Team:ULV-LC-CV/Description
Project Description
♦Background♦
The increasing demand of fossil-based energy, such as petroleum and diesel, has negative consequences on the environment and health, creating the need for an alternative energy. The increasing demand of fossil-based energy, such as petroleum and diesel, has led to an increase in fracking, consequently creating a negative impact on the environment and to the communities nearby. In order to reduce the strain that fracking for fossil fuels cause, researchers are looking towards microalgae to produce biofuels. Microalgae are the ideal chassis for biofuel production because of their photosynthetic ability, the variety of biomass they produce, as well as the ease and the rate at which they can be manipulated and grown. Microalgae naturally produce free fatty acids (FFAs), which serves as the precursors to biofuel products such as alkanes, fatty alcohols, and fatty acid esters.
♦Biodiesel♦
Biodiesel, which is mainly composed of fatty acid methyl esters (FAMEs) from vegetable oil, is renewable, reduces lifecycle carbon emissions, and provides the highest energy balance compared to other commercially available fuels. Currently, FAMEs are synthesized from vegetable oil and more recently from E. coli through a process called transesterification, where a methyl donor, such as methanol, is used along with an alkaline catalyst to speed up the reaction. Transesterification is the most costly step of preparing FAME’s from FFA, largely contributing to the carbon footprint as well as expenses. Preparation of FAMEs from FFA through the process of transesterification has the largest contribution to its high cost and unfavorable carbon footprint. In order to reduce the cost of FAME synthesis, microorganisms can be engineered to overproduce FFAs and use those FFAs toward FAME biosynthesis through the use of methyltransferase enzymes.
♦Previous Work♦
In vivo production of FAMEs in E. coli was achieved through direct methylation of FFAs by S-adenosyl-L-methionine (SAM) dependent bacterial methyltransferase (Nawabi et al., 2011). However, the methyltransferase used only targeted 3-OH FFAs, which is only a small portion of the FFAs produced by the cell. Sherkhanov et al., 2016 furthered improved in vivo production of fatty acid methyl esters in E. Coli, by introducing a Drosophila melanogaster Juvenile Hormone Acid O-methyltransferase (DmJHAMT), which targets a wider range of FFAs, for catalyzing the transesterification reaction.
♦Our Project♦
We propose a whole cell factory which will prepare FFA’s to FAME’s in Cyanobacteria. Cyanobacteria is the ideal chassis for in vivo FAME production because it produces longer chain FFAs (14C-22C) which are ideal for biodiesel production and is able to provide its own energy. We plan on introducing DmJHAMT gene into a strain of heterotrophic cyanobacteria, Synechococcus elongatus PCC 7942, that has been engineered to overproduce free FFA’s (Ruffing et al., 2012).
Another cost associated with FAME production in cells is extraction. In order to reduce this cost, we researched ways to lyse the cells in vivo to release FAME into the solution. However, to optimize the FAME produced by cyanobacteria, we would need to activate lysis at a certain cell density.
To do so, we decided to implement a quorum sensing system in Synechococcus. By varying the strength of the promoter used to produce signaling molecules in the lux quorum sensing system, we can change the optical density at which cell lysis occurs. Theoretically, we could select a promoter that would lyse the cells at the optimal optical density.
To get closer to this theoretical, we plan on using a reporter gene to correlate promoter strength to optical density at which the rate of change of reporter gene expressed is the highest. We plan on expressing our system in both E. coli and Synechococcus. Characterization of this system can help any researchers interested in activating or deactivating gene expression at specific cell densities.
Design
♦Methods♦
♦Parts♦
Results
♦Diagnostic Gel♦
Constructs were digested with EcoRI and run on an agarose gel. The expected band sizes were seen.
Constructs were digested with EcoRI and PstI and run on an agarose gel. The expected band sizes were seen.
♦Characterization Experiments♦
GFP Expression in E. coli. Transformed E. coli containing various constructs were analyzed by FACS. Mean FL-1 fluorescence was graphed. Untransformed E. coli were used as a control. Data was analyzed using Kruskal-Wallace One-way ANOVA test. Error Bars: +/- 2 SE.
GFP Expression of E. coli Containing pLRDmJ Treated with Oleic Acid. Transformed E. coli containing construct pLRDmJ treated with 1 mM oleic acid was analyzed by FACS. Mean Fl-1 fluorescence was graphed. Untreated E. coli containing construct pLRDmJ was used as a control. Unpaired T-test produced a p-value of 0.017. Error Bars: +/- 2 SE.
References
Nawabi, P., Bauer, S., Kyrpides, N., & Lykidis, A. (2011). Engineering E. coli for biodiesel production utilizing a bacterial fatty acid methyltransferase.Applied and environmental microbiology, AEM-05046.
Ruffing, A. M., & Jones, H. D. (2012). Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942.Biotechnology and bioengineering, 109(9), 2190-2199.
Sherkhanov, S., Korman, T. P., Clarke, S. G., & Bowie, J. U. (2016). Production of FAME biodiesel in E. coli by direct methylation with an insect enzyme. Scientific reports, 6.