UCSC 2014: Microbial Engineering of Haloferax volcanii for the production of an alternative biofuel Chris Lee, Kevin Sweeney, Jazel Hernandez, Dominic Schenone
Energy Consumption ● OECD - Organisation for Economic Co-operation and Development ● Non-OECD countries will increase 90 percent in energy demand by 2040 ● OECD countries increase 17 percent ● What type of liquid energy/fuel? http://www.eia.gov/forecasts/ieo/world.cfm
Climate Change: Carbon Cycle ● The geologic carbon cycle takes between 100-200 million years to move carbon dioxide from the atmosphere to energy-rich fossil fuels ● Biofuels such as butanol considerably shorten this cycle down to years as opposed to millions of years (7)
Shortening the Carbon Cycle: Butanol ● Not water soluble, making it fit into current shipping and engine infrastructure without corrosion issues ● Energy density is near that of gasoline ● Switchgrass as a source of cellulose (4)
Starting Substrate - Cellulose Cellulose: ● Most abundant organic compound on Earth ● Composed of linked glucose molecules, an energy currency in biochemistry ● Agricultural waste is an excellent source But… ● Difficult to isolate from lignin proteins in plants ● Requires “cooking” in high temperature, high salt conditions to access (Ionic Liquids) ● (Source 1/Source 5)
A solution: pretreatment in hot ionic liquids Hydrolyzes hemicellulose, solubilizes lignin Solubilizes cellulose, freeing it from other compounds Requires heat energy, water, and salts Our plan: Incorporate a halophile into the pretreatment process, which can be engineered to digest (2) cellulose into biofuel
Haloferax volcanii ● Halophile, facultative anaerobe ● Found in the Dead Sea; optimum growth at 45°C and 18% salinity; chemoorganotroph ● 4-5 hr. doubling time ● Well-studied model organism for archaea ● But how are we going to use Volcanii? (3)
Hijacking a pathway ● Synthetic Pathway assembled bioinformatically ● The Fatty Acid cycle normally continues to add hydrocarbons to the starting chain of acetyl CoA ● The enzymes responsible -> Acyl-CoA dehydrogenase ● To accumulate 4 carbon product(Butanoyl CoA) we knock out the ACD responsible for the 4 carbon to 6 carbon step
Knockout Construct ● 400 bp upstream and downstream homologous sequence (Acd2/4) ● 800 bp upstream and downstream sequence (Acd3) ● 50bp from the 3’ and 5’ ends of each gene Upstream Homologous Seq Nonsense Base Pairs Downstream Homologous Seq
Performing Acyl-CoA Dehydrogenase Gene Knockouts Recombinant Plasmid Vector ● used to perform the various Acyl- CoA dehydrogenase gene knockouts ● assembled this plasmid from the previous plasmid PTA963(Thorsten Allers), via PCR and Gibson Cloning, both have a uracil gene (hdrB) ● Each gene deletion construct was then put separately into the plasmid vector via linearization PCR of PSCKiKo and Gibson cloning of that linear piece with the gene deletion contructs.
Pop-in Pop-out Method ● Method developed by Thorsten Allers to knockout and pop in genes(Source 2) ● Transform Volcanii with a nonsense sequence in middle of desired gene (The Plasmid Vector) ○ Insert has a gene to make uracil ● “Pop-In” phase ○ After first recombination the cells will be Uracil Prototrophs ● “Pop-Out” phase ○ After second recombination there will be four products , Deletion Mutant Uracil auxtroph/prototroph, Wild Type Uracil auxotroph/prototroph ---> Select with 5-FOA, then colony picks
acd Knockout Gels 3kb 3kb 1kb 1kb
Aldehyde Dehydrogenase Overexpression ● After the 4 carbon product has been accumulated via acd gene deletion Overexpress the gene responsible for the following step. ● Create an overexpression plasmid vector, from PSCKiKo, instead inserting the fully functioning gene(aldy5) responsible for that step ● Butanoyl CoA → Butanal
Gas Chromatography ● Gas chromatography is the best way to check for small, organic molecules. ● First need to look for butyric acid instead of butanol ○ More likely to accumulate because it is part of the pathway
Preparation of Culture Samples ● Culture samples first need to be prepared ● We used a liquid-liquid extraction of culture supernatant with ethyl acetate ● After extracting, we ran on an acid-treated wax column. This is the 1.0% Butyric acid in EtOAc standard
Current Work -The project will continue during the year under the supervision of Dr. Bernick -Currently checking for accumulation of substrates in our acd knockouts ● Senior Thesis projects ● Senior Design groups -The iGEM course will pick up the project over summer
Synthetic Biology at UCSC: iGEM Class ● 2-quarter course taught by Dr. David Bernick ● 2 unit class in spring (BME 181) ● 10 unit class in summer (BME 188) ● Satisfies exit requirements for BME ● Will count as an elective/thesis for the physical sciences
Acknowledgments ● University of California, Santa Cruz ● Dean’s Office, Baskin School of Engineering, University of California Santa Cruz ● Undergraduate Research Funding Scholarship, Crown College, University of California Santa Cruz ● Dean’s Office, Division of Physical and Biological Sciences, University of California Santa Cruz ● Minority Access to Research Careers, University of California Santa Cruz ● UCSC School of Physical and Biological Science, Department of Molecular Cell and Developmental Biology, Alan Zahler Chair ● UCSC School of Physical and Biological Sciences, Department of Chemistry, Ilan Benjamin Chair ● UCSC School of Engineering, Department of Biomolecular Engineering, Mark Akeson Chair ● Hartnell Community College, STEM Internship Program ● Experiment.com Supporters
Literature Cited ( 1) Leskinen, Timo, Alistair WT King, and Dimitris S. Argyropoulos. "Fractionation of Lignocellulosic Materials with Ionic Liquids." Production of Biofuels and Chemicals with Ionic Liquids . Springer Netherlands, 2014. 145-168. (2) Allers, Thorsten and Mevarech, Moshe. “Archaeal genetics - the third way.” Nature Reviews Genetics , 2005. (6) 58- 73. (3) National Oceanic and Atmospheric Administration. “Measuring and Analyzing Greenhouse Gases: Behind the Scenes.” Earth System Research Laboratory . www.esrl.noaa. gov/gmd/outreach/behind_the_scenes/meas_analyzers.html. Accessed 8/8/2014. (4) Vaghela, Anish, et al. “Biobutanol: Origins and Prospects.” Biofuels in Bacteria. http://2012.igem.org/Team: Rutgers/BIB. Accessed 8/8/2014. (5) Cho, Hyung Min, Gross, Adam S, and Chu, Jhih-Wei. ”Dissecting Force Interactions in Cellulose Deconstruction Reveals the Required Solvent Versatility for Overcoming Biomass Recalcitrance” J. Am. Chem. Soc. 2011. 133 (35) 14033-14041 (6) Dibrova, Daria V., Michael Y. Galperin, and Armen Y. Mulkidjanian. "Phylogenomic Reconstruction of Archaeal Fatty Acid Metabolism." Environmental Microbiology 16.4 (2014): 907-18. Web.
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