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Methanator

Project Description

Carbon dioxide (CO₂) is notorious for its major contribution to global warming, where one of the impacts brought to the ecosystem is its excessive solvation into the ocean in carbonate form, threatening marine life [1]. This year we would like to utilize and recharge these abundantly available CO₂ by converting to methane (CH₄), an important carbon source for fuel and bio-degradable plastic production. While there are naturally existing methane-generating microorganisms, the convertion involves multi-step metabolic reactions, not to mention that they can only survive in anaerobic environment thus diffcult to manipulate.

A recent research showed that a mutated form of nitrogenase from Azotobacter vinelandii, a nitrogen-fixing bacteria found in soil, has carbon fixation ability [2]. Yang et al. [3] demonstrated that by introducing 70^Ala and 195^Gln mutations on nitrogenase alpha subunit, the nitrogenase enzyme complex reduced CO₂ and CO₃^- to CH₄ instead of converting nitrogen to ammonia. This system provided a one-step reaction to convert CO₂ into CH₄ and other carbon compounds directly. However, since a large electron flux, thus energy, was wasted in producing molecular hydrogen (H₂) from proton during the reaction, we utilized a soluble hydrogenase complex from Aquifex aeolicus to recycle H₂ to proton. To further enhance the efficiency of carbon fixation process, we physically linked both nitrogenase and hydrogenase complexes with SH3 and PDZ ligand-domain pairs to accelerate the H₂ recycling.

To allow regulation of expression of these enzymes in A. vinelandii, we plan to build a T7 protein expression system in this diazotrophic bacteria. Given that this organism produces more nitrogenase complex, which includes the structural gene nifH, in the absence of ammonia [4], we utilized its strong nitrogen-derepressible nifH promoter [4] to control T7 RNA polymerase expression. nifH promoter appeared to be stronger than its counterpart, lacUV5 promoter, in E. coli ([5] and characterization data of iGEM Biobrick K568003). Our system may provide an alternative platform for protein expression, as it is compatible to existing T7 promoter-driven constructs. Expression of enzymes along a particular metabolic pathway becomes possible as stable genome integration of DNA up to several MBps could be done in A. vinelandii [6]. Moreover, we only need ammonia to repress the expression instead of using expensive IPTG in the counterpart, thus lowering the expense.

To sum up, two goals will be achieved: to create a carbon fixation system using a combination of bacterial nitrogenase and hydrogenase enzyme complexes to convert CO₂ to CH₄, and to develop a novel nitrogen-regulated T7 protein expression system.

References

1.    Baldocchi, D., Valentini, R., Running, S., Oechel, W., And Dahlman, R. (1996) Strategies for measuring and modelling carbon dioxide and water vapour fluxes over terrestrial ecosystems. Global change biology 2, 159-168

2.     Seefeldt, L. C., Yang, Z. Y., Duval, S., and Dean, D. R. (2013) Nitrogenase reduction of carbon-containing compounds. Biochim Biophys Acta 1827, 1102-1111

3.     Yang, Z. Y., Moure, V. R., Dean, D. R., and Seefeldt, L. C. (2012) Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase. Proc Natl Acad Sci U S A 109, 19644-19648

4.      Hamilton, T. L., Ludwig, M., Dixon, R., Boyd, E. S., Dos Santos, P. C., Setubal, J. C., Bryant, D. A., Dean, D. R., and Peters, J. W. (2011) Transcriptional profiling of nitrogen fixation in Azotobacter vinelandii. J Bacteriol 193, 4477-4486

5.    Curatti, L., Brown, C. S., Ludden, P. W., and Rubio, L. M. (2005) Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii. Proc Natl Acad Sci U S A 102, 6291-6296

6.    Renaud, C. S., Pasternak, J., and Glick, B. R. (1989) Integration of exogenous DNA into the genome of Azotobacter vinelandii. Archives of microbiology 152, 437-44

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