Project description:Streptomycetes are known as producers of bioactive substances, particularly antibiotics. Streptomyces netropsis IMV Ac-5025 simultaneously produces different classes of antibiotics, including polyene compounds, phytohormones, and sterols, but the metabolic pathways involved in their biosynthesis are largely understudied. The aim of this work was to explore the biosynthesis of polyene antibiotics, sterols, and phytohormones when the producer is cultivated in a nutrient medium supplemented with exogenous β-sitosterol. Gas chromatography and high-performance liquid chromatography were applied to analyze the spectrum of bioactive compounds. The obtained results demonstrated not only an increase in the accumulation of biomass but also polyene antibiotics, intracellular sterols, auxins, and cytokinins, when cultivating S. netropsis IMV Ac-5025 in a liquid medium with the addition of β-sitosterol. The amount of biomass raised 1.5-2-fold, whilst the sum of polyene antibiotics increased 4.5-fold, sterols' sum (ergosterol, cholesterol, stigmasterol, β-sitosterol, and 24-epibrassinolide) by 2.9-fold, auxins' sum (indole-3-acetic acid, indole-3-acetic acid hydrazide, indole-3-carbinol, indole-3-butyric acid, indole-3-carboxaldehyde, and indole-3-carboxylic acid) by 6-fold, and cytokinins' sum (zeatin, isopentyladenine, zeatin riboside, and isopentenyladenosine) by 11-fold. Thus, we put forward the hypothesis that β-sitosterol plays a regulatory role in the network of biosynthetic reactions of S. netropsis IMV Ac-5025.
Project description:Rhizosphere microorganisms play important ecological roles in promoting herb growth and producing abundant secondary metabolites. Studies on the rhizosphere microbes of traditional Chinese medicines (TCMs) are limited, especially on the genomic and metabolic levels. In this study, we reported the isolation and characterization of a Steptomyces netropsis WLXQSS-4 strain from the rhizospheric soil of Clematis manshurica Rupr. Genomic sequencing revealed an impressive total of 40 predicted biosynthetic gene clusters (BGCs), whereas metabolomic profiling revealed 13 secondary metabolites under current laboratory conditions. Particularly, medium screening activated the production of alloaureothin, whereas brominated and chlorinated pimprinine derivatives were identified through precursor-directed feeding. Moreover, antiproliferative activities against Hela and A549 cancer cell lines were observed for five compounds, of which two also elicited potent growth inhibition in Enterococcus faecalis and Staphylococcus aureus, respectively. Our results demonstrated the robust secondary metabolism of S. netropsis WLXQSS-4, which may serve as a biocontrol agent upon further investigation.
Project description:In biosynthesis of natural products, potential intermediates or analogs of a particular compound in the crude extracts are commonly overlooked in routine assays due to their low concentration, limited structural information, or because of their insignificant bio-activities. This may lead into an incomplete and even an incorrect biosynthetic pathway for the target molecule. Here we applied multiple compound mining approaches, including genome scanning and precursor ion scan-directed mass spectrometry, to identify potential pyrrolamide compounds in the fermentation culture of Streptomyces netropsis. Several novel congocidine and distamycin analogs were thus detected and characterized. A more reasonable route for the biosynthesis of pyrrolamides was proposed based on the structures of these newly discovered compounds, as well as the functional characterization of several key biosynthetic genes of pyrrolamides. Collectively, our results implied an unusual "iterative strategy" underlying the pyrrole polymerization in the biosynthesis of pyrrolamide antibiotics.
Project description:tRNAs are encoded by a large gene family, usually with several isogenic tRNAs interacting with the same codon. Mutations in the anticodon region of other tRNAs can overcome specific tRNA deficiencies. Phylogenetic analysis suggests that such mutations have occurred in evolution, but the driving force is unclear. We show that in yeast suppressor mutations in other tRNAs are able to overcome deficiency of the essential TRT2-encoded tRNAThrCGU at high temperature (40°C). Surprisingly, these tRNA suppressor mutations were obtained after whole-genome transformation with DNA from thermotolerant Kluyveromyces marxianus or Ogataea polymorpha strains, but from which the mutations did apparently not originate. We suggest that transient presence of donor DNA in the host facilitates proliferation at high temperature and thus increases the chances for occurrence of spontaneous mutations suppressing defective growth at high temperature. Whole-genome sequence analysis of three transformants revealed only four to five non-synonymous mutations of which one causing TRT2 anticodon stem stabilization and two anticodon mutations in non-threonyl-tRNAs, tRNALysCUU and tRNAeMetCAU, were causative. Both anticodon mutations suppressed lethality of TRT2 deletion and apparently caused the respective tRNAs to become novel substrates for threonyl-tRNA synthetase. LC-MS/MS data could not detect any significant mistranslation and RT-qPCR results contradicted induction of the unfolded protein response. We suggest that stress conditions have been a driving force in evolution for the selection of anticodon-switching mutations in tRNAs as revealed by phylogenetic analysis. Importance of the work In this work we have identified for the first time the causative elements in a eukaryotic organism introduced by applying whole-genome transformation and responsible for the selectable trait of interest, i.e. high temperature tolerance. Surprisingly, the whole-genome transformants contained just a few SNPs, which were unrelated to the sequence of the donor DNA. In each of three independent transformants, we have identified a SNP in a tRNA, either stabilizing the essential tRNAThrCGU at high temperature or switching the anticodon of tRNALysCUU or tRNAeMetCAU into CGU, which is apparently enough for in vivo recognition by threonyl-tRNA synthetase. LC-MS/MS analysis indeed indicated absence of significant mistranslation. Phylogenetic analysis showed that similar mutations have occurred throughout evolution and we suggest that stress conditions may have been a driving force for their selection. The low number of SNPs introduced by whole-genome transformation may favor its application for improvement of industrial yeast strains.