BioBeer BioBeer: : Resv esver eratr trol-Pr ol-Producing - PowerPoint PPT Presentation

BioBeer BioBeer: : Resv esver eratr trol-Pr ol-Producing oducing Yeast f east for F or Fermenta ermentation tion Houston, TX Houston, TX

A history of the phytoalexin resveratrol 1940 - Isolated from Veratrum grandiflorum (white hellebore) 1963 - Found to be responsible for the anti- inflammatory properties of the Polygonum cuspidatum (Japanese Knotweed) 1992 - Proposed to underlie the “French Paradox” Resveratrol Citatitions on PubMed V. grandiflorum trans- resveratrol

Resveratrol imparts diverse health benefits Resveratrol Inhibits carcinogenesis Improves cardiovascular function Improves insulin sensitivity Extends lifespan Reduces neurodegeneration

Unfortunately, only a limited number of foods provide resveratrol Raw f w foods oods 0.02 – 1.92 µg/g 0.032 µg/g 0.16 – 3.54 µg/g 0.5–1 µg/g Other dietary sources Other dietary sour ces [active] = ? 0.2 µg/mL <0.1–2.1 µg/mL 0.1–14.3 µg/mL µg/g ≈ µg/mL

Resveratrol-enriched beer could provide health benefits for a wider populace $10.28/L $8.69/L 5.5% 5.5% 8.5% 8.5% U.S .S. Be . Bever erage Consumption e Consumption (percent of gallons 2005) 86% 86% $2.51/L Sources: National Institute on Alcohol Abuse and Alcoholism & US Dept. of Labor

Like wine, beer is predicted to maintain resveratrol in a bioactive form Packaging Fermenta ermentation tion Malted Malted grains ains CO 2 CO 2 + Hops/Yeast Sw Sweet w eet wor ort t Pyr Pyruv uvate te Ethanol Ethanol glycolysis alcoholic (Glucose) (Glucose) fermentation

Like wine, beer is predicted to maintain resveratrol in a bioactive form Packaging Fermenta ermentation tion Resv esver eratr trol ol Malted Malted Tyr yrosine osine grains ains CO CO 2 2 +Hops/Yeast Sw Sweet w eet wor ort t Pyruv Pyr uvate te Ethanol Ethanol glycolysis alcoholic (Glucose) (Glucose) fermentation

Brewer’s yeast used in hefewiezen production is a good chassis • Used to make unfiltered beer --- “hefe” means yeast • Was similar to model organism --- is a top-fermenting strain of Saccharomyces • Easy to obtain --- collected cultures from a fermenter at local brewery • Grew readily in the lab --- formed colonies in standard medium (YPG and YPD)

Challenges of engineering brewer’s yeast for hefeweizen production • Strain is polyploid and prototrophic, requiring the use of selectable markers • Transformation efficiency has not been characterized

The pathway introduced into yeast Step 1: Tyrosine Ammonia Lyase (TAL) p -coumaric acid TAL NH NH 3 4-coumarate CoA L-tyrosine resveratrol 3 Rhodotorula glutinis TAL is expected to be functional in our brewing yeast. TAL also catalyzes the synthesis of a flavor enhancer. PDB ID: 1T6P

The pathway introduced into yeast Step 2: 4-Coumarate CoA Ligase (4CL) p -coumaric acid TAL 4CL 4-coumarate CoA L-tyrosine resveratrol CoA + A CoA + ATP TP Arabidopsis thaliana 4CL is expected to be functional in our brewing yeast PDB ID: 1CQJ

The pathway introduced into yeast Step 3: Stilbene Synthase (STS) p -coumaric acid STS TAL 4CL 4-coumarate CoA L-tyrosine resveratrol 4 CO 4 CO 2 2 3 Malonyl-CoA Vitis vinifera stilbene synthase is predicted to be functional in our brewing yeast. PDB ID: 1Z1F

The first expression cassette synthesizes coumaric acid from tyrosine pPGK1 4CL::STS tCYC1 pGAL1 + ZeoR tADH1 pADH1 TAL tPGK1 K122000 K122010 K122003 K122002 K122005 K122013 K122001 K122003 Phosphog Phosphogly lycer cerate te Alcohol dehy Alcohol dehydr drog ogenase enase kinase inase termina terminator tor promoter pr omoter • Truncated promoter • Constitutively active • Lacks glucose repression L-tyrosine p-coumaric acid

The second expression cassette synthesizes resveratrol from coumaric acid pPGK1 4CL::STS tCYC1 pGAL1 + ZeoR tADH1 pADH1 TAL tPGK1 K122000 K122010 K122003 K122002 K122005 K122013 K122001 K122003 Cytoc Cytochr hrome ome C termina C terminator tor Phosphog Phosphogly lycer cerate te • bidirectional yeast kinase inase pr promoter omoter 4CL:STS fusion pr 4CL:STS fusion protein otein terminator • constitutively active • 20-fold higher activity • further induced by fermentation p-coumaric acid ATP TP resveratrol 3 x malonyl-CoA

The third expression cassette confers resistance to bleocin antibiotics pPGK1 4CL::STS tCYC1 pGAL1 + ZeoR tADH1 pADH1 TAL tPGK1 K122000 K122010 K122003 K122001 K122003 K122002 K122005 K122013 Galactose inducib Galactose inducible selection mar le selection marker er • allows for integration selection Zeo Zeo R +G +GAL ALACT CTOSE OSE +GL +GLUCOSE UCOSE Zeo R Zeo R

Our strategy was to create a circuit that is comprised of three expression cassettes Our genetic circuit pPGK1 4CL::STS tCYC1 pGAL1 + ZeoR tADH1 pADH1 TAL tPGK1 K122000 K122010 K122003 K122001 K122003 K122002 K122005 K122013 pPGK1 PGK1 tPGK1 Brewer’s Yeast Genomic Genomic of Desired Strain pPGK1 4CL::STS tCYC1 pGAL1 + ZeoR tADH1 pADH1 TAL tPGK1 K12200 K122010 K122003 K122001 K122003 K122002 K122005 K122013 0

New yeast parts submitted to registry pPGK1 4CL::STS tCYC1 K122010 K122003 K122000 tADH1 pGAL1 + ZeoR K122003 K122001 pADH1 tPGK1 K122002 K122013 pPGK1 4CL::STS tCYC1 pGAL1 + ZeoR tADH1 K122000 K122010 K122003 K122001 K122003 pPGK1 4CL::STS tCYC1 pGAL1 + ZeoR tADH1 K122000 K122010 K122003 K122001 K122003

Our strategy was to create a circuit that is comprised of three expression cassettes Our feeding circuit pPGK1 PGK1 tPGK1 Brewer’s Yeast Genomic Incorporation of feeding circuit pPGK1 4CL::STS tCYC1 pGAL1 + ZeoR tADH1 pADH1 TAL tPGK1 K12200 K122010 K122003 K122001 K122003 K122002 K122005 K122013 0

The feeding circuit has been transformed into Hefeweizen Strain WT Hefe Lab Strain Transformed Hefe strain YPG media with 0.2 µg/mL Bleocin

The feeding circuit has been transformed into Hefeweizen strain WT Hefe Lab Strain Transformed Hefe strain YPG media with 0.2 µg/mL bleocin

PCR screen results confirm genomic integration PCR PCR 900 bp 700 bp C1 C1 C2 C2 C3 C3 C4 C4 P1 P1 G1 G1 C1 C1 C2 C2 C3 C3 C4 C4 1.0 kb –– ––1.0 kb 0.5 kb –– ––0.5 kb P1 = plasmid containing integration circuit G1 = Genomic DNA from parent strain

HPLC can be used to quantify resveratrol concentrations

HPLC can be used to quantify resveratrol concentrations 300 250 20 ug/ml 200 mAU (290nm) 10 ug/mL 150 5 ug/mL 2.5 ug/mL 100 50 0 30 30.5 31 31.5 32 32.5 33 Retention Time (min)

We have performed analytical HPLC on several wines 200 180 mAU (290nm) 160 140 120 100 200 80 10 ug/mL Standard 60 180 40 Shiraz Chardonnay 20 160 0 Merlot Primitivo 21 23 25 27 29 31 33 140 Primitivo Retention Time (min) Merlot mAU (290nm) Suavignon Blanc 120 Sauvignon Blanc 10 ug/mL STD 100 Shiraz Chardonnay 80 60 40 20 0 30 30.5 31 31.5 32 32.5 33 Retention Time (min)

Our current HPLC assay can quantify low levels of resveratrol in wine 4.5 ) mL) 4.0 g/mL 3.5 ol ( µ g/ 3.0 trol ( 2.5 eratr esver 2.0 -resv 1.5 ans -r trans 1.0 0.5 tr * 0.0 Primitivo Primitiv Primitivo (Italy) o Merlot (Australia) Mer Merlot lot Shiraz (Australia) Shiraz Shir az Savignon Blanc (France) Chardonnay (N. California) Sauvignon Sauvignon Chardonna Char donnay Mer Merlot lot Shiraz Shir az Sauvignon Sauvignon Char Chardonna donnay y Italy Italy Australia Austr alia Austr Australia alia Blanc Blanc N. Calif . California ornia Austr Australia alia Australia Austr alia Blanc Blanc N. Car . Carolina olina France ance France ance Note: all wine sample handling was performed by Rice team members who are over 21.

Cur Currently perf ently performing orming p -coumaric coumaric acid acid feeding trials on r eeding trials on recombinants ecombinants

HPLC analysis shows no trans -resveratrol after 24 hours of fermentation 300 standards 250 WT strain 1 200 strain 2 U (290nm) mAU (290nm) 150 mA 100 50 0 27 28 29 30 31 32 33 Retention T etention Time (min) ime (min) Taken @ 10:00 pm Thursday night

Future Work • Construct full TAL containing circuit • Replace selection in favor of screen • Examine utility in other products

Beer as a drinkable bioreactor…