| The purpose of this dissertation is to construct a genetically engineered microorganism for CO2 fixation and to enhance its ability to fix CO2. Therefore, the pollution of CO2 can be decreased, so that the global environmental problem, green house effect, can be solved.The genetically engineered microorganism (GEM) for C02 fixation is constructed by introducing a recombinant Rhodopseudomonas expression plasmid pHM-CBBM, containing the form II ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) gene (cbbM), into the Rhodopseudomonaspalustris (R. palustris) ASI.23 52The growth characteristics of the parent strain AS1.23 52 are studied, in order to lay a foundation for the construction of the GEM. The strain AS1.2352 is resistant to ampicillin, and sensitive to tetracycline and kanamycin. The optimal anaerobic growth condition for the strain AS1.2352 is obtained as follows: illumination intensity 2000LX, temperature 30℃, pH 7.5. Under photoheterotrophic condition, the strain AS1.2352 can grow using some substrates as nutrition, including peptone, beef extract, yeast extract, malate, glucose and sodium acetate, among which peptone is the best substrate of the study and the optimal concentration of the substrate is 0.4-0.5%. Under photolithoautotrophic condition, the optimal growth condition for the strain AS 1.2352 is obtained as follows: gas phase, CO2:H2=1:9; liquid phase, 0.5g/LNH4Cl, 5g/LK2HP04, initial pH 8.5.The measurement of RubisCO enzyme activity with gas chromatography is established. Compared with the spectrophotometer method, the gas chromatography method is simple, rapid, accuracy, and better repeated. Crude RubisCO enzyme is extracted from the strain AS 1.2352 cells under photolithoautotrophic growth, and the purified RubisCO is obtained. The purification fold is 14.9, the recovery rate is 3.5%, and the specific activity of RubisCO is 2.347umolCO2/mg prot.min, whose subunit molecular weight is about 50kDa.On the basis of plasmid pBMP-2, a 3.0-kb DNA fragment from R. palustris DH1 is subcloned and sequenced. Sequencing of the DNA fragment revealed an open reading frame encoding 461 amino acids, homologous to known cbbM genes, with a ribosome binding site (RBS) upstream of cbbM and a terminator downstream of cbbM, without promoter. Then cbbM gene is obtained from genome of the strain AS1.2352 using PCR technique. Sequence of the PCR fragment reveals an open read frame encoding 461-amino-acid polypeptide showed 99% sequence identity to form II RubisCO from thesame species reported by Genbank, and showed 100% identity with corresponding sequence of R. palustris DHL The cbbM gene is expressed in E. coli, analyzed with SDS-PAGE, the molecular weight of the expressed protein is about 50kDa.On the basis of plasmid pBMP-2, the sequence required for plasmid replication in R. palustris, pMG101, is cloned into the plasmid pHSG299, constructing a Rhodopseudomonas/E. coli shuttle vector, pHSG-MG, which can replicate in E. Coli and R. palustris, respectively.Using PCR technique, the promoter of pckA gene from R. palustris is introduced the upstream of cbbM. Then, the target fragment, including the promoter, RBS, cbbM and the terminator, is cloned into the Rhodopseudomonas/E. coli shuttle vector pHSG-MG, constructing a Rhodopseudomonas expression plasmid, pHM-CBBM. The recombinant plasmid pHM-CBBM is transformed into R. palustris AS 1.23 52 by electroporation to construct a genetically engineered microorganism (GEM) for CO2 fixation. The stability of pMG-CBBM in the GEM is 80% after 100 generations.Under photoheterotrophic and photolithoautotrophic conditions, the growth of the GEM is compared with the parent strain AS 1.2352. The results show that the cbbM gene is expressed in the GEM under photolithoautotrophic growth but not under photoheterotrophic growth. The ability to fix CO2 of the GEM is as twice as that of the parent strain AS 1.2352. At the initial concentration of 10% CO2 (under optimal condition), the GEM growth kinetic formula is deduced. In addition, some characteristics of t...
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