Robust reporter system based on chalcone synthase rppA gene from Saccharopolyspora erythraea

Magdevska, Vasilka; Gaber, Rok; Goranovič, Dušan; Kuščer, Enej; Boakes, Steven; Alonso, María Beatriz Durán; Santamaría, Ramón I.; Raspor, Peter; Leadlay, Peter F.; Fujs, Štefan; Petković, Hrvoje

doi:10.1016/j.mimet.2010.08.001

Cited by 15 publications

(20 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Constitutive transcription and relatively high strength of the ermE * promoter from Saccharopolyspora erythraea in the S. tsukubaensis NRRL 18488 strain was demonstrated previously in our work based on a reporter system, using the chalcone synthase rppA gene [41]. …”

Section: Methodssupporting

confidence: 57%

“…Interestingly, double “knock-out” mutants complemented with fkbN , reached comparable FK506 production levels (43%) to the Δ fkbN complemented strains (Figure 3). Therefore, although ermE * promoter (and heterologous RBS) is expressed strongly in S. tsukubaensis , as demonstrated previously by our group [41], it does not seem to be a suitable promoter to match “native” activity, which might require a specific mechanism of gene regulation, possibly also binding of a potential co-inducer. It is possible that the complementation experiment in the case of fkbN gene may have been influenced by the fact that although the transcription of fkbN gene was inactivated by deleting a big part of the CDS and inserting a kanamycin resistance cassette, the remaining parts of the gene, including the C-terminal DNA binding domain could still be expressed (see Additional file 2).…”

Section: Resultsmentioning

confidence: 88%

“…We therefore amplified both versions of the gene, the longer fkbN and 9 bp shorter fkbN-1 and carried out over-expression experiments using both PCR-amplified fkbN variants. The second copy of each version of the fkbN gene was introduced into the S. tsukubaensis wild type strain under the control of the strong ermE * promoter and Streptomyces ribosomal binding site (RBS) [38], a combination which was previously observed to enable high-level protein expression in this strain [41]. Overexpression of either version of fkbN resulted in improved FK506 production.…”

Section: Resultsmentioning

confidence: 99%

“…To study the promoter activity of selected genes from the FK506 gene cluster, which might be under control of FkbN and FkbR transcriptional regulators, we constructed reporter plasmids based on the chalcone synthase gene rppA from Saccharopolyspora erythraea , which was employed successfully in previous studies [20,41]. A series of plasmids were constructed containing the rppA gene as a reporter under the control of different promoters.…”

Section: Methodsmentioning

confidence: 99%

“…Selected apramycin-resistant conjugants of S. tsukubaensis were cultivated in the PG3 production medium as described above until approximately 140 hours post inoculation. The culture broth was then centrifuged and the supernatant diluted 10 times and quantification of water-soluble dark-red flaviolin products of the chalcone synthase was carried out spectrophotometrically using the same conditions as described previously [41]. 270 nm was identified as the most appropriate wavelength for sample analysis and the expression of the rppA gene is presented as absorbance units (AU), taking into account the dilution factor.…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis

et al. 2012

View full text Add to dashboard Cite

BackgroundFK506 (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.ResultsThree 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.ConclusionsOur 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.

show abstract

Section: Methodssupporting

confidence: 57%

Section: Resultsmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis

et al. 2012

View full text Add to dashboard Cite

show abstract

The Genetic System of Actinobacteria

Horbal

Luzhetskyy

2017

Biology and Biotechnology of Actinobacteria

View full text Add to dashboard Cite

An efficient blue-white screening based gene inactivation system for Streptomyces

Guo

et al. 2015

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

Streptomyces is studied intensively for its outstanding ability to produce bioactive secondary metabolites and for its complicated morphological differentiation process. A classical genetic manipulation system for Streptomyces has been developed and widely used in the community for a long time, using antibiotic resistance markers to select for double-crossover mutants. The screening process is always laborious and time-consuming. However, the lack of a suitable chromogenic reporter for Streptomyces has limited the use of color-based screening system to simplify the selection process for double-crossover mutants. In this study, a blue reporter system for Streptomyces has been established by mining an indigoidine synthetase gene (idgS) from Streptomyces lavendulae CGMCC 4.1386, leading to the development of a time-saving gene inactivation system for Streptomyces by simple blue-white screening. A series of Streptomyces suicide and temperature-sensitive plasmids containing the idgS reporter cassette were constructed and used successfully to inactivate genes in Streptomyces, allowing a simple and efficient screening method to differentiate the colonies for double-crossover (white) and single-crossover (blue) mutants. Inactivation of the putative γ-butyrolactone synthase gene afsA-y via the idgS-based blue-white screening method revealed that the paulomycin production is negatively controlled by afsA-y in Streptomyces sp. YN86.

show abstract

Robust reporter system based on chalcone synthase rppA gene from Saccharopolyspora erythraea

Cited by 15 publications

References 32 publications

FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis

FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis

The Genetic System of Actinobacteria

An efficient blue-white screening based gene inactivation system for Streptomyces

Contact Info

Product

Resources

About