Dataset Information

AcnSP - A Novel Small Protein Regulator of Aconitase Activity in the Cyanobacterium Synechocystis sp. PCC 6803.

ABSTRACT: Synechocystis sp. PCC 6803 is a widely used model cyanobacterium whose genome has been well annotated. However, several additional small protein coding sequences (sORFs) have been recently identified, which might play important roles, for example in the regulation of cellular metabolism. Here, we analyzed the function of a sORF encoding a 44 amino acid peptide showing high similarity to the N-terminal part of aconitase (AcnB). The expression of the gene, which probably originated from a partial gene duplication of chromosomal acnB into the plasmid pSYSA, was verified and it was designated as acnSP. The protein-coding part of acnSP was inactivated by interposon mutagenesis. The obtained mutant displayed slower growth under photoautotrophic conditions with light exceeding 100 ?mol photons m-2 s-1 and showed significant changes in the metabolome compared to wild type, including alterations in many metabolites associated to the tricarboxylic acid (TCA) cycle. To analyze a possible direct impact of AcnSP on aconitase, the recombinant Synechocystis enzyme was generated and biochemically characterized. Biochemical analysis revealed that addition of equimolar amounts of AcnSP resulted in an improved substrate affinity (lower K m) and lowered V max of aconitase. These results imply that AcnSP can regulate aconitase activity, thereby impacting the carbon flow into the oxidative branch of the cyanobacterial TCA cycle, which is mainly responsible for the synthesis of carbon skeletons needed for ammonia assimilation.

SUBMITTER: de Alvarenga LV

PROVIDER: S-EPMC7336809 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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AcnSP - A Novel Small Protein Regulator of Aconitase Activity in the Cyanobacterium <i>Synechocystis</i> sp. PCC 6803.

de Alvarenga Luna V LV Hess Wolfgang R WR Hagemann Martin M

Frontiers in microbiology 20200626

<i>Synechocystis</i> sp. PCC 6803 is a widely used model cyanobacterium whose genome has been well annotated. However, several additional small protein coding sequences (sORFs) have been recently identified, which might play important roles, for example in the regulation of cellular metabolism. Here, we analyzed the function of a sORF encoding a 44 amino acid peptide showing high similarity to the N-terminal part of aconitase (AcnB). The expression of the gene, which probably originated from a p ...[more]

PMID: 32695088

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Project description:BACKGROUND:Cyanobacteria 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. RESULTS:Here, 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. CONCLUSIONS:Our 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.

Project description:Cyanobacteria are phototrophic prokaryotes that can convert inorganic carbon as CO2 into organic carbon compounds at the expense of light energy. In addition, they need only a few inorganic nutrients and can be cultivated in high densities using non-arable land and seawater. This features qualified cyanobacteria as attractive organisms for the production of third generation biofuels as part of the development of future CO2-neutral energy production. Synechocystis sp. PCC 6803 represents one of the most widely used cyanobacterial model strains. On the basis of its available genome sequence and genetic tools, many strains of Synechocystis have been generated that produce different biotechnological products. Efficient isoprene production is an attractive goal, since this compound represents not only an energy-rich biofuel but is also used as chemical feedstock. Here, we report on our attempts to generate isoprene-producing strains of Synechocystis. The cDNA of a codon-optimized plant isoprene synthase (IspS) was cloned under the control of different Synechocystis promoters, which ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR, whereas the amount of isoprene was quantified using GC-MS. Incubation of our strains at different salt conditions had marked impact on the isoprene production rates. Under low salt conditions, a good correlation was found between ispS expression and isoprene production rate. However, the cultivation of isoprene production strains under salt-supplemented conditions decreased isoprene production despite the fact that ispS expression was salt-stimulated. The characterization of the metabolome of isoprene producing strains indicated that isoprene production might be limited by insufficient precursor levels. Our isoprene production rates under low salt conditions were 2 - 6.5times higher compared to the previous report of Lindberg et al. (2010). These results can be used to guide future attempts establishing the isoprene production with cyanobacterial host systems.

Dataset Information

AcnSP - A Novel Small Protein Regulator of Aconitase Activity in the Cyanobacterium Synechocystis sp. PCC 6803.

Publications

AcnSP - A Novel Small Protein Regulator of Aconitase Activity in the Cyanobacterium <i>Synechocystis</i> sp. PCC 6803.

Similar Datasets

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