Project description:Streptomyces sp. are a rich source for natural products with recognized industrial value, explaining the high interest to improve and streamline production in these microbes. Here, we studied the production of pamamycins, macrodiolide homologues with a high activity against multi-resistant pathogenic microbes, using recombinant S. albus J1074/R2. Talc particles of micrometer size added to submerged cultures of the recombinant strain tripled pamamycin production up to 50 mg L-1. Furthermore, they strongly affected morphology, reduced the size of cell pellets, formed by the filamentous microbe during the process, up to six-fold, and shifted the pamamycin spectrum to larger derivatives. Integrated analysis of transcriptome and metabolome of particle-enhanced and control cultures provided detailed insights into the underlying molecular changes. The microparticles affected the expression of 3341 genes (56%), revealing a global and fundamental impact on metabolism. Morphology-associated genes, encoding major regulators such as SsgA, RelA, EshA, Factor C, as well as chaplins and rodlins, were found massively upregulated, indicating that the particles caused a substantially accelerated morphogenesis. In line, the pamamycin cluster was strongly upregulated (up to log2 10-fold). Furthermore, the microparticles perturbed genes encoding for central catabolism and CoA-ester metabolism, which were mainly activated. The altered expression resulted in changes in the availability of intracellular CoA-esters, the building blocks of pamamycin. Notably, the ratio between methylmalonyl CoA and malonyl-CoA was increased four-fold. Both metabolites compete for incorporation into pamamycin so that the altered availability explained the pronounced preference for larger derivatives in the microparticle-enhanced process. Our findings are straightforward to further develop pamamycins into antituberculosis leads. The novel insights into the behavior of S. albus in response to talc appears of general relevance to further explore and upgrade the concept of microparticle enhanced cultivation, widely used for filamentous microbes.
Project description:On the example of the biosynthetically exhausted landomycin A cluster we demonstrate unbalancing of gene transcription as an efficient method for the generation of new compounds. Decoupled from the native regulators LanI and LanK, all landomycin A structural genes were set under the control of a single synthetic promoter and expressed in a heterologous host Streptomyces albus J1074. Previously being both temporarily and quantitatively regulated, these genes were transcribed as a single polycistronic mRNA leading to the production of four novel and two known compounds. No glycosylated landomycins were detected though the entire biosynthetic cluster was transcribed, showing the crucial role of the balanced gene expression for the production of landomycin A. Two new compounds, fridamycin F and G, isolated in this study were shown to originate from the interplay between the expressed biosynthetic pathway and metabolic network of the heterologous host. Structure activity studies of the isolated compounds as well as results of transcriptome sequencing are discussed in this article. Comparison of gene expression of the H2-26 cosmid (encoding landomycin A biosynthetic genes) with H2-26-act, where an additional constitutive promoter cassette was integrated to drive biosynthetic genes transcription.
Project description:On the example of the biosynthetically exhausted landomycin A cluster we demonstrate unbalancing of gene transcription as an efficient method for the generation of new compounds. Decoupled from the native regulators LanI and LanK, all landomycin A structural genes were set under the control of a single synthetic promoter and expressed in a heterologous host Streptomyces albus J1074. Previously being both temporarily and quantitatively regulated, these genes were transcribed as a single polycistronic mRNA leading to the production of four novel and two known compounds. No glycosylated landomycins were detected though the entire biosynthetic cluster was transcribed, showing the crucial role of the balanced gene expression for the production of landomycin A. Two new compounds, fridamycin F and G, isolated in this study were shown to originate from the interplay between the expressed biosynthetic pathway and metabolic network of the heterologous host. Structure activity studies of the isolated compounds as well as results of transcriptome sequencing are discussed in this article.
Project description:Streptomyces albus S12, TK and Tet30Chl25 are the parental strain , low-yield and high-yield of salinomycin mutant obtained by ARTP and ribosome engineering ,respectively. There are total 1602 differentially expressed genes (DEGs) show differences in expression between the mutant strain TK, Tet30Chl25 and the initial strain S12. KEGG pathway analysis of differentially expressed genes (DEGs) between the mutant strain TK, Tet30Chl25 and the initial strain S12 show that the relevant differential pathways affecting salinomycin production were mainly related to butanoate metabolism, starch and sucrose metabolism, glyoxylate metabolism. Besides , the transcription of genes in the salinomycin biosynthesis gene cluster and the transcription level of related genes in the precursors biosynthesis pathway were more active in the high-yield salinomycin production strain Tet30Chl25. Furthermore, the transcription level ribosomal protein, string response, two component system and sigma factors are more active in high-yield of salinomycin mutants and that may involve in regulation of salinomycin biosynthesis and may account for the high-yield of salinomycin.
Project description:To evaluate the DNA binding of FscRI in Streptomyces albus S4 in vivo ChIP-Seq experiments were carried out on dusing anti-FLAG antibodies against 3xFLAG-FscRI protein using two biological replicates. The wild type strain was used as a control in experiments using the anti-FLAG antibodies as well as a DNA only control.
Project description:To evaluate the DNA binding of AntA in Streptomyces albus S4, in vivo ChIP-Seq experiments were performed 3xFLAG-AntA and anti-FLAG antibodies using two biological replicates. The wild type strain was used as a control in experiments using the anti-FLAG antibodies as well as a DNA only control (which were published previously, E-MTAB-5122).
Project description:We describe a lasso peptide, albusnodin, that is post-translationally modified with an acetyl group, the first example of a lasso peptide with this modification. Using heterologous expression, we further show that the acetyltransferase colocalized with the albusnodin gene cluster is required for the biosynthesis of this lasso peptide. This type of lasso peptide is widespread in Actinobacteria with 44 examples found in currently sequenced genomes.
Project description:BACKGROUND: The Streptomyces albus J1074 strain is one of the most widely used chassis for the heterologous production of bioactive natural products. The fast growth and an efficient genetic system make this strain an attractive model for expressing cryptic biosynthetic pathways to aid drug discovery. RESULTS: To improve its capabilities for the heterologous expression of biosynthetic gene clusters, the complete genomic sequence of S. albus J1074 was obtained. With a size of 6,841,649 bp, coding for 5,832 genes, its genome is the smallest within the genus streptomycetes. Genome analysis revealed a strong tendency to reduce the number of genetic duplicates. The whole transcriptomes were sequenced at different time points to identify the early metabolic switch from the exponential to the stationary phase in S. albus J1074. CONCLUSIONS: S. albus J1074 carries the smallest genome among the completely sequenced species of the genus Streptomyces. The detailed genome and transcriptome analysis discloses its capability to serve as a premium host for the heterologous production of natural products. Moreover, the genome revealed 22 additional putative secondary metabolite gene clusters that reinforce the strain's potential for natural product synthesis.
Project description:The genus Streptomyces is a promising source of biologically active secondary metabolites. Here, we report the complete genome sequence of Streptomyces albus strain G153. The assembled genome comprised a single linear chromosome of 6.9 Mbp with a G+C content of 73.3%.