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Sample Set Information

ID TSE1339
Title Genetic engineering of group 2 sigma factor SigE widely activates expressions of sugar catabolic genes in Synechocystis species PCC 6803.
Description Metabolic engineering of photosynthetic organisms is required for utilization of light energy and for reducing carbon emissions.Control of transcriptional regulators is a powerful approach for changing cellular dynamics, because a set of genes is concomitantly regulated. Here, we show that overexpression of a group 2 σ factor, SigE, enhances the expressions of sugar catabolic genes in the unicellular cyanobacterium, Synechocystis sp. PCC 6803. Transcriptome analysis revealed that genes for the oxidative pentose phosphate pathway and glycogen catabolism are induced by overproduction of SigE. Immunoblotting showed that protein levels of sugar catabolic enzymes, such as glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glycogen phosphorylase, and isoamylase, are increased. Glycogen levels are reduced in the SigE-overexpressing strain grown under light. Metabolome analysis revealed that metabolite levels of the TCA cycle and acetyl-CoA are significantly altered by SigE overexpression. The SigE-overexpressing strain also exhibited defective growth under mixotrophic or dark conditions. Thus, SigE overexpression changes sugar catabolism at the transcript to phenotype levels, suggesting a σ factor-based engineering method for modifying carbon metabolism in photosynthetic bacteria.
Authors Osanai, T., Oikawa, A., Azuma, M., Tanaka, K., Saito, K., Hirai, M. Y. and Ikeuchi, M.
Reference J Biol Chem. 2011 Sep 2;286(35):30962-71. doi: 10.1074/jbc.M111.231183.

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Sample Information

ID S01
Title Synechocystis sp. PCC 6803
Organism - Scientific Name Synechocystis sp. PCC 6803
Organism - ID NCBI:txid1148
Compound - ID
Compound - Source
Preparation The glucose-tolerant (GT) strain of Synechocystis sp. PCC 6803, isolated by Williams, and the SigE-overexpressing strain were grown in BG-110 liquid medium with 5 mM NH4Cl (buffered with 20 mM Hepes-KOH (pH 7.8)), termed modified BG-11 medium. Liquid cultures were bubbled with 1% (v/v) CO2 in air at 30 °C under continuous white light (∼50–70 μmol photons m−2 s−1). For plate cultures, modified BG-110 (the concentration of NH4Cl was 10 mM instead of 5 mM in liquid medium) was solidified using 1.5% (w/v) agar (BD Biosciences) and incubated in air at 30 °C under continuous white light (∼ 50–70 μmol photons m−2 s−1). The null mutant of sigE, named G50, was generated as published previously. For the SigE-overexpressing strain and the sigE null mutant, 20 μg/ml kanamycin (Sigma) was supplemented in the modified BG-11 liquid medium. Dark conditions were achieved by wrapping culture plates with aluminum foil. Growth and cell densities were measured at A730 with a Beckman DU640 spectrophotometer.
Sample Preparation Details ID

Analytical Method Information

ID M01
Title CE-MS
Method Details ID MS01
Sample Amount 20 μl

Analytical Method Details Information

Title Capillary Electrophoresis/Mass Spectrometry (CE/MS) Analysis
Instrument Agilent CE capillary electrophoresis system, Agilent G3250AA LC/MSD TOF system, Agilent 1100 series binary HPLC pump,G1603A Agilent CE-MS adapter and G1607A Agilent CE-ESI-MS sprayer kit
Instrument Type
Ionization ESI
Ion Mode positive and negative
Description Cells of mid-exponential phase cultures of Synechocystis 6803 (A730, 0.5–0.7) grown in modified BG-11 were collected by centrifugation at 9,800 × g for 3 min, followed by freezing in liquid nitrogen. 50–200 mg of cells (fresh weight) were suspended in 600 μl of 60% (v/v) methanol, including 27 μM 10-camphorsulfonic acid as an internal standard, and vortexed by a MicroSmash (Tomy) eight times for 1 min at 4 °C, followed by centrifugation at 20,400 × g for 5 min at 4 °C. 400 μl of the supernatant was then centrifuged through a Millipore 5-kDa cutoff filter at 10,000 × g for 90 min. 300 μl of the filtrate was dried for 120 min by a centrifugal concentrator. The residue was dissolved in 20 μl of water and used for CE/MS analysis. The CE/MS system and conditions were as described previously.

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