Phycocyanin

The strain E412 presented in the current study was initially isolated from Lotus Lake hot springs in the Ganzi region of Sichuan Province, China. Information about the sampling site and preliminary taxonomic allocation of the strain was reported in our previous studies (Tang et al., 2018a ,b (link)). The unicyanobacterial culture of the strain was cryopreserved as 10% DMSO in BG11 stocks in −80°C. Final precultures for experiments were established as previously described (Tang et al., 2018b (link)) and cultivated at 45°C in 150 mL BG-11 medium in 500 mL Erlenmeyer flasks agitated at 100 rpm under 12L:12D photoperiod at 45 μmol m^–2 s^–1 provided by fluorescent tubes unless stated otherwise. The strain initially denoted as PKUAC-SCTE412 has been recently deposited in the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB-collection) with an accession number FACHB-2490. Physiological assessment of the strain was performed using BG-11 medium free of an essential nutrient (nitrogen or sulfur) supplemented with 17 mM NaNO₂, 85 mM NaNO₃, 10 mM Na₂SO₄, and 10 mM NaHSO₃. The nitrogen fixation capacity during 72 h was performed using the methodology described by Li et al. (2021) (link).
The chromatic adaptation capacity of strain E412 was assessed by culturing the cells at constant LED illumination using either white light (6,500 K) or far-red light (730 nm) at the intensity of 250 and 25 μmol m^–2 s^–1, respectively. After 15 days, cyanobacterial cells were collected to measure chlorophyll a, chlorophyll b, carotenoids, allophycocyanin, phycocyanin, and phycoerythrin, according to previously published protocols (Bennett and Bogorad, 1973 (link); Dere et al., 1998 ). Lipophilic and water-soluble pigments from 15 mg of lyophilized biomass were extracted with 100% methanol and phosphate-buffered saline (PBS), respectively. The absorbance of the supernatant at 470, 562, 615, 652, 653, and 666 nm were recorded against the relative blank using a UV-Vis spectrophotometer (Shimadzu UV-1,800, Japan). The concentration of pigments was calculated using respective formulae for chlorophylls and carotenoids (Dere et al., 1998 ) and phycobiliproteins (Bennett and Bogorad, 1973 (link)).

Twenty-one water samples were collected in the Kattegat, the Baltic Proper and the Gulf of Bothnia using a FerryBox system installed in the ship TransPaper during 13th–19th of July 2013. The ship followed the route: Gothenburg (Sweden)—Kemi (Finland)—Oulu (Finland)—Lübeck (Germany)—Gothenburg. The FerryBox system consists of a pump with a water inlet at 3 m depth, a circuit of multiple sensors for temperature, conductivity, chlorophyll and phycocyanin fluorescence, turbidity, and oxygen as well as automated water sampling devices. A detailed description of the FerryBox system is found in Karlson et al. (in press ). Manual water sampling for DNA analysis was carried out both on the Northward and Southward legs. Approximately, 10 L of seawater were collected in a polycarbonate carboy. Subsamples of 200–500 mL were filtered onto 0.22 μm pore-size mixed cellulose ester membrane filters (Merck Millipore co., Cat. No. GSWP04700) to capture plankton. The filters were frozen in liquid nitrogen on board and kept at −20 to −80°C until DNA extraction.

All clonal Planktothrix strains were isolated by cutting out single filaments migrating on agar as described (Kurmayer et al., 2004 (link)), and cultivated at 15 °C under low light conditions (5–10 μmol m⁻² s⁻¹) in BG11 medium (Rippka, 1988 (link)). In total, 138 strains were analyzed, including 62 isolated previously (Christiansen et al., 2008a (link)); Table 1; Supplementary Table S1). Strains were analyzed for (i) the presence of the full mcy gene cluster and the mcyT gene, which occurred in toxic strains and as a remainder in nontoxic strains (Christiansen et al., 2008a (link)), (ii) the genetic variation within seven housekeeping gene loci and intergenic spacer regions (IGS): 16S rDNA, 16S rDNA-internal transcribed spacer region (ITS), phycocyanin (PC)-IGS, photosystem I (PSA)-IGS (in between photosystem I related psaA and psaB), RNaseP, rbcLX-IGS (in between the large subunit of the ribulose bisphosphate carboxylase/oxygenase and rbcX), and rpoC. In addition, the ability to express gas vesicle protein variants of 28, 20 and 16 kDa was revealed by the presence of the genes gvpC²⁸, gvpC²⁰ and gvpC¹⁶ as described (Beard et al., 2000 ; D'Alelio et al., 2011 ).

The dried biomass (1 g) was homogenized with 0.1 M sodium phosphate buffer (pH 7) and repeated freezing (at −20°C for 3 h) and thawing (at 4°C for 5 min) were done in dark. Subsequently, the mixture was centrifuged at 10,000g and 4°C for 20 min to separate clear supernatant that contains phycobiliproteins [44 ]. A supernatant sample was run on 12.5% SDS-PAGE. The absorbance of supernatant samples was evaluated at wavelengths 600, 610, 615, 620, 630, 640, 650, and 652 nm (OPTIZEN Scan UV/VIS spectrophotometer, KLab Co., Daejeon, Republic of Korea) for C-phycocyanin.
The concentration of C-phycocyanin was calculated as previously reported by Siegelman and Kycia [45 ] as follows: $\begin{array}{c}C-\mathrm{p}\mathrm{h}\mathrm{y}\mathrm{c}\mathrm{o}\mathrm{c}\mathrm{y}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{n}=\frac{\left(A_{615}–0.474\times A_{652}\right)}{5.34}\text{,}\end{array}$ where A₆₁₅ is the absorbance measured at 615 nm and A₆₅₂ is the absorbance measured at 652 nm.

The extreme alkaliphilicArthrospira fusiformis strain used in this study was isolated from the brackish Lake Mariout at the southwest of Alexandria city (Latitude 31.08011° or 31°4′48″ north, longitude 29.79562° or 29° 47′44″ east, Elevation −26 feet, Open location code 8G3F3QJW +26, GeoNames ID 352723). Arthrospira fusiformis was identified via sequencing and analysis of the phycocyanin intergenic spacer region (PC-IGS) in the phycocyanin gene (Accession CBA13040, phycocyanin alpha subunit, partial from Limnospira fusiformis LM) by Sharaf et al. [20 (link)].

The pACYCDuet-1 Vector was digested with restriction endonucleases (Thermo Fisher Scientific) BamHI and SalI, and then the genes pcBA and apcAB were respectively connected to the linear vector with T4 ligase (TransGen Biotech) to obtain plasmids pACYCDuet-pcBA and pACYCDuet-apcAB. The plasmid pACYCDuet-pcBA was then digested with restriction endonucleases NdeI and XhoI, and the gene apcAB was connected to it to obtain the plasmid pACYCDuet-pcBA-apcAB. The above three plasmids were respectively transformed into E. coli BL21 (DE3) to obtain a strain expressing phycocyanin (E. coli PC), a strain expressing allophycocyanin (E. coli APC), and a strain expressing both phycocyanin and allophycocyanin (E. coli PC-APC).
The genes ho (digested by BamHI and SacI), pcyA (digested by SacI and PstI) were inserted into the pETDuet-1 Vector to obtain the plasmid pETDuet-ho-pcyA, and transformed into E. coli BL21 (DE3) to obtain the strain E. coli HP.
Then the chromophore lyase genes cpcU (digested by PstI and SalI), cpcS (digested by SalI and HindIII), cpcT (digested by HindIII and NotI), cpcE (digested by NdeI and AatII), cpcF (digested by AatII and KpnI) were inserted into the plasmid pETDuet-ho-pcyA to obtain the plasmid pETDuet-ho-pcyA-cpcU-cpcS-cpcT-cpcE-cpcF.
The three strains E. coli PC, E. coli APC, and E. coli PC-APC were transformed with the plasmid pETDuet-ho-pcyA-cpcU-cpcS-cpcT-cpcE-cpcF respectively to obtain three kinds of double plasmid expression strains E. coli PC-APC-HPUSTEF, E. coli PC-HPUSTEF, and E. coli APC-HPUSTEF.