| Global warming, contributed largely by the increasing level of anthropogenic greenhouse gases(GHG) in the atmosphere, has received great concern worldwide. Carbon dioxide(CO2) is a major component of GHG. According to the statistics released by World Meteorological Organization(WMO), the level of atmospheric CO2 is rising alarmingly and has reached to above 400 ppm in 2015, approximately 43% higher than the pre-industrial level of 280 ppm. It is, therefore, urgent and imperative to develop low carbon technologies for CO2 mitigation. Up to now, many efforts have been made to develop technologies for CO2 capture and sequestration. Recently, microalgae-mediated biomitigation has garnered a surge of attention due to their superior ability in converting sunlight and CO2 into chemical energy such as lipids, proteins, starch and carotenoids. In addition, microalgae is of particular interest owing to their unique traits such as great photosynthetic efficiency, rapid growth, low cost and ease of culture for industrial integration. Hence, the microalgae-mediated biomitigation is considered as an efficient and economic approach, which may have a promising prospect in future. However, the CO2 fixation ability of microalgae still remains limited for practical use. Finding ways to overcome its inherent photosynthetic capacity is of substantial importance for promoting the CO2 fixation ability.In the present study, we used Chlorella vulgaris, a robust microalga with good CO2 fixation rate and tolerance to CO2, for our research. We have for the first time proposed the idea of genetic engineering of C. vulgaris for the substantially increased CO2 fixation capacity for CO2 biomitigation. An attempt was therefore made by overexpressing the key enzyme involved in the Calvin cycle for enhanced CO2 fixation rate. Briefly, we first developed a stable and efficient genetic system for C. vulgaris by using the reporter gene, enhanced green fluorescent protein(EGFP) gene. Then we cloned the plastid transit peptide(c TP) sequence from ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit gene(rbc S) and verified its function by subcellular localization. On this basis, we cloned the key enzyme, fructose 1,6-bisphosphate aldolase gene(FBA) involved in the Calvin cycle, and introduced it into the C. vulgaris genome. The FBA gene has been successfully expressed in the plastids of C. vulgaris, which significantly enhanced the cell growth and photosynthesis(CO2 fixation rate). Besides, we also studied the possible mechanisms for increased photosynthesis in transformants caused by overexpressed FBA in plastids. Our results will, to some extent, lay a good foundation for studies concerning genetic engineering and metabolic engineering of Chlorella, and on the other hand will provide a valuable implication for further possible success of substantially increased CO2 fixation capacity in microalgae and plant in future. Our results could be summarized as follows:(1) The antibiotic sensitivity spectrum of C. vulgaris was evaluated, and the npt II gene was selected as a dominant selectable marker for genetic transformation. First, the sensitivity of C. vulgaris to four common antibiotics, including spectinomycin, geneticin(G418), kanamycin and chloramphenicol, was investigated in agar plates. We found that C. vulgaris was highly sensitive to G418, with a median lethal concentration(LC50) value of as low as 11.74 μg /m L. To test the efficiency of selected antibiotic in liquid media, C. vulgaris was then exposed to G418 at various concentrations. We found that a strong growth inhibition(over 90%) was achieved when G418 concentration was increased to 30 μg/m L or more. Therefore, 30 μg/m L of G418 was effective for the selection of transformants. Since the npt II gene encoding neomycin phosphotransferase confers excellent resistance to G418, it was therefore chosen as a dominant selectable marker for subsequent C. vulgaris transformation.(2) A stable and efficient genetic system for C. vulgaris was developed by using the reporter gene, EGFP gene. The EGFP gene was cloned and introduced into C. vulgaris genome by using a polyethylene glycol(PEG)-mediated method, giving a transformation efficiency of 356 ± 30 cfu per μg vector DNA. Molecular characterization(PCR, southern blot and RT-PCR) and live-cell fluorescence microscopy demonstrated that the EGFP gene was stably integrated into C. vulgaris genome and expressed in the cytoplasm of transformed cells. Besides, we found that the EGFP was also detectable in transformants after 16 consecutive subcultures by genetic stability test. Therefore, we have developed a stable and efficient genetic system for C. vulgaris.(3) The plastid transit peptide(c TP) sequence from rbc S gene was cloned and functionally analyzed, and the feasibility of expressing nuclear-encoded transgene in plastids of C. vulgaris was proved. The c TP sequence from rbc S gene of C. vulgaris was predicted and synthesized, and fused to the N-terminal of EGFP gene and then introduced into C. vulgaris. By fluorescence microscopy, we found that transformed cells with fused c TP::EGFP gene exhibited green fluorescence in plastids, while transformed cells with only EGFP gene exhibited green fluorescence in cytoplasm, indicating that the c TP sequence functions targeting the chimeric gene into the plastids and that it was feasible to express nuclear-encoded transgene in plastids of C. vulgaris. Also, the success use of EGFP in C. vulgaris efficiently expands the use of fluorescent protein technology in microalgal molecular biology.(4) The FBA gene was overexpressed in C. vulgaris, and the cell growth and photosynthesis were significantly increased. We first cloned the FBA gene from the model organism of cyanobacteria, Synechocystis sp. PCC6803. It was fused to the c TP transit peptide sequence, and then introduced into C. vulgaris by using the PEG-mediated transformation method. Molecular characterization(PCR, southern blot, RT-PCR and western blot) showed that the FBA gene was successfully integrated into the genome of C. vulgaris transformants and expressed in the plastids. Besides, we found the aldolase activities of both transfomants #3 and #5 were 1.27-1.30-fold significantly higher than those of WT cells(p<0.05). Also, their biomass at the middle and late culture periods both significant increased, with both oxygen evolution rate 1.18-1.21-fold and CO2 fixation rate 1.15-1.18-fold higher than that of WT, respectively(p<0.05). This indicated that the increased FBA in plastids could effectively increased the growth and photosynthesis of C. vulgaris.(5) The possible mechanisms for increased photosynthesis by overexpressed FBA in transformants were also studied by comparison of physiochemical traits of transformants and wild type(WT). Compared with WT, we found that both transformants #3 and #5 had a significantly higher chlorophyll concentration(p<0.05). By using chlorophyll fluorescence analysis, we also found that both transformants #3 and #5 had a higher photochemical quenching coefficient(q P) and quantum yield of photosystem II electron transport(ΦPSII)(p<0.05), but a significantly lower nonphotochemical quenching coefficient(NPQ) than WT(p<0.05), indicating a faster energy transport in photoreaction of transformants. In addition, we also compared the gene expression level and enzyme activity of key enzymes involved in the Calvin cycle in transformants and WT. We found that both transformants had a higher gene expression levels of Rubisco and Rubisco initial activity than that of the WT cells. But we found that the enzyme activities of other key enzymes remained no significant changes although their gene expression level increased significantly. Taken together, we may infer that the enhanced photosynthesis of transformants caused by overexpressed FBA in plastids may be attributed to the increased energy transport in photoreaction and the accelerated carbon turnover rate in the Calvin cycle that was caused by increased activation of Rubisco induced by enhanced level of Ru BP. |
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