purify active phytase AppA in S. rimosus. We thus demonstrate that S. rimosus can be used as a potential alternative protein expression system.This study was funded by the Ministry of Higher Education, Science and Technology, Slovenian Research Agency (grant no. L4-7117), grant no. C3330-17-529038 ‘Raziskovalci-2.0-UL-BF-529038’ by the Ministry of Higher Education, Science and Technology—Republic of Slovenia, Slovene Human Resources, Development and Scholarship Funds (grant no. 58-T-003) and ‘Augusto González Linares’ program (University of Cantabria, Spain)

SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea

Background Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. Results We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. Conclusions SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bldD, SACE_5599 is involved in morphological development of S. erythraea, suggesting a very close relationship between secondary metabolite biosynthesis and morphological differentiation in this organism. While the mode of action of SACE_5599 remains to be elucidated, the manipulation of this gene clearly shows potential for improvement of erythromycin production in S. erythraea in industrial setting. We have also demonstrated the applicability of the comparative proteomics approach for identifying new regulatory elements involved in biosynthesis of secondary metabolites in industrial conditions

Multiple copies of the oxytetracycline gene cluster in selected Streptomyces rimosus strains can provide significantly increased titers.

Background: Natural products are a valuable source of biologically active compounds that have applications in medicine and agriculture. One disadvantage with natural products is the slow, time-consuming strain improvement regimes that are necessary to ensure sufficient quantities of target compounds for commercial production. Although great efforts have been invested in strain selection methods, many of these technologies have not been improved in decades, which might pose a serious threat to the economic and industrial viability of such important bioprocesses. Results: In recent years, introduction of extra copies of an entire biosynthetic pathway that encodes a target product in a single microbial host has become a technically feasible approach. However, this often results in minor to moderate increases in target titers. Strain stability and process reproducibility are the other critical factors in the industrial setting. Industrial Streptomyces rimosus strains for production of oxytetracycline are one of the most economically efficient strains ever developed, and thus these represent a very good industrial case. To evaluate the applicability of amplification of an entire gene cluster in a single host strain, we developed and evaluated various gene tools to introduce multiple copies of the entire oxytetracycline gene cluster into three different Streptomyces rimosus strains: wild-type, and medium and high oxytetracycline-producing strains. We evaluated the production levels of these engineered S. rimosus strains with extra copies of the oxytetracycline gene cluster and their stability, and the oxytetracycline gene cluster expression profiles; we also identified the chromosomal integration sites. Conclusions: This study shows that stable and reproducible increases in target secondary metabolite titers can be achieved in wild-type and in high oxytetracycline-producing strains, which always reflects the metabolic background of each independent S. rimosus strain. Although this approach is technically very demanding and requires systematic effort, when combined with modern strain selection methods, it might constitute a very valuable approach in industrial process development

FK506 biosynthesis is regulated by two positive regulatory elements in <it>Streptomyces tsukubaensis</it>

<p>Abstract</p> <p>Background</p> <p>FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various <it>Streptomyces</it> 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.</p> <p>Results</p> <p>Three putative regulatory elements, namely <it>fkbR</it>, belonging to the LysR-type family, <it>fkbN</it>, a large ATP-binding regulator of the LuxR family (LAL-type) and <it>allN</it>, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from <it>Streptomyces tsukubaensis</it> NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of <it>fkbN</it> caused a complete disruption of FK506 biosynthesis, while inactivation of <it>fkbR</it> resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the <it>allN</it> gene. Using RT-PCR and a reporter system based on a chalcone synthase <it>rppA</it>, we demonstrated, that in the wild type as well as in <it>fkbN</it>- and <it>fkbR</it>-inactivated strains, <it>fkbR</it> is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas <it>fkbN</it> expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of <it>fkbN</it> (or <it>fkbR</it>) 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 <it>fkbR</it> or <it>fkbN</it> genes under the control of the strong <it>ermE</it>* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively.</p> <p>Conclusions</p> <p>Our results clearly demonstrate the positive regulatory role of <it>fkbR</it> and <it>fkbN</it> genes in FK506 biosynthesis in <it>S. tsukubaensis</it> 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 <it>S. tsukubaensis</it> for improving the yield of fermentative processes for production of FK506.</p

MOESM2 of Integrated omics approaches provide strategies for rapid erythromycin yield increase in Saccharopolyspora erythraea

Additional file 2. An integrated table of all obtained genomic, transcriptomic and proteomic data as well as data obtained and published in previous studies (Peano et al., 2012; Marcellin et al., 2013, Li et al., 2011)

MOESM8 of Integrated omics approaches provide strategies for rapid erythromycin yield increase in Saccharopolyspora erythraea

Additional file 8. Results of the 2-D PAGE experiments: representation of 2-D gels of WT and HP strain in different time points (t1-t4), comparison of 2-D images between HP and WT in t1 and t2