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

▶
genetically modified yeast breaks down gluten in beer
SMART_READER_LITE
LIVE PREVIEW

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

Oct 29, 2023 231 likes •441 views

Genetically Modified Yeast Breaks Down Gluten in Beer CSU iGEM Guy Stewart, Steven Denham CSU Oval Our Gluten-Free Mission About Celiac Disease Brewing Enzymes Secretion Assembly Results Discussion Safety

slide-1
SLIDE 1

Genetically Modified Yeast Breaks Down Gluten in Beer

CSU iGEM Guy Stewart, Steven Denham

slide-2
SLIDE 2

CSU Oval

slide-3
SLIDE 3

Our Gluten-Free Mission

  • About Celiac Disease
  • Brewing
  • Enzymes
  • Secretion
  • Assembly
  • Results
  • Discussion
  • Safety
  • Contributors
slide-4
SLIDE 4

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

slide-5
SLIDE 5
slide-6
SLIDE 6

Gluten and Beer

  • Yeast enter Fermenter
  • pH 5.2-5.5
  • 1. Lag phase
  • 2. Growth phase
  • 3. Fermentation phase
  • 4. Sedimentation phase
slide-7
SLIDE 7

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

– 2011 UW iGEM team – pH 4 – Cleaved PQPQLP peptide – Codon bias (bacterial to yeast)

  • A. niger

labmed.ucsf.edu Kumamolisin-As

http://2011.igem.org/Team:Washington

slide-8
SLIDE 8

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.

slide-9
SLIDE 9

Summary of the System

slide-10
SLIDE 10

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.

slide-11
SLIDE 11

pCM190+aK

10327 bps

2000 4000 6000 8000 10000

EcoRI BamHI PstI NotI PstI BglII NheI tTA a-SPEK AmpR URA3

Episomal

slide-12
SLIDE 12

Integrative

pCM189-aK-Kan

10478 bps

2000 4000 6000 8000 10000

EcoRI BamHI NotI BglII NheI tTA a-SPEK AmpR URA3 Kan

slide-13
SLIDE 13

Screening For Gluten

  • Equinox Donated Wort
  • Yeast carrying pCM 190+αK
  • Tet-off promoter
  • Test batches of beer

– Lab strain (tet) – Lab strain (no tet) – Industrial strain (tet) – Industrial strain (no tet) – Unaltered wort

  • ELISA

pct-1.com

slide-14
SLIDE 14

ELISA Standard Curve

G lia d in C o m p e ta tiv e E L IS A

C o n c e n tra tio n (n g /m l)

1 0 0 2 0 0 3 0 0 0 .0 0 .5 1 .0 1 .5 2 .0

A b s o rb a n c e

slide-15
SLIDE 15

Wort LS (tet) LS (no tet) IS (tet) IS (no tet)

20 40 60 80 100 120 140

1 2 3 4 5

Gliadin (ng/ml) Sample

Concentration of Gliadin in Wort

LS=lab strain, IS=industrial strain

slide-16
SLIDE 16

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

slide-17
SLIDE 17

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
slide-18
SLIDE 18

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

slide-19
SLIDE 19
  • 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.