GENETICALLY ENGINEERING Haloferax Volcanii FOR BUTANOL PRODUCTIONMax 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

Analytical ChemistryAbstract

● Butanol is a low-emission biofuel whose

energy per volume is near that of

gasoline and can be used in unmodified

gasoline engines.(4)

● Butanol shortens the carbon cycle down

from millions of years to years.

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.

Table of strengths of ionic liquids’ ability to deconstruct cellulose (5)

Introduction

Synthetic Biology

(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 AcidMetabolism." Environmental Microbiology 16.4 (2014): 907-18. Web.

Literature Cited

Bioinformatically Assembled Synthetic Pathway Fatty Acid Synthesis Cycle (6)

● 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

Acknowledgments

Results

● Cellulose is difficult to isolate from

lignin in plants. Requires “cooking” in

high temperature, high salt conditions

to access (Ionic Liquids)(4).

● Salt-loving halophile H. volcanii was

chosen.

Synthetic Pathway

Conclusion

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.

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.

Cartoon Schematic of the Carbon Cycle. Red arrows indicate fossil fuels, purple arrows indicate biofuel. Credit: Paige Welsh

Deletion Sequence

3ʹ Homologous Region

5ʹ Homologous Region

Acd3 popout colony PCR result. Red arrow indicates size of knockout band. Blue arrow indicates size of wild-type band.

Acd2 popout colony PCR result. Red arrow indicates position of band showing knockout.

A. Plasmid Vector pSCKiKo

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.

B. Pop-in pop-out

A. Using PCR and Gibson cloning, we assembled the above plasmid. Knockout constructs containing were Gibson assembled into the region shown.

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 5-Fluoroorotic acid (5-FOA), which is toxic to uracil prototrophs. (2)

Growth of ACD 2 Knockout Strain on 5-FOA containing selective media

We have submitted plasmid vector pSCKiKo, KiKo+ACD2, KiKo+ACD3, and KiKo+ACD4 as biobricks:: BBa_K1560001, BBa_K1560002, BBa_K1560003, BBa_K1560004

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

knockout of ACD stops fatty acid synthesis at butanoyl-CoA

Figure credit: (4)

pSCKiKo3,558 bp

3 Kb1 Kb

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.