UCSC 2014: Microbial Engineering of Haloferax volcanii for the - - PowerPoint PPT Presentation

Microbial Engineering of Haloferax volcanii for the production of an alternative biofuel

UCSC 2014:

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
• n 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 cellulose into biofuel

(2)

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 1kb 1kb 3kb

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
• f 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
• ur 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.

Questions…?