scholarly journals Identification of AflR Binding Sites in the Genome of Aspergillus flavus by ChIP-Seq

2020 ◽  
Vol 6 (2) ◽  
pp. 52
Author(s):  
Qing Kong ◽  
Perng-Kuang Chang ◽  
Chunjuan Li ◽  
Zhaorong Hu ◽  
Mei Zheng ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Binding Sites ◽  
Biosynthetic Gene Cluster ◽  
Binding Motif ◽  
Gene Ontology Analysis ◽  
Biosynthetic Gene ◽  
Upstream Promoter ◽  
Upstream Promoter Region ◽  
Translational Start ◽  
First Time

We report here the AflR binding motif of Aspergillus flavus for the first time with the aid of ChIP-seq analysis. Of the 540 peak sequences associated with AflR binding events, 66.8% were located within 2 kb upstream (promoter region) of translational start sites. The identified 18-bp binding motif was a perfect palindromic sequence, 5′-CSSGGGWTCGAWCCCSSG’3′ with S representing G or C and W representing A or T. On closer examination, we hypothesized that the 18-bp motif sequence identified contained two identical parts (here called motif A and motif B). Motif A was in positions 8–18 on the upper strand, while motif B was in positions 11-1 on the bottom strand. The inferred length and sequence of the putative motif identified in A. flavus were similar to previous findings in A. parasiticus and A. nidulans. Gene ontology analysis indicated that AflR bound to other genes outside the aflatoxin biosynthetic gene cluster.

scholarly journals LmbU directly regulates SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780, which are located outside the lmb cluster and inhibit lincomycin biosynthesis in Streptomyces lincolnensis

2021 ◽  
Author(s):  
Bingbing Hou ◽  
Xianyan Zhang ◽  
Yue Mao ◽  
Ruida Wang ◽  
Jiang Ye ◽  
...  
Keyword(s):  
Binding Sites ◽  
Biosynthetic Gene Cluster ◽  
Regulatory Mechanisms ◽  
Whole Genome ◽  
Biosynthetic Gene ◽  
Qrt Pcr ◽  
Dna Binding Sites ◽  
Reporter Assays ◽  
Streptomyces Lincolnensis

The productions of antibiotics are usually regulated by cluster-situated regulators (CSRs), which can directly regulate the genes within the corresponding biosynthetic gene cluster (BGC). However, few studies have looked into the regulation of CSRs on the targets outside the BGC. Here, we screened the targets of LmbU in the whole genome of S. lincolnensis, and found 14 candidate targets, among of which, 8 targets can bind to LmbU by EMSAs. Reporter assays in vivo revealed that LmbU repressed transcription of SLINC_RS02575 and SLINC_RS05540, while activated transcription of SLINC_RS42780. In addition, disruptions of SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 promoted the production of lincomycin, and qRT-PCR showed that SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 inhibited transcription of the lmb genes, indicating that all the three regulators can negatively regulate lincomycin biosynthesis. What's more, the homologues of LmbU and its targets SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 are widely found in actinomycetes, while the distributions of DNA-binding sites (DBS) of LmbU are diverse, indicating the regulatory mechanisms of LmbU homologues in various strains are different and complicated.

scholarly journals Cell type-specific protein-DNA interactions in the human zeta-globin upstream promoter region: displacement of Sp1 by the erythroid cell-specific factor NF-E1.

1990 ◽  
Vol 10 (1) ◽  
pp. 282-294 ◽  
Author(s):  
C Y Yu ◽  
J Chen ◽  
L I Lin ◽  
M Tam ◽  
C K Shen
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Promoter Region ◽  
Globin Gene ◽  
Cell Type ◽  
Upstream Promoter ◽  
Protein Dna Interactions ◽  
Cell Type Specific ◽  
Upstream Promoter Region ◽  
Zeta Globin Gene

The protein-DNA interactions of the upstream promoter region of the human embryonic zeta-globin gene in nuclear extracts of erythroid K562 cells and nonerythroid HeLa cells were analyzed by DNase I footprinting, gel mobility shift assay, methylation interference, and oligonucleotide competition experiments. There are mainly two clusters of nuclear factor-binding sites in the zeta promoter. The proximal cluster spans the DNA sequence from -110 to -60 and consists of binding sites for CP2, Sp1, and NF-E1. NF-E1 binding is K562 specific, whereas CP2 binding is common to both types of cells. Overlapping the NF-E1- and CP2-binding sites is a hidden Sp1-binding site or CAC box, as demonstrated by binding studies of affinity-purified Sp1. In the distal promoter region at -250 to -220, another NF-E1-binding site overlaps a CAC box or Sp1-binding site. Extract-mixing experiments demonstrated that the higher affinity of NF-E1 binding excluded the binding of Sp1 in the K562 extract. NF-E1 factors could also displace prebound Sp1 molecules. Between the two clusters of multiple-factor-binding sites are sequences recognized by other factors, including zeta-globin factors 1 and 2, that are present in both HeLa and K562 extracts. We discuss the cell type-specific, competitive binding of multiple nuclear factors in terms of functional implications in transcriptional regulation of the zeta-globin gene.

Cell type-specific protein-DNA interactions in the human zeta-globin upstream promoter region: displacement of Sp1 by the erythroid cell-specific factor NF-E1

1990 ◽  
Vol 10 (1) ◽  
pp. 282-294
Author(s):  
C Y Yu ◽  
J Chen ◽  
L I Lin ◽  
M Tam ◽  
C K Shen
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Promoter Region ◽  
Globin Gene ◽  
Cell Type ◽  
Upstream Promoter ◽  
Protein Dna Interactions ◽  
Cell Type Specific ◽  
Upstream Promoter Region ◽  
Zeta Globin Gene

The protein-DNA interactions of the upstream promoter region of the human embryonic zeta-globin gene in nuclear extracts of erythroid K562 cells and nonerythroid HeLa cells were analyzed by DNase I footprinting, gel mobility shift assay, methylation interference, and oligonucleotide competition experiments. There are mainly two clusters of nuclear factor-binding sites in the zeta promoter. The proximal cluster spans the DNA sequence from -110 to -60 and consists of binding sites for CP2, Sp1, and NF-E1. NF-E1 binding is K562 specific, whereas CP2 binding is common to both types of cells. Overlapping the NF-E1- and CP2-binding sites is a hidden Sp1-binding site or CAC box, as demonstrated by binding studies of affinity-purified Sp1. In the distal promoter region at -250 to -220, another NF-E1-binding site overlaps a CAC box or Sp1-binding site. Extract-mixing experiments demonstrated that the higher affinity of NF-E1 binding excluded the binding of Sp1 in the K562 extract. NF-E1 factors could also displace prebound Sp1 molecules. Between the two clusters of multiple-factor-binding sites are sequences recognized by other factors, including zeta-globin factors 1 and 2, that are present in both HeLa and K562 extracts. We discuss the cell type-specific, competitive binding of multiple nuclear factors in terms of functional implications in transcriptional regulation of the zeta-globin gene.

NovG, a DNA-binding protein acting as a positive regulator of novobiocin biosynthesis

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 1949-1961 ◽  
Author(s):  
Alessandra S. Eustáquio ◽  
Shu-Ming Li ◽  
Lutz Heide
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Binding Sites ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Gene Clusters ◽  
Binding Motif ◽  
Multicopy Plasmid ◽  
Biosynthetic Gene ◽  
Positive Regulator

The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e. novE and novG. The predicted gene product of novG shows a putative helix–turn–helix DNA-binding motif and shares sequence similarity with StrR, a well-studied pathway-specific transcriptional activator of streptomycin biosynthesis. Here functional proof is provided, by genetic and biochemical approaches, for the role of NovG as a positive regulator of novobiocin biosynthesis. The entire novobiocin cluster of the producer organism Streptomyces spheroides was expressed in the heterologous host Streptomyces coelicolor M512, and additional strains were produced which lacked the novG gene within the heterologously expressed cluster. These ΔnovG strains produced only 2 % of the novobiocin formed by the S. coelicolor M512 strains carrying the intact novobiocin cluster. The production could be restored by introducing an intact copy of novG into the mutant. The presence of novG on a multicopy plasmid in the strain containing the intact cluster led to almost threefold overproduction of the antibiotic, suggesting that novobiocin biosynthesis is limited by the availability of NovG protein. Furthermore, purified N-terminal His6-tagged NovG showed specific DNA-binding activity for the novG–novH and the cloG–cloY intergenic regions of the novobiocin and clorobiocin biosynthetic gene clusters, respectively. By comparing the DNA sequences of the fragments binding NovG, conserved inverted repeats were identified in both fragments, similar to those identified as the binding sites for StrR. The consensus sequence for the StrR and the putative NovG binding sites was GTTCRACTG(N)11CRGTYGAAC. Therefore, NovG and StrR apparently belong to the same family of DNA-binding regulatory proteins.

scholarly journals Cyclofaulknamycin with the Rare Amino Acid D-capreomycidine Isolated from a Well-Characterized Streptomyces albus Strain

2021 ◽  
Vol 9 (8) ◽  
pp. 1609
Author(s):  
Liliya Horbal ◽  
Marc Stierhof ◽  
Anja Palusczak ◽  
Nikolas Eckert ◽  
Josef Zapp ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Pyridoxal Phosphate ◽  
Cyclic Peptide ◽  
Genome Mining ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Streptomyces Albus ◽  
Cluster Biosynthesis ◽  
First Time

Targeted genome mining is an efficient method of biosynthetic gene cluster prioritization within constantly growing genome databases. Using two capreomycidine biosynthesis genes, alpha-ketoglutarate-dependent arginine beta-hydroxylase and pyridoxal-phosphate-dependent aminotransferase, we identified two types of clusters: one type containing both genes involved in the biosynthesis of the abovementioned moiety, and other clusters including only arginine hydroxylase. Detailed analysis of one of the clusters, the flk cluster from Streptomyces albus, led to the identification of a cyclic peptide that contains a rare D-capreomycidine moiety for the first time. The absence of the pyridoxal-phosphate-dependent aminotransferase gene in the flk cluster is compensated by the XNR_1347 gene in the S. albus genome, whose product is responsible for biosynthesis of the abovementioned nonproteinogenic amino acid. Herein, we report the structure of cyclofaulknamycin and the characteristics of its biosynthetic gene cluster, biosynthesis and bioactivity profile.

scholarly journals Characterization of the biosynthetic gene cluster for the ribosomally synthesized cyclic peptide ustiloxin B in Aspergillus flavus

2014 ◽  
Vol 68 ◽  
pp. 23-30 ◽  
Author(s):  
Myco Umemura ◽  
Nozomi Nagano ◽  
Hideaki Koike ◽  
Jin Kawano ◽  
Tomoko Ishii ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Aspergillus Flavus ◽  
Cyclic Peptide ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene

scholarly journals Direct Rbs Engineering of the Biosynthetic Gene Cluster for Efficient Productivity of Violaceins in E. Coli

2020 ◽  
Author(s):  
Yuyang Zhang ◽  
Hongping Chen ◽  
Yao Zhang ◽  
Huifang Yin ◽  
Chenyan Zhou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Gene Cluster ◽  
Large Scale ◽  
Value Added ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Fermentation Time ◽  
Engineering Strategy ◽  
First Time

Abstract Background: Violaceins have attracted much attention as potential targets used in medicines, food additives, insecticides, cosmetics and textiles, but low productivity was the key factor to limit their large-scale applications. This work put forward a direct RBS engineering strategy to engineer the violacein biosynthetic gene cluster cloned from Chromobacterium violaceum ATCC 12472 to efficiently improve the fermentation titers.Results: Through four rounds of engineering of the native RBSs within the violaceins biosynthetic operon vioABCDE, this work apparently broke through the rate-limiting steps of intermediates conversion, resulting in 2.41-fold improvement of violaceins production compared to the titers of the starting strain Escherichia coli BL21(DE3) (Vio12472). Furthermore, by optimizing the batch-fermentation parameters including temperature, concentration of IPTG inducer and fermentation time, the maximum yield of violaceins from (BCDE)m (tnaA-) reached 3269.7 μM at 2 mM tryptophan in the medium. Interestingly, rather than previous reported low temperature (20 ℃), we for the first time found the RBS engineered Escherichia coli strain (BCDE)m worked better at higher temperature (30 ℃ and 37 ℃), leading to a higher-level production of violaceins. Conclusion: To our knowledge, this is the first time that a direct RBS engineering strategy is used for the biosynthesis of natural products, having the potential for a greater improvement of the product yields within tryptophan hyperproducers and simultaneously avoiding the costly low temperature cultivation for large-scale industrial production of violaciens. This direct RBS engineering strategy could also be easily and helpfully used in engineering the native RBSs of other larger and value-added natural product biosynthetic gene clusters by widely used site-specific mutagenesis methods represented by inverse PCR or CRISPR-Cas9 techniques to increase their fermentation titers in the future.

scholarly journals Exploring novel herbicidin analogues by transcriptional regulator overexpression and MS/MS molecular networking

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Yuanyuan Shi ◽  
Renjie Gu ◽  
Yihong Li ◽  
Xinwei Wang ◽  
Weicong Ren ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Consensus Sequence ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Binding Motif ◽  
Biosynthetic Gene ◽  
Structural Genes ◽  
Biosynthetic Gene Clusters ◽  
Molecular Networking ◽  
Overexpression Strain

Abstract Background Herbicidin F has an undecose tricyclic furano-pyrano-pyran structure with post-decorations. It was detected from Streptomyces mobaraensis US-43 fermentation broth as a trace component by HPLC–MS analysis. As herbicidins exhibit herbicidal, antibacterial, antifungal and antiparasitic activities, we are attracted to explore more analogues for further development. Results The genome of S. mobaraensis US-43 was sequenced and a herbicidin biosynthetic gene cluster (hcd) was localized. The cluster contains structural genes, one transporter and three potential transcription regulatory genes. Overexpression of the three regulators respectively showed that only hcdR2 overexpression significantly improved the production of herbicidin F, and obviously increased the transcripts of 7 structural genes as well as the transporter gene. After performing homology searches using BLASTP in the GenBank database, 14 hcd-like clusters were found with a cluster-situated hcdR2 homologue. These HcdR2 orthologues showed overall structural similarity, especially in the C-terminal DNA binding domain. Based on bioinformatics analysis, a 21-bp consensus binding motif of HcdR2 was detected within 30 promoter regions in these genome-mined clusters. EMSA results verified that HcdR2 bound to the predicted consensus sequence. Additionally, we employed molecular networking to explore novel herbicidin analogues in hcdR2 overexpression strain. As a result, ten herbicidin analogues including six new compounds were identified based on MS/MS fragments. Herbicidin O was further purified and confirmed by 1H NMR spectrum. Conclusions A herbicidin biosynthetic gene cluster (hcd) was identified in S. mobaraensis US-43. HcdR2, a member of LuxR family, was identified as the pathway-specific positive regulator, and the production of herbicidin F was dramatically increased by overexpression of hcdR2. Combined with molecular networking, ten herbicidin congeners including six novel herbicidin analogues were picked out from the secondary metabolites of hcdR2 overexpression strain. The orthologues of herbicidin F pathway-specific regulator HcdR2 were present in most of the genome-mined homologous biosynthetic gene clusters, which possessed at least one consensus binding motif with LuxR family characteristic. These results indicated that the combination of overexpression of hcdR2 orthologous regulator and molecular networking might be an effective way to exploit the “cryptic” herbicidin-related biosynthetic gene clusters for discovery of novel herbicidin analogues.

scholarly journals Direct RBS Engineering of the biosynthetic gene cluster for efficient productivity of violaceins in E. coli

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Yuyang Zhang ◽  
Hongping Chen ◽  
Yao Zhang ◽  
Huifang Yin ◽  
Chenyan Zhou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Gene Cluster ◽  
Large Scale ◽  
Value Added ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Fermentation Time ◽  
Engineering Strategy ◽  
First Time

Abstract Background Violaceins have attracted much attention as potential targets used in medicines, food additives, insecticides, cosmetics and textiles, but low productivity was the key factor to limit their large-scale applications. This work put forward a direct RBS engineering strategy to engineer the violacein biosynthetic gene cluster cloned from Chromobacterium violaceum ATCC 12,472 to efficiently improve the fermentation titers. Results Through four-rounds of engineering of the native RBSs within the violaceins biosynthetic operon vioABCDE, this work apparently broke through the rate-limiting steps of intermediates conversion, resulting in 2.41-fold improvement of violaceins production compared to the titers of the starting strain Escherichia coli BL21(DE3) (Vio12472). Furthermore, by optimizing the batch-fermentation parameters including temperature, concentration of IPTG inducer and fermentation time, the maximum yield of violaceins from (BCDE)m (tnaA−) reached 3269.7 µM at 2 mM tryptophan in the medium. Interestingly, rather than previous reported low temperature (20 ℃), we for the first time found the RBS engineered Escherichia coli strain (BCDE)m worked better at higher temperature (30 ℃ and 37 ℃), leading to a higher-level production of violaceins. Conclusions To our knowledge, this is the first time that a direct RBS engineering strategy is used for the biosynthesis of natural products, having the potential for a greater improvement of the product yields within tryptophan hyperproducers and simultaneously avoiding the costly low temperature cultivation for large-scale industrial production of violaciens. This direct RBS engineering strategy could also be easily and helpfully used in engineering the native RBSs of other larger and value-added natural product biosynthetic gene clusters by widely used site-specific mutagenesis methods represented by inverse PCR or CRISPR-Cas9 techniques to increase their fermentation titers in the future.

scholarly journals Functional Interaction between the Fab-7 and Fab-8 Boundaries and the Upstream Promoter Region in the Drosophila Abd-B Gene

2008 ◽  
Vol 28 (12) ◽  
pp. 4188-4195 ◽  
Author(s):  
Olga Kyrchanova ◽  
Stepan Toshchakov ◽  
Yulia Podstreshnaya ◽  
Alexander Parshikov ◽  
Pavel Georgiev
Keyword(s):  
Binding Sites ◽  
Regulatory Region ◽  
Upstream Region ◽  
Functional Interaction ◽  
Long Distance ◽  
Functional Interactions ◽  
Upstream Promoter ◽  
Upstream Promoter Region

ABSTRACT Boundary elements have been found in the regulatory region of the Drosophila melanogaster Abdominal-B (Abd-B) gene, which is subdivided into a series of iab domains. The best-studied Fab-7 and Fab-8 boundaries flank the iab-7 enhancer and isolate it from the four promoters regulating Abd-B expression. Recently binding sites for the Drosophila homolog of the vertebrate insulator protein CTCF (dCTCF) were identified in the Fab-8 boundary and upstream of Abd-B promoter A, with no binding of CTCF to the Fab-7 boundary being detected either in vivo or in vitro. Taking into account the inability of the yeast GAL4 activator to stimulate the white promoter when its binding sites are separated by a 5-kb yellow gene, we have tested the functional interactions between the Fab-7 and Fab-8 boundaries and between these boundaries and the upstream promoter A region containing a dCTCF binding site. It has been found that dCTCF binding sites are essential for pairing between two Fab-8 insulators. However, a strong functional interaction between the Fab-7 and Fab-8 boundaries suggests that additional, as yet unidentified proteins are involved in long-distance interactions between them. We have also shown that Fab-7 and Fab-8 boundaries effectively interact with the upstream region of the Abd-B promoter.

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