SE179:/S01/M03

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	M03
Title	HPLC
Method Details ID	MS03
Sample Amount
Comment

Analytical Method Details Information

ID	MS03
Title	Measurement of organic acids by high-performance liquid chromatography (HPLC)
Instrument	LC-2000Plus Systems (JASCO, Tokyo, Japan)
Instrument Type
Ionization
Ion Mode
Description	Freeze-dried supernatants were resolved in 100 μL of filtered 3 mM perchloric acid. The resolved samples were analyzed by HPLC using a LC-2000Plus Systems (JASCO, Tokyo, Japan) with a photodiode array detector and two RSpak KC-811 columns (Showa Denko, Tokyo, Japan). Organic acids were quantified with 0.2 mM bromothymol blue in 15 mM sodium phosphate buffer; peaks were detected at 445 nm. The column temperature was 60°C, and the flow rates of 3 mM perchloric acid and 0.2 mM bromothymol blue solutions were 1.0 and 1.5 mL/min, respectively.
Comment_of_details