Streptomycin production by Streptomyces griseus can be modulated by a mechanism not associated with change in the adpA component of the A-factor cascade

Hong, Bin; Phornphisutthimas, Somkiat; Tilley, Emma; Baumberg, Simon; McDowall, Kenneth J.

doi:10.1007/s10529-006-9216-2

Cited by 33 publications

(45 citation statements)

References 35 publications

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“…The PCR fragments were initially cloned into pUC19 and their DNA sequence confirmed by sequencing. Further, the selected DNA fragments were excised from pUC19 using NdeI and XbaI restriction enzymes, gel purified and subcloned into the phiC31-based integrative expression vector pSET152, containing the constitutive ermE * promoter and a Streptomyces ribosome binding site [38], via NdeI and XbaI restriction sites, thus generating plasmids pDG1 ( allN ), pDG2 ( allN + mgl ), pDG3 ( fkbR ) and pDG4 ( fkbN ) (Table 1). …”

Section: Methodsmentioning

confidence: 99%

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

et al. 2012

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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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2012

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“…The fragment was also ligated into an integrative vector pSET152 [30] to give pSETR3. The sgcR3 coding region (1,188 bp) amplified by PCR was introduced to pL646 [37], displacing atrAc gene under the control of a strong constitutive promoter ermE *p, to give pLR3.…”

Section: Methodsmentioning

confidence: 99%

“…RNA was isolated from S. globisporus mycelia scraped from cellophane laid on the surface of S5 agar plates, treated with DNaseI (Promega, WI, USA) and quantitated as described previously [37,38]. The first strand synthesis of cDNA was performed with SuperScript III First-strand Synthesis System (Inivitrogen, CA, USA) using 2 μg total RNA and the random hexamers as primers following the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Role of sgcR3 in positive regulation of enediyne antibiotic C-1027 production of Streptomyces globisporus C-1027

Wang

Zhang

et al. 2009

BMC Microbiol

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BackgroundC-1027, produced by Streptomyces globisporus C-1027, is one of the most potent antitumoral agents. The biosynthetic gene cluster of C-1027, previously cloned and sequenced, contains at least three putative regulatory genes, i.e. sgcR1, sgcR2 and sgcR3. The predicted gene products of these genes share sequence similarities to StrR, regulators of AraC/XylS family and TylR. The purpose of this study was to investigate the role of sgcR3 in C-1027 biosynthesis.ResultsOverexpression of sgcR3 in S. globisporus C-1027 resulted in a 30–40% increase in C-1027 production. Consistent with this, disruption of sgcR3 abolished C-1027 production. Complementation of the sgcR3-disrupted strain R3KO with intact sgcR3 gene could restore C-1027 production. The results from real time RT-PCR analysis in R3KO mutant and wild type strain indicated that not only transcripts of biosynthetic structural genes such as sgcA1 and sgcC4, but also putative regulatory genes, sgcR1 and sgcR2, were significantly decreased in R3KO mutant. The cross-complementation studies showed that sgcR1R2 could functionally complement sgcR3 disruption in trans. Purified N-terminal His10-tagged SgcR3 showed specific DNA-binding activity to the promoter region of sgcR1R2.ConclusionThe role of SgcR3 has been proved to be a positive regulator of C-1027 biosynthesis in S. globisporus C-1027. SgcR3 occupies a higher level than SgcR1 and SgcR2 in the regulatory hierarchy that controls C-1027 production and activates the transcription of sgcR1 and sgcR2 by binding directly to the promoter region of sgcR1R2.

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“…To investigate the contribution of ssaA to the regulation of sansanmycin biosynthesis, the coding region of ssaA was cloned into a pSET152-derived expression plasmid, pL646 (28), under the control of a strong constitutive promoter, ermE*p, to give pL-ssaA. The plasmid was introduced into the wild-type strain by conjugation, and the resulting ssaA overexpression strain was designated SS/pL-ssaA.…”

Section: Identification and Analysis Of The Sansanmycin Gene Cluster mentioning

confidence: 99%

SsaA, a Member of a Novel Class of Transcriptional Regulators, Controls Sansanmycin Production in Streptomyces sp. Strain SS through a Feedback Mechanism

Wang

Xie

et al. 2013

J Bacteriol

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Sansanmycins, produced by Streptomyces sp. strain SS, are uridyl peptide antibiotics with activities against Pseudomonas aeruginosa and multidrug-resistant Mycobacterium tuberculosis. In this work, the biosynthetic gene cluster of sansanmycins, comprised of 25 open reading frames (ORFs) showing considerable amino acid sequence identity to those of the pacidamycin and napsamycin gene cluster, was identified. SsaA, the archetype of a novel class of transcriptional regulators, was characterized in the sansanmycin gene cluster, with an N-terminal fork head-associated (FHA) domain and a C-terminal LuxR-type helix-turnhelix (HTH) motif. The disruption of ssaA abolished sansanmycin production, as well as the expression of the structural genes for sansanmycin biosynthesis, indicating that SsaA is a pivotal activator for sansanmycin biosynthesis. SsaA was proved to directly bind several putative promoter regions of biosynthetic genes, and comparison of sequences of the binding sites allowed the identification of a consensus SsaA binding sequence, GTMCTGACAN 2 TGTCAGKAC. The DNA binding activity of SsaA was inhibited by sansanmycins A and H in a concentration-dependent manner. Furthermore, sansanmycins A and H were found to directly interact with SsaA. These results indicated that SsaA strictly controls the production of sansanmycins at the transcriptional level in a feedback regulatory mechanism by sensing the accumulation of the end products. As the first characterized regulator of uridyl peptide antibiotic biosynthesis, the understanding of this autoregulatory process involved in sansanmycin biosynthesis will likely provide an effective strategy for rational improvements in the yields of these uridyl peptide antibiotics.

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Streptomycin production by Streptomyces griseus can be modulated by a mechanism not associated with change in the adpA component of the A-factor cascade

Cited by 33 publications

References 35 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

Role of sgcR3 in positive regulation of enediyne antibiotic C-1027 production of Streptomyces globisporus C-1027

SsaA, a Member of a Novel Class of Transcriptional Regulators, Controls Sansanmycin Production in Streptomyces sp. Strain SS through a Feedback Mechanism

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