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분야
docx2) Uptake Hydrogenase
3) Bidirectional Hydrogenase
1. Uptake Hydrogenase
1) Definition of Uptake Hydrogenase and Mechanism of Hydrogen Production
2) Limitation of Hydrogen Production
3) Our Group’s idea
4) Method
2 .Bidirectional hydrogenase
1) Definition of bidirectional hydrogenase
2) Production of hydrogen from bidirectional hydrogenase
3) Limitations of hydrogen production by bidirectional hydrogenase : Oxygen sensitivity
4) Our idea
References
1) Definition of Uptake Hydrogenase and Mechanism of Hydrogen Production
Uptake hydrogenases are Ni-Fe enzymes that catalyze the conversion of hydrogen gas to hydrogen ions and electrons. They are found in the heterocysts and not in the vegetative cells of nitrogen-fixing strains of cyanobacteria. They remove the hydrogen gas produced by nitrogenase when they carry out nitrogen fixation.
Uptake hydrogenase is encoded by hup genes. It is a heterodimeric enzyme with at least 2 subunits, HupL and HupS. HupL is a large subunit of size 60kDa containing the active site while HupS is the small subunit of size 35kDa which is involved in electron transfer. HupL gene is expressed only in heterocysts of cyanobacteria. In vegetative cells, hupL gene is interrupted by a 9.5kb element. Therefore functional HupL subunits are not able to be produced in vegetative cells. However, a recombinase XisC that is within the element, excises the element when there is differentiation of a vegetative cell to a heterocyst. XisC excises the element, leding to the formation of a complete hupL gene. This way the hupL gene is properly transcribed and translated in heterocysts and HupL subunit is formed and functional in heterocysts.
Based on present research findings, it can be seen that hydrogen production in cyanobacteria can be increased by blocking the function of uptake hydrogenase.
2) Limitation of Hydrogen Production
When the 9.5kb element is excised, the heterocyst form of the cell will produce uptake hydrogenase. Therefore we must inhibit the 9.5kb element that has the XisC unit or just the XisC enzyme.
3) Our Group’s idea
a) Approach 1
Our first approach is to prevent the formation of complete hupL gene by mutating xisC gene. Functional XisC recombinase and HupL subunit will not be produced. Without the HupL subunit, there will be no functional uptake hydrogenase and the active site for converting hydrogen gas will not be present. The function of HupL is more crucial than HupS. This is the reason for targeting HupL.
b) Approach 2
Our second approach is to target HupL gene directly by mutating it. This is done just in case the mutation of xisC is not sufficient to prevent formation of hupL subunit.
For two of our approaches, 10 bases are deleted for the mutations to take place
4) Method
The method we are going to use is “Homologous recombination” to knock out hupL and xicS genes. Homologous recombination is type of genetic recombination which DNA sequences are exchanged between two similar or identical strands. It can be used for deletion of the target gene.
First, design and fabricate the similar DNA construct you want to insert into the chromosome in place of the wild-type allele. This constructs may contain selective marker that help isolate the knockout cells. Insert the reporter genes into the bacteria. Treat selective agents and isolate the knockout cells.
2 .Bidirectional hydrogenase
1) Definition of bidirectional hydrogenase
Unlike uptake hydrogenase, bidirectional hydrogenases are helpful in hydrogen production. The biological role of bidirectional hydrogenase is thought to control ion levels in the organism. Bidirectional hydrogenase is associated with the cytoplasmic membrane and likely
• Savage F, Afonso B, Chen Anna, Silver Pamela: Spatially Ordered Dynamics of the Bacterial Carbon Fixation Machinery. Science 307, 2010
• Liang Yi, Wu Xiabing, Gan Lihui, Xu Huijuan, Hu Zhong, Long Minnan: Increased biological hydrogen production by deletion of hydrogen-uptake system in photosynthetic bacteria. Microbiological Research 164 (2009) 674-679
• Yu Jian-Wei, Price G, Song Lirong, Badger M: Isolation of a Putative Carboxysomal Carbonic Anhydrase Gene from the Cyanobacterium Synechococcus PCC7942. Plant Physiol. (1002) 100, 794-800
• Ghirardi M, Posewitz M, Maness P, Dubini A, Yu J, Seibert M: Hydrogenases and Hydrogen Photoproduction in Oxygenic Photosynthetic Organisms. Annu. Rev. Plant Biol. 2007. 58:71-91
• Dutta D, De Debojyoti, C Surabhi, Bhattacharya S: Hydrogen production by Cyabacteria. Microbial Cell Factories 2005
• Savage F, Afonso B, Chen Anna, Silver Pamela: Spatially Ordered Dynamics of the Bacterial Carbon Fixation Machinery. Science 307, 2010
• Liang Yi, Wu Xiabing, Gan Lihui, Xu Huijuan, Hu Zhong, Long Minnan: Increased biological hydrogen production by deletion of hydrogen-uptake system in photosynthetic bacteria. Microbiological Research 164 (2009) 674-679
• Yu Jian-Wei, Price G, Song Lirong, Badger M: Isolation of a Putative Carboxysomal Carbonic Anhydrase Gene from the Cyanobacterium Synechococcus PCC7942. Plant Physiol. (1002) 100, 794-800
• Ghirardi M, Posewitz M, Maness P, Dubini A, Yu J, Seibert M: Hydrogenases and Hydrogen Photoproduction in Oxygenic Photosynthetic Organisms. Annu. Rev. Plant Biol. 2007. 58:71-91
