Strains, plasmids construction and culture condition
elongatus UTEX 2973 and E. coli BL21(DE3) were respectively engineered and applied to the construction of the artificial consortium system. The essential genes for sucrose metabolism, permease coding gene cscB, invertase coding gene cscA and fructokinase coding gene cscK were derived from E. coli W [53], while the malonyl-CoA reductase gene mcr was from Chloroflexus aurantiacus [70]. Super strong promoter Pcpc560 was used to direct the gene expression in S. elongatus UTEX 2973 [71]. All plasmids are prepared in E. coli DH5α. Sucrose hydrolysis system was integrated into pET-30a in E. coli with kanamycin resistance, and mcr was integrated into pACYC184 under the constitutive promoter PJ23100 in E. coli with spectinomycin resistance, respectively.
elongatus UTEX 2973 and resulting engineered strains were cultivated in BG-11 medium (pH7.5) under a light intensity of about 100 μmol photons m−2s−1 in an illuminating shaking incubator (HNYC-202T, Honour, Tianjin, China) at 130 rpm and 37°C or on BG-11 agar plates in incubator (SPX-250B-G, Boxun, Shanghai, China) [72]. In order to maintain the stable phenotype of sucrose secretion, appropriate antibiotic(s) were added when necessary. E. coli strains were grown on LB medium or agar plates with appropriate antibiotic(s) added to maintain plasmids at 37°C in shaking incubator (HNY-100B, Honour, Tianjin, China) at 200 rpm or in an incubator, respectively. All strains used in this study were listed in Table 1.
Conjugation of S. elongatus UTEX 2973
Constructs were delivered into S. elongatus UTEX 2973 through conjugation [73]. E. coli HB101 harboring pRL443 and pRL623 (named “helper”) and E. coli DH5α harboring the plasmid with target gene were cultivated overnight and then transferred separately into fresh liquid LB medium with appropriate antibiotic(s) at 1:50 ratio. When both strains grew to exponential phase (OD600=0.3~0.5), 10 mL cells of each strain were collected by centrifugation and washed with fresh LB medium three times to remove all the antibiotics. Then 0.1 mL fresh LB was used to re-suspend each strain, and they were mixed together and incubated at 37℃ for 30 min. During this time, 10 mL of the S. elongatus UTEX 2973 cells at exponential phase (OD750≈1) were collected by centrifugation and re-suspended in 0.2 mL fresh BG11 medium. S. elongatus UTEX 2973 cells was mixed with the E. coli mixture mentioned above and incubated at 37℃ under light for 30 min. Then, the mixture was spread on BG11 agar plates which was then covered by sterile cellulose filters (0.45 μm pore size). The plates were incubated under light at intensity of approximately 100 μmol photons m−2s−1 for 24 h, and then the cellulose filters were transferred onto new BG11 agar plates with appreciate antibiotics [74].
Construction of artificial consortium system
Co-culture medium (named CoBG-11) was designed based on BG-11 medium and optimized for E. coli growth by supplementing 150 mM NaCl, 4 mM NH4Cl and 3 g/L 2-[[1,3-dihydroxy-2-(hydroxymethyl) propan-2-yl] amino] ethanesulfonic acid (TES). The pH value is adjusted with NaOH to 8.3. NaCl and NH4Cl were used to maintain the cell survival of E. coli, and NaCl was used as a stress inducer for sucrose accumulation in S. elongatus UTEX 2973, respectively.
Before two strains were cultivated together, S. elongatus UTEX 2973 was propagated in BG-11 at 37°C with appropriate antibiotics to exponential phase (OD750≈1.0), and then collected by centrifugation and inoculated into 25 mL CoBG-11 medium and grown at 30℃ for 48 h to OD750 of 0.5. E. coli was incubated in CoBG-11 with 1 g/L sucrose for 48 h and then collected by centrifugation, re-suspended with deionized water and inoculated into the 25 mL S. elongatus culture above at initial OD600 of 0.01.
Quantification of cyanobacteria and E. coli
For pure culture of S. elongatus UTEX 2973 and E. coli, cell density was measured at OD750 and OD600 using a UV-1750 spectrophotometer (Shimadzu, Kyoto, Japan), respectively. For co-culture, serial dilutions were made and solid LB agar plates were used to determine E. coli viability and cell number by colony-forming units (CFU) after 24 h incubation at 37°C. Cell number of S. elongatus UTEX 2973 was determined by hemocytometer under microscopy (BX43, Olympus, Shinjuku, Tokyo, Japan).
Determination of extracellular sucrose content
Supernatant of S. elongatus UTEX 2973 pure culture was collected and analyzed for sucrose content via a colorimetric Glucose-Sucrose Assay (Megazyme, Ireland) that employs high purity glucose oxidase, peroxidase and β-fructosidase (invertase). At pH 4.6, sucrose is hydrolyzed by invertase to D-glucose and D-fructose, and then free D-glucose is determined by converting to a red colored quinoneimine dye compound through the action of glucose oxidase and peroxidase at pH 7.4, and employing p-hydroxybenzoic and 4-aminoantipyrine. Measurements were conducted at 510 nm.
Quantification of 3-HP
3-HP concentration was quantified according to a previous method [37]. 3-HP standard of analytical purity was purchased from Tokyo Chemical Industry (Tokyo, Japan). The supernatant containing 3-HP was collected from the co-culture medium by centrifuging at 12,000 rpm for 2 min at room temperature (Eppendorf 5430R, Hamburg, Germany) and used for 3-HP analysis. Sample derivatization was carried out according to the two stage technique described previously [75]. GC-MS analysis was conducted on a GC-MS system-GC 7890 coupled to an MSD 5975 (Agilent Technologies, Inc., Santa Clara, CA) equipped with a HP-5MS capillary column (30 mm×250 mm id).
Quantitative real-time RT-PCR analysis
Approximately 4×106 cells of S. elongatus UTEX 2973 cells of pure or co-culture were collected by centrifugation at 12,000 rpm, 4°C for 1 min. The supernatant was removed and the cells were used for RNA extraction and RT-qPCR analysis using methods described previously [76]. The relative abundance of different mRNA molecules could be estimated using 2-△△CT [77].
Analysis of hydrogen peroxide (H2O2) concentration
H2O2 content in supernatant was analyzed via H2O2 Quantitative Assay Kit (Sangon Biotech, Shanghai, China). In the reaction, Fe2+ is oxidized to Fe3+ by H2O2 when pH is less than 7.0, and then Fe3+ generated combines with dye molecules to form claret-colored Fe3+-dye complex with maximum absorption wavelength at 560 nm or 595 nm, and the absorption value is directly proportional to the concentration of hydrogen H2O2 in cells.