SE179:/S01/M01

Sample Set Information

ID	TSE1337
Title	Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium.
Description	Succinate is a building block compound that the U.S. Department of Energy (DOE) has declared as important in biorefineries, and it is widely used as a commodity chemical. Here, we identified the two genes increasing succinate production of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Succinate was excreted under dark, anaerobic conditions, and its production level increased by knocking out ackA, which encodes an acetate kinase, and by overexpressing sigE, which encodes an RNA polymerase sigma factor. Glycogen catabolism and organic acid biosynthesis were enhanced in the mutant lacking ackA and overexpressing sigE, leading to an increase in succinate production reaching five times of the wild-type levels. Our genetic and metabolomic analyses thus demonstrated the effect of genetic manipulation of a metabolic enzyme and a transcriptional regulator on succinate excretion from this cyanobacterium with the data based on metabolomic technique.
Authors	Osanai, T., Shirai, T., Iijima, H., Nakaya, Y., Okamoto, M., Kondo, A. and Hirai, M.Y.
Reference	Front Microbiol. 2015 Oct 6;6:1064. doi: 10.3389/fmicb.2015.01064.
Comment

PubMed

Sample Information

ID	S01
Title	Synechocystis sp. PCC 6803
Organism - Scientific Name	Synechocystis sp. PCC 6803 substr. GT-I
Organism - ID	NCBI:txid1080228
Compound - ID
Compound - Source
Preparation	The glucose-tolerant strain of Synechocystis sp. PCC 6803, isolated by Williams (Williams, 1988), was grown in modified BG-11 medium, consisting of BG-110 liquid medium (Rippka, 1988) supplemented with 5 mM NH4Cl (buffered with 20 mM HEPES–KOH, pH 7.8). The GT-I strain, among GT substrains, was used in the current study (Kanesaki et al., 2012). Liquid cultures were bubbled with 1% (v/v) CO2 in air and incubated at 30°C under continuous white light (~50–70 μmol photons m−2 s−1). For the mutant strains, 10, 0.3, and 10 μg/mL of kanamycin, gentamycin and chloramphenicol, respectively, were added for preculturing. Modified BG-11 medium (containing 10 mM NH4Cl in liquid medium) was solidified with agar (1.5% w/v) for plate cultures, and similarly incubated in air at 30°C under continuous white light (~50–70 μmol photons m−2 s−1). Cell densities were measured at A730 using a Hitachi U-3310 spectrophotometer (Hitachi High-Tech., Tokyo, Japan). For succinate production, cells grown in 70 mL modified BG-11 medium (started from A730 = 0.4) for 3 days were concentrated into 10 mL HEPES buffer (20 mM HEPES–KOH, pH 7.8) or modified BG-11 medium to A730 = 20 in a GC vial. The vial was sealed using butyl rubber, and N2 gas was introduced using syringes for 1 h to produce anaerobic conditions. After removing the syringes, the vial was wrapped with aluminum foil and shaken at 30°C. Cell cultures were then centrifuged at 5800 × g for 2 min, the supernatant was filtrated, and 1 mL supernatant was freeze-dried for 1 day. The dried sample was used for high-performance liquid chromatography analysis.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M01
Title	LC-MS/MS analysis
Method Details ID	MS01
Sample Amount	20 μl
Comment

Analytical Method Details Information

ID	MS01
Title	LC-MS/MS analysis
Instrument	LCMS‐8040 triple quadrupole LC/MS/MS spectrometer; Shimadzu
Instrument Type
Ionization	ESI
Ion Mode
Description	Equal amounts of cells (10 mL cell culture with A730 = 1.0) were harvested by rapid filtration, and metabolites were extracted using a previously described method (Osanai et al., 2014b). Briefly, the cells were filtrated, and then the intermediate metabolites were quenched and extracted in 1.2 mL of solvent mixture (CHCl3:CH3OH:H2O, 2.5:2.5:1, v/v/v) containing 10 μg/L D-(+)-camphor-10-sulfonic acid as an internal standard. After centrifugation at 15,000 × g at 4°C for 5 min, 400 μL of the upper phase was transferred to a new tube and vacuum-dried.
Comment_of_details

PubMed