20.109(F11): Research Proposal Brian Djaja and Robin Yeo

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For our research proposal, we are interested in optimizing a cyanobacterial system for biofuel production. Given the worldwide energy crisis and the recent interest in alternative energy sources, biofuel production by plants, microbes and cyanobacteria have begun to receive more attention as a potential solution (Lu, 2010). Our group is interested in the use of cyanobacteria because they combine the ease of genetic manipulation seen in bacterial systems with the phototrophic capabilities of plants. Historically, attempts to create cyanobacterial systems that harvest light and CO2 for biofuel production have suffered from low yield compared to other means of producing biofuels; we hope to design an experiment that could increase the efficiency of a cyanobacterial system's ability to produce biofuel. Of the possible biofuels that can be produced by biologically engineered cyanobacteria, we chose to pursue the production of 1-butanol since there remains much to be done in the optimization of 1-butanol production by cyanobacteria. We also considered trying to optimize a cyanobacterial system to produce bioethanol (Deng & Coleman, 1998) more efficiently but we found that Joule Unlimited obtained a patent in July 2011 to do just that, obtaining more than 90% direct conversion of CO2 to bioethanol by genetic engineering of their cyanobacterial system!

Just recently, a paper was published describing the first genetically engineered cyanobacterial system that produced 1-butanol from CO2 and light (Lan & Lia, 2011). One reason the design of this system has been so challenging is that the expression of the metabolic enzymes in the CoA-dependent pathway necessary for 1-butanol production is derived from anaerobes and yet the cyanobacterial system produces oxygen through its phototrophic process which inhibits the 1-butanol production pathway. Other problems limiting the efficiency of 1-butanol production include competing metabolic pathways, insufficient enzymatic activity in the 1-butanol production pathway and limited reduced carbon storage in cyanobacteria for 1-butanol production. Some of these problems are addressed by Lan & Lia in their paper but of course the efficiency of biofuel production in their system remains low compared to the commercial standard.

We hope to design an experiment to tackle the problem of oxygen sensitivity in cyanobacterial systems since this appears to be one of the major limiting factors to increased efficiency of 1-butanol production. One solution to this problem would be to optimize growth conditions such that the cyanobacteria are cultured in a tightly controlled anaerobic environment with a means to quickly diminish the levels of oxygen as they are produced by photosynthesis. Another more interesting solution to the problem of oxygen sensitivity would be to genetically engineer this same pathway of 1-butanol production in green or purple sulfur bacteria instead of the cyanobacterial strain used by Lan & Lia. Green and purple bacteria perform anoxygenic photosynthesis using hydrogen sulfide instead of oxygen as the ultimate reducing agent thus avoiding the problem of enzymatic inhibition by high oxygen levels produced through photosynthesis. We could design plasmids to integrate the metabolic enzymes necessary for the 1-butanol production pathway into a green or purple sulfur bacterial host's genome and then optimize the new system for maximum production of 1-butanol.

References:

Ethan I. Lan, James C. Liao, Metabolic engineering of cyanobacteria for 1-butanol production from carbon dioxide, Metabolic Engineering, Volume 13, Issue 4, July 2011, Pages 353-363, ISSN 1096-7176, 10.1016/j.ymben.2011.04.004. (http://www.sciencedirect.com/science/article/pii/S1096717611000474)

Deng MD, Coleman JR (1999) Ethanol synthesis by genetic engineering in cyanobacteria. Appl Environ Microbiol 65:523–528. http://aem.asm.org/content/65/2/523.abstract

Xuefeng Lu, A perspective: Photosynthetic production of fatty acid-based biofuels in genetically engineered cyanobacteria, Biotechnology Advances, Volume 28, Issue 6, November-December 2010, Pages 742-746, ISSN 0734-9750, 10.1016/j.biotechadv.2010.05.021.(http://www.sciencedirect.com/science/article/pii/S0734975010000728)

http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=7,981,647.PN.&OS=PN/7,981,647&RS=PN/7,981,647

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