Genetically Modified Yeast Breaks Down Gluten in Beer CSU iGEM Guy - PowerPoint PPT Presentation

Genetically Modified Yeast Breaks Down Gluten in Beer CSU iGEM Guy Stewart, Steven Denham

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Our Gluten-Free Mission • About Celiac Disease • Brewing • Enzymes • Secretion • Assembly • Results • Discussion • Safety • Contributors

Celiac Disease • Autoimmune disorder • Affects the digestive system • Severe reaction when exposed to gluten, a protein found in wheat, rye, and barley. • Antigenicity of gluten due to proline and glutamine rich peptides

Gluten and Beer • Yeast enter Fermenter • pH 5.2-5.5 1. Lag phase 2. Growth phase 3. Fermentation phase 4. Sedimentation phase

Enzymes • Aspergillus niger- prolyl endoprotease(AN-PEP) – Cleaves after proline residues and is – Stable at low pH. – Used in Brewers Clarex – Protected by patent • KumaMax A. niger – 2011 UW iGEM team labmed.ucsf.edu – pH 4 – Cleaved PQPQLP peptide – Codon bias (bacterial to yeast) Kumamolisin-As http://2011.igem.org/Team:Washington

Secretion • Mating Factor alpha (MF- α ) – Tag’s sequence was placed directly upstream of KumaMax. – Signal sequence is cleaved in the golgi before protein export. – The DNA synthesized by IDT, but we were also able to extract it from the yeast genome by PCR.

Summary of the System

Assembly of the System • Expression vectors – pCM189: centromeric yeast plasmid, low copy number, tet-off promoter – pCM190: episomal yeast plasmid, high copy number, tet-off promoter – Integrative plasmid constructed by adding the resistance gene for geneticin to pCM189/MF- alpha/Kuma-max.

Episomal EcoRI tTA NheI BglII 10000 BamHI pCM190+aK 2000 PstI 8000 URA3 10327 bps 4000 6000 a-SPEK AmpR NotI PstI

Integrative EcoRI tTA Kan NheI BglII 10000 2000 pCM189-aK-Kan BamHI 8000 URA3 10478 bps 4000 6000 a-SPEK AmpR NotI

Screening For Gluten • Equinox Donated Wort • Yeast carrying pCM 190+ α K • Tet-off promoter • Test batches of beer – Lab strain (tet) – Lab strain (no tet) – Industrial strain (tet) – Industrial strain (no tet) pct-1.com – Unaltered wort • ELISA

ELISA Standard Curve G lia d in C o m p e ta tiv e E L IS A 2 .0 1 .5 A b s o rb a n c e 1 .0 0 .5 0 .0 0 1 0 0 2 0 0 3 0 0 C o n c e n tra tio n (n g /m l)

Concentration of Gliadin in Wort 140 IS (tet) Gliadin (ng/ml) 120 Wort 100 LS (tet) 80 60 LS (no tet) 40 IS (no tet) 20 0 1 2 3 4 5 Sample LS=lab strain, IS=industrial strain

Discussion • ELISA demonstrated that the engineered yeast were capable of breaking down gluten during fermentation • Further work: – optimization of the enzyme – completion of an integrative plasmid – Insertion of integrative plasmid into brewing yeast – Shift the focus from proof of concept in the laboratory to a potentially marketable gluten-free beer New Belgium Brewery

Safety • Saccharomyces cerevisiae and E. coli K-12 were the organisms used, which are exempt Host-Vector Systems as per EXEMPTIONS UNDER SECTION III-F-6 of the NIH Guidelines. • BS L-1 safety practices were enforced to ensure proper safety and containment. • Inspection and approval of the laboratory by University Director of Biosafety, Dr. Bob Ellis. • Alcohol consumed only by members at least 21 yrs • Consumption restricted to taste-testing

Contributors • Founder of CSU iGEM: Dr. June Medford • Thank you to the following for their support: Dr. Lucas Argueso, CSU; Dr. Brian Geiss, CSU; Dr. Carol Wilusz, CSU

• Celiac disease-national digestive diseases information clearinghouse . (2008, September). Retrieved from http://digestive.niddk.nih.gov/ddiseases/pubs/celiac/ • Structural Basis for Gluten Intolerance in Celiac Sprue. Lu Shan, Øyvind Molberg, Isabelle Parrot, Felix Hausch, Ferda Filiz, Gary M. Gray, Ludvig M. Sollid, and Chaitan Khosla. Science 27 September 2002: 297 (5590), 2275-2279. [DOI:10.1126/science.1074129] • Celiac disease-national digestive diseases information clearinghouse . (2008, September). Retrieved from http://digestive.niddk.nih.gov/ddiseases/pubs/celiac/ • Goldammer, T. (2012). The brewer's handbook . Apex Publishers. Retrieved from http://www.beer-brewing.com/beer- brewing/brewers_yeast/yeast_life_cycle.htm • Anthony J. Brake, James P. Merryweather . (1984). α -factor-directed synthesis and secretion of mature foreign proteins in saccharomyces cerevisiae. Biochemistry , 81 (August 1984), 4642-4646. • Waters, M. G. (1987). Prepro-a-factor has a cleavable signal sequence. Journal of Biological Chemistry, 263(May 5), 6209-6214 .