Bacteria strains, plasmids and culture conditions
The bacteria strains and plasmids used in this study are listed in Table 1. Spirulina platensis FACHB-904 was obtained from the Fresh water Algae Culture Collection at the Institute of Hydrobiology in WuHan, and cultured in Zarrouk medium (pH = 9.0) at 28ºC and 120 rpm/min, under a light intensity of 2000 Lx with a photoperiod of 12 light/12 dark. Luria-Bertani (LB) medium was used for seed cultivation of E. coli. The E. coli was cultured on a rotary shaker at 200 rpm and 37°C.
Table 1
Bacterial strains and plamids.
Bacterial strains and plasmidsDescription Source
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Strains
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Spirulina. platensis FACHB-904
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Wild type,
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Purchased from FACHB-collection
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E. coli DH5α
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Cloning host and expression host
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Purchased from TransGen Biotech, China
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Plasmids
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|
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pEASY-Blunt
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Cloning vector
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Purchased from TransGen Biotech, China
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pGEX-6p-1
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Expression vector
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Laboratory preservation
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pEASY-Blunt-SpcrtR
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Recombinant cloning vector, SpcrtR gene cloned into the pEASY-Blunt vector
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Developed in this study
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pGEX-6p-1-SpcrtR
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Recombinant expression vector, SpcrtR gene cloned into the pGEX-6p-1 vector
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Developed in this study
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pACCAR16△crtX
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harbor the carotenoid biosynthetic genes for producing β-carotene
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KunMing institute of zoology, Chinese academy of sciences
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RT-PCR detection of Spirulina platensis gene expression
After 18 h of dark starvation, Spirulina platensis in the logarithmic phase was cultured in 12,000 Lx strong light for 0, 6, 12, 24 and 48 h at 120 rpm in a shaker to obtain algal solution. Total RNA was extracted from algal solution at different time points. The transcript levels of SpcrtR were analyzed using quantitative real-time PCR in an iCycler iQ5 Real-Time PCR instrument (BioRad, USA). The forward and reverse primers were: 5'-ATGTCGGAGGGCCAGAAGCT-3' and 5'-ATCCTCTGAGTTAGAC-3', respectively.
1.3. Determination of zeaxanthin in Spirulina platensis and bacteria
Carotenoid content in E. coli culture solution was determined by high-performance liquid chromatography (HPLC) on a C-18 reversed-phase (250*4.6 mm, 5 µm) column (Kromasil®, Sweden) with methanol/acetonitrile/dichloromethane (70:20:10, by vol.) as the mobile phase at a 2 mL/min flow rate. The wavelength of maximum absorption of zeaxanthin and β-carotene was 450 nm. The column was calibrated with available zeaxanthin and β-carotene (Sigma-Aldrich, USA).
Two mL of fresh Spirulina platensis solution in the logarithmic growth period were harvested by centrifugation at 1200 rpm for 10 min to remove supernatant, then organic extracts containing different proportions of organic solvents were added: petroleum ether-acetone (4:1 v/v), petroleum ether-acetone (1:4 v/v), methanol-acetone (3:1 v/v), methanol-acetone (1:1 v/v), or methanol-acetone (1:3 v/v), and then incubated in an ultrasonic cleaner for 15 min at 40ºC. The supernatant was centrifuged for 5 min at 1200 rpm to detect zeaxanthin content in Spirulina platensis by HPLC.
5 mL of co-transformed bacteria solution was cultured in a shaker at 37ºC, 150 rpm for 24 h, and then centrifuged at 12,000 rpm. After discarding the supernatant, 2 mL of acetone, ethanol, ethyl acetate, petroleum ether-acetone (1:1 v/v), methanol-acetone (1:3 v/v), ethyl acetate-ethanol (1:1 v/v) organic solvent were added for ultrasonic extraction of carotenoids at 40ºC for 15 min, followed by centrifugation for 5 min at 12,000 rpm to obtain supernatant. The OD value was detected using a spectrophotometer at 450 nm, and the zeaxanthin concentration in bacteria was detected by HPLC.
PCR amplification and sequence analysis
The Spirulina platensis FACHB-904 filaments were harvested at log phase (OD560 = 0.8-1.0), collected by centrifugation, and washed with cold water at least twice. After removing moisture from the algal groups, the cells were ground with liquid nitrogen for five freeze-thaw cycles in a mortar, then all filaments were transferred into a centrifuge tube, in accordance with the instructions of the RNeasy® Plant Mini Kit (QIANGEN, Shanghai, China) to extract the total DNA. The genomic DNA was then stored at -80ºC. SpcrtR was amplified by PCR using the forward primer, 5'-CGGGATCCATGTCGGAGGGCCAGAAGCT-3', which contained a BamHI site (underlined) and the reverse primer, 5'-CGGAATTCATCCTCTGAGTTAGAC-3', which contained an EcoRI site (underlined). The PCR products were cloned into pEASY-Blunt vector (Takara Biotechnology) at 16ºC overnight, and transformed in E. coli DH5α sensory state (TIANGEN, Beijing, China). The recombinant vectors carrying the PCR products were extracted from the E. coli transformants and purified. The gene identification was conducted by Beijing Genewiz Institute-Suzhou.
Bioinformatics and homology analysis
The homology analysis and comparison of the amino acids encoded by SpcrtR and other SpCRTR protein sequences was conducted using the BLAST program from NCBI and DNAMAN, respectively. To deduce the evolutionary relationships among sequences, the neighbor-joining(NJ) method was used to perform phylogenetic analysis in MEGA 5.0. The basic characteristics of the amino acid sequence encoded by SpcrtR were analyzed using ProtParam software (http://web.expasy.org/protparam/). The transmembrane regions of SpcrtR encoded protein was predicted using the TMH MM 2.0 server (http://www.cbs.dtu.dk/services/TMH mM/).
Expression of SpcrtR in E. coli
The purified PCR product was double digested with BamHI and EcoRI, and then inserted into the same sites in pGEX-6p-1 (ampicillin resistance) to construct pGEX-6p-1-SpcrtR. The plasmid pGEX-6p-1-SpcrtR was purified and ligated with the plasmid pACCAR16ΔcrtX (which harbors the carotenoid biosynthetic genes for producing β-carotene and chloramphenicol resistance) and co-transformed into competent cells of the E. coli DH5α host for synthesis of zeaxanthin.
The pACCAR16ΔcrtX and pGEX-6p-1SpcrtR were co-transformed into E. coli, and an empty plasmid pGEX-6p-1 and the pACCAR16ΔcrtX were co-transformed into E. coli as the control, these were spread on an LB plate containing 50 µg/mL of ampicillin and 34 µg/mL chloramphenicol and incubated for 10 h at 37ºC. Then, a single colony was picked and cultured in 5 mL LB medium for 10 h at 37ºC in a shaking incubator (200 rpm/min). The cells were induced with 0.4 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) when the OD600 of the culture had reached about 0.6, and these cultures were further shaken in the dark at 28ºC overnight. Cells were harvested and analyzed by SDS-PAGE and protein expression was confirmed by western blot analysis.
SDS-PAGE and western blot analysis
The extracted cells were combined with 100 µL protein extraction solution and heated in a metal bath (Thermo Fisher, China) at 95ºC for 5 min. The samples were then centrifuged at 12,000 rpm for 2 min, and the supernatant was collected to obtain protein solution. The expression level and molecular mass of the CRTR protein were determined by SDS-PAGE analysis.
The SDS-PAGE gel was transferred to a new polyvinylidene difluoride membrane (PVDF, Bio-Rad, USA) by a wet film transfer device (Bio-Rad, USA), and the membrane current was set at 300 mA for 40 min. After the membrane was transferred, the protein membrane was put into Tris-buffered saline (TBS) andrinsed for 1–2 min to fully wash the transfer membrane liquid. The membrane was then moved to the plate containing the sealing liquid, and decolorized at room temperature with shaking for 1 h. Afterwards, the PVDF membrane was incubated for 3 h at room temperature with anti-gsttag antibodies (Qiagen, USA). The membrane was washed three times with TBS-0.05% Tween 20 (TBST), then added into TBS liquid, diluting the secondary antibody for 1 h at room temperature. The protein band was detected by DAB solution (Sigma, USA) after three washes.
Zeaxanthin accumulation in different conditions
Four parts of 50 mL medium were measured into a 100 mL triangular flask, and 1% of the activated recombinant strain was inoculated in LB medium containing different carbon sources(1% glucose, maltose, sucrose and glycerol), different nitrogen sources (1% NH4Cl, (NH4)2SO4, urea and ammonium acetate) and different metal ions (1% K2HPO4, CaCl2, MgSO4 and Na2HPO4). Fermentation culture was conducted for 24h at 37ºC with 150 rpm shaking. Samples were extracted by optimal organic solvents, and zeaxanthin yield was detected by HPLC.
Statistical analyses
All results were expressed as mean ± SD. Prism 6.0 software (GraphPad Software, La Jolla, CA, USA) was used for data analysis.