Improved Biohydrogen Production With Expression Of Codon-optimized HemH And Lba Genes In Chloroplast Of Chlamydomonas Reinhardtii

Chlamydomonas reinhardtii, with simple structure, clear molecular genetic background, easy operation and low cost on culturation, especially with high activity of hydrogenase which can produce hydrogen using solar energy and water, is considered as the most potential algal species for development of biological hydrogen production in the future.Hydrogenase of C. reinhardtii has high catalytic activity, but particularly sensitive to oxygen which is the main byproduct of algal photosynthesis. Therefore, the process of hydrogen production and hydrogenase activity is very susceptible to oxygen inhibition and inactivation, which is the maximum obstacle for hydrogen production of C. reinhardtii. To improve the biohydrogen production of C. reinhardtii, the yield of oxygen must be reduced in C. reinhardtii cells to ensure the activity of hydrogenase. The most common way at present is to remove sulfur in the culture medium or by adding some of inhibitors to inhibit the activity of photosystem II. But at the same time it will also reduce the electronic source from the water photolysis process and result in low hydrogen production. Therefore, how to reduce the oxygen content in algal cells without affecting the electronic supply for hydrogenase is a key issue to increase hydrogen production of C. reinhardtii.Leghemoglobin (Lb) has high oxygen affinity and rapid turnover rate. It helps to reduce the oxygen concentration inside the nodule cells and ensures the oxygen-sensitive nitrogenase activity. No Lbs, no nitrogen fixation activities in the soybean nodules.We have tried to transfer soybean Lb protein genes lba and hemH-lba into the chloroplast of C. reinhardtii in our previous work. Since the expression of protein in the C. reinhardtii chloroplast has AT bias, in this experiment, the codon-optimized Lb genes lbac and hemHc-lbac were transfered into the chloroplast of C. reinhardtii in order to increase recombinant protein expression levels and hydrogen production. The contents and results of this thesis are as follows:1. The codon-optimized soybean Lb genes lbac and hemHc were synthezed in the company and the chloroplast expression vector cg401-1-hemHc-lbac with the design of the polycistronic hemHc and lbac genes was successfully constructed.2. The plasmid DNA of cg401-1-hemHc-lbac was transfered into C. reinhardtii chloroplasts with the particle bombartment method. Transformants were screened by the resistance of spectinomycin and the well-growth one was picked up and named as transgenic alga hemHc-lbac after screening.3. PCR assay based on DNA templates of the transgenic alga hemHc-lbac showed that the hemHc-lbac gene fragments had been heteroplasmicly integrated into the chloroplast genome DNA of C. reinhardtii. RT-PCR assay based on RNA templates of the transgenic algae showed that the hemHc and lbac genes transcripted successfully in algal chloroplasts.4. By the method of Western blotting, the recombinant hemHc and lbac proteins had been successfully expressed in the chloroplasts of transgenic algae hemHc-lbac under both normal culture condition and hydrogen production condition. On the fifth days, the expression of recombinant protein reached to the maximum level and the expression level of the codon-optimized recombinant proteins was 6.8 times of the non-optimzied one. The Western blotting result on the native gel electrophoresis showed that the two Lb subunits, hemH and Lba were in the same hybridyzation band with their own specific antibodies .5. By the quantitative real-time PCR assay, the transcriptions of hydrogenase genes in both codon-optimized and non-optimized transgenic algae were increased than those in the control algal strain cc849.6. The growth curves of the control algae 849 and transgenic algae hemH-lba, and hemHc-lbac showed that the growth of control algae 849 reached saturation with the maximum OD750 value of 3.2, while that of the transgenic algae hemH-lba was 3.5 and transgenic alga hemHc-lbac was 3.3. At saturation phase, the maximum chlorophyll contents of the transgenic alga hemHc-lbac and hemH-lba were all about 41mg / L, while it was about 35mg / L for the control algae 849. The results showed the growth of the transgenic algae was not inhibited. Moreover, their chlorophyll contents were increased slightly.7. Hydrogen production and oxygen consumption of both transgenic algae and the control alga were detected. The results showed that the hydrogen production of transgenic algae hemHc-lbac and hemH-lba were higher than that of the control algae nomatter under full argon condition or under the dark anaerobic condition.8. Heme was extracted with ether from both transgenic algae and scaned by using UV spectrophotometer. There was a small absorption peak at 550nm for both transgenic algae hemH-lba and hemHc-lbac, indicating that a trace heme might be synthesized.9. Respiratory rate and photosynthetic rate of the control and transgenic algae were measured. The results showed that net photosynthetic oxygen evolution rate of transgenic algae hemHc-lbac was lower than transgenic algae hemH-lba and both were significantly lower than that of the control algae. On the other hand, the respiration rates of both transgenic algae were higher than that of the control algae, particularly for that of the codon-optimized algae hemHc-lbac.