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Analysis of RNA-dependent proteins in the cyanobacterium Synechocystis by RNase sensitive gradient profiling (Grad-R) Analysis of RNA-dependent proteins in the cyanobacterium Synechocystis by RNase sensitive gradient profiling (Grad-R)

ABSTRACT: RNA-binding proteins (RBPs) are an essential component of ribonucleoprotein complexes and are crucial for post-transcriptional regulation by regulatory sRNAs. Well-studied examples in different groups of bacteria include RBPs such as various ribosomal and RRM-domain containing proteins, or the RNA chaperones Hfq, ProQ and CsrA. However, the knowledge about RBPs and possible RNA chaperones in photosynthetic cyanobacteria is very limited, although there is extensive evidence for the presence of post-transcriptional regulation. Here, we analyzed cellular protein complexes by gradient profiling with or without RNase treatment (Grad-R) in Synechocystis sp. PCC 6803.

INSTRUMENT(S):

ORGANISM(S): Synechocystis Sp. Pcc 6803

SUBMITTER: Sandra Maass

LAB HEAD: Dörte Becher

PROVIDER: PXD045848 | Pride | 2025-08-26

REPOSITORIES: Pride

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			Action	DRS
	220517_OV3_P5_PG_SK_LH_GradR_Synech_BR1_RNase_01.raw	Raw
	220519_OV3_P8_PG_SK_LH_GradR_Synech_BR1_RNase_02.raw	Raw
	220519_OV3_P8_PG_SK_LH_GradR_Synech_BR1_RNase_03.raw	Raw
	220519_OV3_P8_PG_SK_LH_GradR_Synech_BR1_RNase_04.raw	Raw
	220519_OV3_P8_PG_SK_LH_GradR_Synech_BR1_RNase_05.raw	Raw

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Project description:BackgroundCyanobacteria are phototrophic prokaryotes that convert inorganic carbon as CO2 into organic compounds at the expense of light energy. They need only inorganic nutrients and can be cultivated to high densities using non-arable land and seawater. This has made cyanobacteria attractive organisms for the production of biofuels and chemical feedstock. Synechocystis sp. PCC 6803 is one of the most widely used cyanobacterial model strains. Based on its available genome sequence and genetic tools, Synechocystis has been genetically modified to produce different biotechnological products. Efficient isoprene production is an attractive goal because this compound is widely used as chemical feedstock.ResultsHere, we report on our attempts to generate isoprene-producing strains of Synechocystis using a plasmid-based strategy. As previously reported, a codon-optimized plant isoprene synthase (IspS) was expressed under the control of different Synechocystis promoters that ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR and Western blotting, while the amount of isoprene was quantified using GC-MS. In addition to isoprene measurements in the headspace of closed culture vessels, single photon ionization time-of-flight mass spectrometry (SPI-MS) was applied, which allowed online measurements of isoprene production in open-cultivation systems under various conditions. Under standard conditions, a good correlation existed between ispS expression and isoprene production rate. The cultivation of isoprene production strains under NaCl-supplemented conditions decreased isoprene production despite enhanced ispS mRNA levels. The characterization of the metabolome of isoprene-producing strains indicated that isoprene production might be limited by insufficient precursor levels. Transcriptomic analysis revealed the upregulation of mRNA and regulatory RNAs characteristic of acclimation to metabolic stress.ConclusionsOur best production strains produced twofold higher isoprene amounts in the presence of low NaCl concentrations than previously reported strains. These results will guide future attempts to establish isoprene production in cyanobacterial hosts.

Dataset Information

Analysis of RNA-dependent proteins in the cyanobacterium Synechocystis by RNase sensitive gradient profiling (Grad-R) Analysis of RNA-dependent proteins in the cyanobacterium Synechocystis by RNase sensitive gradient profiling (Grad-R)

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