UCSC Championship Poster

GENETICALLY ENGINEERING Haloferax Volcanii FOR BUTANOL PRODUCTION
Max Genetti, Kevin Sweeney, Christopher Lee, Dominic Schenone, Jazel Hernandez, Renee Jocic,
Kaylee Walker, Charles Paine, Vinay Poodari, Lenore Pafford, Saumya Singh, Wade Dugdale, Rolando Perez,
Hossein Amiri, Logan Mulroney, Jeff Nivala, Sandra Dreisbach, David L. Bernick
University of California, Santa Cruz
Abstract
Analytical Chemistry
Synthetic Pathway
Due to increased use of petroleum-based fuels they have become increasingly difficult to obtain and have
resulted in increased pollution due to their long carbon-cycles. The development of energy- dense biofuels
has gained increased research interest. One such biofuel is butanol, a four carbon alcohol that can be
metabolized naturally from glucose and potentially from cellulose, a glucose polymer. Conditions used to
produce butanol from cellulose in bulk require exposure to ionic liquids, a class of high salinity organic
solvents that can be used to extract cellulose from plant materials; to which many mesophilic bacterial
enzymes and hosts are not accustomed. Many archaeal organisms thrive naturally under high salinity
conditions and thus provide a distinct advantage, such as the halophile Haloferax volcanii. By modifying the
pathway responsible for fatty acid synthesis in Haloferax volcanii we intend to produce butanol from
cellulose. Within this pathway we have identified seven possible paralogous Acyl-CoA dehydrogenase
genes (ACD 1-ACD 7), each thought to favor activity on specific fatty acid carbon chain lengths. By
interrupting the ACD responsible for the conversion of the four-carbon to six-carbon product, we can
accumulate butyryl-CoA, which may be converted to butanol through the overexpression of certain
aldehyde dehydrogenases and alcohol dehydrogenases.
We designed a synthetic pathway that hijacks fatty acid synthesis in H. volcanii
based upon hypothetical genes. Seven copies of the Acyl-CoA dehydrogenase
(ACD) gene control fatty acid synthesis by adding two carbons to a growing
chain each time the chain enters the pathways’ cycle (6). We hypothesize that
deletion of the gene responsible for adding carbons onto a 4-carbon chain
would disrupt the continuation of the pathway and accumulate a 4-carbon
product, Butyryl-CoA. Of the seven paralogous ACD genes for fatty acid
synthesis, bioinformatic evidence identified ACD2 and ACD3 as the two genes
most likely to allow carbons to be added to the 4-carbon product.
To test for the presence of butanol in our
cultures, we developed a method using gas
chromatography (GC). Although the end
product we are looking for is butanol, we need
to test for butyric acid as well. Butyric acid is
the four-carbon acid we predict will accumulate
as a result of our proposed knockouts in the
fatty acid synthesis pathway. We used a
column containing polyethylene glycol treated
with nitroterephthalic acid.
To the right is the butyric acid standard, with
the relevant peak just after 10 min. In order to
extract target compounds (nonpolar/ organic)
from liquid culture, we ran a liquid-liquid
extraction using an equal amount of Ethyl
Acetate.
knockout of ACD stops
fatty acid synthesis at
butanoyl-CoA
Introduction
The goal of this project is to develop a clean, efficient
and economically competitive biofuel that can be
used in place of gasoline and coal. To achieve this
we intend to metabolically engineer H. Volcanii to
produce butanol from cellulose found in plant waste.
Results
Bioinformatically Assembled Synthetic Pathway
Fatty Acid Synthesis Cycle (6)
Colony polymerase chain reaction (PCR) after treating with 5-Fluoroorotic acid (5-FOA) resulted in
one ACD 2 knockout (Lane 2), two ACD 3 knockouts (Lanes 3 and 10), two potential ACD 2
knockouts (Lanes 4 and 8), and 3 wild-type ACD 3 colonies (Lanes 4, 8, 3, and 5). Unfortunately
primer annealing conditions were not ideal and thus for the colonies that did not show any bands we
are unable to make a conclusion about the success of our knockout experiment.
Synthetic Biology
A. Plasmid Vector pSCKiKo
B. Pop-in pop-out
3 Kb
1 Kb
Figure credit: (4)
● Butanol is a low-emission biofuel whose
energy per volume is near that of
gasoline and can be used in unmodified
pSCKiKo
3,558 bp
gasoline engines.(4)
Acd2 popout colony PCR result.
Red arrow indicates position of
band showing knockout.
● Butanol shortens the carbon cycle down
Cartoon Schematic of the Carbon Cycle. Red arrows indicate
fossil fuels, purple arrows indicate biofuel. Credit: Paige Welsh
Acd3 popout colony PCR result.
Red arrow indicates size of
knockout band. Blue arrow
indicates size of wild-type band.
Growth of ACD 2 Knockout
Strain on 5-FOA containing
selective media
from millions of years to years.
Conclusion
● Cellulose is difficult to isolate from
We have submitted plasmid vector pSCKiKo, KiKo+ACD2, KiKo+ACD3, and KiKo+ACD4 as
biobricks:: BBa_K1560001, BBa_K1560002, BBa_K1560003, BBa_K1560004
lignin in plants. Requires “cooking” in
high temperature, high salt conditions
to access (Ionic Liquids)(4).
3ʹ Homologous
Region
A.
● Salt-loving halophile H. volcanii was
chosen.
(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.
B.
After the pop-in stage, cells will be uracil
prototrophs; after the pop-out, the desired product
will be the broken gene of interest and and a uracil
auxotroph, allowing for selective screening via 5Fluoroorotic acid (5-FOA), which is toxic to uracil
prototrophs. (2)
Acknowledgments
Literature Cited
(2) Allers, Thorsten and Mevarech, Moshe. “Archaeal genetics - the third way.” Nature Reviews Genetics, 2005. (6) 58-73.
5ʹ Homologous
Region
Using PCR and Gibson cloning, we assembled the above
plasmid. Knockout constructs containing were Gibson
assembled into the region shown.
Table of strengths of ionic liquids’ ability to deconstruct cellulose (5)
(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.
Deletion
Sequence
We successfully designed and implemented knock-in knock-out vector, pSCKikO, yet there still
remains much to be done. The research will continue throughout the year. Planned tasks include:
● incorporating cellulases into H. Volcanii
● increasing H. Volcanii ionic liquid tolerance
● test for accumulation of butyric acid in acd knockouts
● creating a synthetic protein for last two steps of pathway by combining genes with a linker
sequence or continue to attempt to overexpress AldY5
●
●
●
●
●
●
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
● Experiment.com Supporters

Download Report