Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Daniel Vallejo - California State Polytechnic University
The development of renewable energy sources is necessary to substitute for diminishing natural energy resources1. A renewable energy source of great interest is biofuel, which is energy produced from biological sources through various carbon fixation processes. Biofuels are commonly manufactured from food crops, but ideally would be produced from nonfood crops because the degradation of starch into fermentable sugars is time-consuming and expensive. We will test the hypothesis that yeast expressing and secreting the starch digesting alphaamylase enzyme will be able to synthesize bioethanol from a starchy non-edible feedstock2,3. We used the budding yeast, Saccharomyces cerevisiae (D-213-1B, lab culture #118), which cannot degrade starch. However, when these cells carried the mouse-alpha-amylase-containing plasmid pMS12, they were tested for starch degradation from fermentation processes4. S. diastaticus (KK2-R1, lab culture #1611), a glucoamylase producing yeast strain, was used to determine if a dual enzyme starch digestion process would yield higher bioethanol yields5. The pMS12 plasmid contains an ADH promoter region that includes an ethanol-sensitive repressor, which was excised from pMS12 to produce pMS12deltaR. Buffalo gourd root (Cucurbita foetidissima) was the primary starch source for the fermentations. 118/pMS12 deltaR, 118/pMS12, and 1611/ pMS12 were allowed to ferment for 72 hours in 2% dextrose YPD, 4% dextrose YPD, and Root Starch/2% dextrose YPD. Aliquots of supernatant and suspended yeast were taken to determine any changes in bioethanol concentration based on colligative properties. Ethanol was extracted from the yeast aliquot via headspace liquid-phase microextraction using 1octanol suspended on a 10 uL analytical SGE syringe tip, and was measured on an Agilent 5890 GC with FID.
Our findings show that 118/pMS12 produced bioethanol from the starch containing media. Additionally, it was observed that the excision of the ADH-repressor region in the plasmid allowed for increased bioethanol production and that 1611/pMS12 produced comparable bioethanol concentrations to 118/ pMS12deltaR. These results indicate that the addition of pMS12 allows S. cerevisiae to degrade starch into fermentable sugars, and S. diastaticus is able to produce higher yields of bioethanol due to its natural glucoamylase expression. Future research involves the stable integration of alpha-amylase into the genomic DNA of both yeast strains and optimization of the fermentation and GC-FID method.
References: 1. Monthly Energy Review. (U.S. Energy Information Administration, 2015). at http://www.eia.gov/totalenergy/data/ monthly/pdf/mer.pdf.
2. Kim, T. G. & Kim, K. The construction of a stable starchfermenting yeast strain using genetic engineering and raremating. Appl. Biochem. Biotechnol. 59, 39–51 (1996).
3. Kim, J. H. et al. Construction of a direct starch-fermenting industrial strain of Saccharomyces cerevisiae producing glucoamylase, -amylase and debranching enzyme. Biotechnol. Lett. 32, 713–719 (2010).
4. Hutchings, G. G. & Key, S. C. S. Gene Knockout / Gene Therapy in Yeast Using Homologous Recombination. Association Biol. Lab. Educ. 30, 49–62 (2008).
5. Lambrechts, M. G., Pretorias, I. S., Sollitti, P. & Marmur, J. Primary structure and regulation of a glucoamylase-encoding gene (STA2) in Saccharomyces diastaticus. Gene 100, 95–103 (1991).
Funder Acknowledgement(s): I thank W. Haggren and J. Owens as well as all of the UCCS faculty for their support throughout the REU program. I would also like to thank the Faculty at Cal Poly Pomona for allowing me to pursue this opportunity by administering my course material early. This study was supported, in part, by a REU grant from NSF awarded to the Department of Chemistry & Biochemistry, University of Colorado Colorado Spring, Colorado Springs, CO 80918.
Faculty Advisor: Wendy Haggren,