Reagents and enzymes
The restriction enzymes, T4 ligase and DNA polymerase enzymes were obtained from New England BioLabs (Ipswich, MA, USA) or from Thermo-Scientific Fermentas (Finland). Oligonucleotides were from Eurofins MWG Operon (Germany). All chemicals were purchased from Sigma-Aldrich (Finland) if not mentioned otherwise.
Organisms and growth conditions
Escherichia coli strain DH5α was used as the host for the plasmid construction in this study. The cells were grown in Luria-Bertani (LB) medium at 37 °C in a shaker at 150–200 rpm or on solid LB plates containing 1.5% (w/v) agar. When necessary, LB medium was supplemented with appropriate antibiotics at the following concentrations: 50 µg/mL spectinomycin (Spec) and 20 µg/mL streptomycin (Str).
The glucose tolerant substrain of Synechocystis sp. PCC 6803 was used for all the cyanobacterial experiments. The cells were grown in liquid BG-11 medium containing 17 mM NaNO3 (J. B. Baker) and 20 mM TES-KOH pH 8.0. The AT and CS strains were cultivated in the presence of 25 µg/mL Spec and 12.5 µg/mL Str, and the cultures were supplied with 0.5 mM of isopropyl-β-d-thiogalactopyranoside (IPTG) and 0–30 mM sodium acetate when necessary. All flask cultures were carried out in Algaetron 230 growth chambers (Photon Systems Instruments) at 30 °C under continuous light of 20–50 µmol photons m−2 s−1, and ambient CO2 in ~120 rpm orbital shaking. BG-11 plates containing additional 1.5% (w/v) Bactoagar (Difco, USA) and 0.3% (w/v) sodium thiosulfate were used for solid plate cultivations, incubated under corresponding conditions in 1% CO2 in SANYO growth chambers.
ActP construct and expression strains
The acetate transporter gene yjcG (actP) was amplified by PCR using E. coli K-12 substrain MG1655 genomic DNA  as a template, and primers gctc
GGTACCATGAAAAGAGTTCTGACGGCGCTTG and tcagCTGCAGTTAATGCGCGCGGCCTTGCT carrying the KpnI and PstI restriction sites (underlined), respectively. The amplified DNA fragment (1670 bp) was subcloned (KpnI–PstI) into an autonomously replicating expression shuttle vector pDF-lac under the control of an inducible lac-promoter  and transformed into E. coli DH5α strain for propagation. Wild type Synechocystis was transformed with the resulting pDF-lac-yjcG shuttle vector by natural transformation , and plated on BG-11 plates supplemented with increasing amount of Spec and Str. The presence of the pDF-lac-yjcG in the antibiotic resistance clones was confirmed by a colony PCR using primer set of forward primer (GCTGCAACTCTCTCAGGGCCAG) and reverse primer (GCGCTTATGGCAGAGCAGGG). The control strain (CS) used in all the experiments was prepared by transforming a corresponding empty plasmid (pDF-trc)  into the WT strain.
Comparison of the Synechocystis strains was carried out using a photobioreactor Multi-Cultivator MC1000 (Photon Systems Instrument, Czech Republic), with eight parallel 100 mL culture tubes immersed in a temperature-controlled water bath, each equipped with individual LED lights, aeration, and capacity for automated periodical measurement of optical density at 730 nm. Before each experiment, the MC1000 lights were calibrated with Li-COR (Li-250A Light Meter, Biosciences), and aeration was adjusted (based on bubbling) between the replicates. Precultures prepared in Erlenmeyer flasks were diluted to the optical density of 0.05 or 0.1 at 750 nm, measured in 10 mm single-use cuvettes using a Thermo-Scientific Genesys 10S UV–Vis spectrophotometer, and transferred into MC1000 tubes in the final culture volume 80 mL. To induce expression, 0.5 mM of IPTG was added to the cultures, with 15 mM sodium acetate unless mentioned otherwise. At the end of the cultivations, pH and the wavescans were recorded, and aliquots were streaked on LB-plates to confirm that the cultures were free of contamination. The cultures were typically carried out in at least three biological replicates with two repetitions in each case.
Evaluating cell size and morphology
For the microscopic evaluation of cell size and morphology, the Synechocystis strains were cultured in MC1000 under continuous 20 μmol photons m−2 s−1 light with supplemented 15 mM acetate for 7 days. The cells were examined under a light microscope (Leitz Orthoplan Large Field Research Microscope) and photographed with a digital microscope camera (Leica DFC420C). The cells were visualized at 100-fold magnification with Leica Application Suite V 4.1, and the horizontal diameter of 115 AT and CS cells were measured form the digital captures (in arbitrary units) using Adobe Photoshop CS4 Ruler Tool.
Determining the dry cell weight of the Synechocystis strains
In order to compare the dry cell weight of AT and CS, the strains were cultivated in MC1000 under 20 μmol photons m−2 s−1 constant light for 8 days. Aliquots of four biological replicate cultures of each strain were concentrated to the same optical density (OD750 = 1), and 5 mL of the samples were filtered through pre-weighted microfiber membranes (VWR). The membranes were washed once with deionized water, oven-dried at 98 °C, and weighted using an analytical scale (Sartorius MC1 Research RC 210P).
Measuring the use of acetate
The consumption of acetate was measured from 20 mL batch cultures (three biological replicates for each strain) grown in 100 mL Erlenmeyer bottles. The cultures were diluted to the OD750 of 0.05, induced with 0.5 mM IPTG, and grown in BG-11 supplemented with antibiotics at ambient air under continuous low light (20 μmol photons m−2 s−1). Samples were collected on days 0, 2, 4, 6, 8, 10 and 12 followed by sample collection (0.5 mL each) and storage at −80 °C until analysis. Acetate quantitation was carried out from the growth media with Acetate Colorimetric Assay kit (Sigma-Aldrich) according to manufacturer’s instructions.
Quantification of glycogen
For quantitative glycogen analysis, 1 mL cell samples (three biological replicates for each strain) were collected from MC1000 cultivations grown under 20 μmol photons m−2 s−1 at the time points 0, day 4, day 8 and day 10. The quantitation was carried out with a commercial Total Starch Assay Kit (Megazyme) using a modified method . The collected samples were pelleted and resuspended in 100 µL of 50 mM NaOAc + 5 mM CaCl2 pH 5.0. The cells were lysed by addition of 900 µL ethanol (96%) and acid washed glass beads (Sigma), followed by eight cycles of vortexing (1 min) and incubating on ice (2 min). The lysates were incubated at 90 °C for 10 min, on ice for 30 min, and centrifuged at 16000g for 30 min. The supernatant was used for chlorophyll a quantitation, while the pellets were incubated at 45 °C to remove the ethanol. Subsequently, the pellets were resuspended into 100 µL of 50 mM NaOAc + 5 mM CaCl2 pH 5.0-buffer with 0.75 U of α-amylase and 5 U of amyloglucosidase to break down the glycogen into glucose. The samples were mixed well and incubated at 60 °C for 2 h, followed by the addition of 150 µL of supplied GOPOD-reagent into the samples. The amount of released glucose in the cell was determined by colourimetric method by reading the absorbance at 510 nm with a PlateReader (Tecan infinite 200 PRO).
Quantification of PHB
For quantitative PHB analysis, aliquots of AT and CS cells were collected at the beginning of the main culture and on day 8 of MC1000 cultivation grown under 20 μmol photons m−2 s−1 with supplemented 15 mM acetate. The PHB quantitation was carried out for four biological replicates for each strain, using a commercial D-3-Hydroxybutyric Acid Assay Kit (Megazyme) according to manufacturer’s protocol. The samples were first adjusted to correspond to OD750 = 0.1 in 50 mL total volume. After centrifugation, the pellets were suspended to 400 µL of 0.5 M NaOH and incubated for 1 h at 85 °C in shaking. The samples were then cooled down and neutralized with 100 µl of 1 M HCl, and the generated D-3-hydrozybutyric acid (corresponding to the amount of PHB in the cells) was analysed colourimetrically from the supernatant by recording the absorbance at 492 nm with a PlateReader (Tecan infinite 200 PRO).
Determination of in vivo light-saturated photosynthetic activity
Samples for the oxygen evolution experiments (three biological replicates for each strain) were collected from MC1000 cultures cultivated under continuous light 20 μmol photons m−2 s−1 for 8 days, followed by concentration of the cells to OD750 1.5. A Clark-type oxygen electrode (Hansatech Ltd) was used to measure the oxygen evolution capacity of photosystem II (PSII) from 1 mL samples under saturating light (2000 μmol photons m−2 s−1) at 30 °C in the presence of an electron acceptor 0.5 mM 2,6-dichloro-p-benzoquinone (DCBQ) and 0.5 mM ferricyanide to maintain DCBQ in oxidized form. The light-saturated whole chain net photosynthesis was measured similarly in the presence of 10 mM NaHCO3 as carbon source. The values were calculated as µmol O2 produced h−1 mL−1 (cell culture) at OD750 = 1.
Absorption spectra (400–800 nm) of all cultures were recorded at the end of each MC1000 cultivation batch using a microplate reader (TECAN Infinite M200PRO). The data was normalized to 750 nm.
Measuring the cellular ROS content
For determining the overall ROS content of the cells, the AT mutant and CS (four biological replicates for each strain) were cultured in MC1000 under 20 μmol photons m−2 s−1 with supplemented 15 mM acetate for 8 days. The analysis was carried out using a commercial membrane-permeable fluorescence indicator 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, CM-H2DCFDA (Invitrogen), which detects hydrogen peroxide, hydroxyl radicals, peroxyl radicals, and peroxynitrite, with a protocol modified from Hakkila et al. 2014 . The cells were washed once and resuspended in 1 mL BG-11 supplemented with 15 mM acetate, to OD750 1. Samples were incubated with and without 25 µM CM-H2DCFDA for 90 min under darkness at 30 °C with shaking. After washing cells twice the samples were resuspended to the final volume of 0.5 mL. Fluorescense (485 nm excitation and emission 535 nm) and autofluorescence (680 nm; chlorophyll) were measured using a PlateReader (Tecan infinite 200 PRO) on a 96-well microtiter plate (Perkin Elmer Isoplate™ −96 F, Black frame and Clear well). Subsequently, samples were incubated further for 45 min under 20 μmol m−2 s−1 of light with shaking, and fluorescence was re-measured. Autofluorescence was used for normalization, and changes in CM-H2DCFDA fluorescence were used to calculate the overall ROS content of the cells.
Singlet oxygen detection
The amount of singlet oxygen in the cells was determined by measuring His-mediated oxygen uptake, which is based on the removal of oxygen from the suspension due to oxidation of histidine by 1O2 . Samples of AT and CS (three biological replicates for each strain) were taken from MC1000 cultures grown under 20 μmol photons m−2 s−1 with supplemented 15 mM acetate on day 8. The cells were washed once and concentrated to OD750 1.5. The rate of singlet oxygen-induced oxygen uptake was measured using Hansatech DW2 O2 electrode. Measurements were performed in the presence and absence of 5 mM His under 3000 μmol photons m−2 s−1 constant light.