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

BioBeer BioBeer: :

Resv esver eratr trol-Pr

• l-Producing
• ducing

Yeast f east for F

• r 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

Inhibits carcinogenesis Extends lifespan Improves insulin sensitivity Improves cardiovascular function Reduces neurodegeneration

Resveratrol

Unfortunately, only a limited number of foods provide resveratrol

0.1–14.3 µg/mL 0.5–1 µg/g 0.032 µg/g

Raw f w foods

• ods

Other dietary sour Other dietary sources ces

0.2 µg/mL <0.1–2.1 µg/mL [active] = ? 0.02 – 1.92 µg/g 0.16 – 3.54 µg/g

µg/g ≈ µg/mL

Resveratrol-enriched beer could provide health benefits for a wider populace

Sources: National Institute on Alcohol Abuse and Alcoholism & US Dept. of Labor

U.S .S. Be . Bever erage Consumption e Consumption

(percent of gallons 2005)

8.5% 8.5%

86% 86%

5.5% 5.5%

$2.51/L $10.28/L $8.69/L

Like wine, beer is predicted to maintain resveratrol in a bioactive form

Fermenta ermentation tion

Packaging

Sw Sweet w eet wor

• rt

t (Glucose) (Glucose) Pyr Pyruv uvate te Ethanol Ethanol

glycolysis alcoholic fermentation CO CO2

2

Malted Malted grains ains

+Hops/Yeast

Like wine, beer is predicted to maintain resveratrol in a bioactive form

Packaging

Resv esver eratr trol

• l

Sw Sweet w eet wor

• rt

t (Glucose) (Glucose) Pyr Pyruv uvate te Ethanol Ethanol

glycolysis alcoholic fermentation CO CO2

2

Malted Malted grains ains

+Hops/Yeast

Tyr yrosine

• sine

Fermenta ermentation tion

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

Rhodotorula glutinis TAL is expected to be functional in our brewing yeast. TAL also catalyzes the synthesis of a flavor enhancer.

PDB ID: 1T6P

NH NH3

3

TAL

L-tyrosine resveratrol p-coumaric acid 4-coumarate CoA

The pathway introduced into yeast

Step 1: Tyrosine Ammonia Lyase (TAL)

The pathway introduced into yeast

Step 2: 4-Coumarate CoA Ligase (4CL)

L-tyrosine resveratrol p-coumaric acid 4-coumarate CoA

4CL

Arabidopsis thaliana 4CL is expected to be functional in our brewing yeast

PDB ID: 1CQJ CoA + A CoA + ATP TP

TAL

The pathway introduced into yeast

Step 3: Stilbene Synthase (STS)

L-tyrosine resveratrol

Vitis vinifera stilbene synthase is predicted to be functional in our brewing yeast.

3 Malonyl-CoA PDB ID: 1Z1F

STS

4 CO 4 CO2

2

4-coumarate CoA p-coumaric acid

4CL TAL

The first expression cassette synthesizes coumaric acid from tyrosine

pPGK1 K122000 4CL::STS K122010 tCYC1 K122003 pGAL1 + ZeoR K122001 tADH1 K122003 pADH1 K122002 TAL K122005 tPGK1 K122013

L-tyrosine p-coumaric acid

• Truncated promoter
• Constitutively active
• Lacks glucose repression

Alcohol Alcohol dehy dehydr drog

• genase

enase pr promoter

• moter

Phosphog Phosphogly lycer cerate te kinase inase termina terminator tor

The second expression cassette synthesizes resveratrol from coumaric acid

pPGK1 K122000 4CL::STS K122010 tCYC1 K122003 pGAL1 + ZeoR K122001 tADH1 K122003 pADH1 K122002 TAL K122005 tPGK1 K122013

ATP TP

p-coumaric acid 3 x malonyl-CoA resveratrol

• constitutively active
• further induced by

fermentation

Phosphog Phosphogly lycer cerate te kinase inase pr promoter

• moter

Cytoc Cytochr hrome

• me C termina

C terminator tor

• 20-fold higher activity

4CL:STS fusion pr 4CL:STS fusion protein

• tein
• bidirectional yeast

terminator

The third expression cassette confers resistance to bleocin antibiotics

Galactose Galactose inducib inducible selection mar le selection marker er

pPGK1 K122000 4CL::STS K122010 tCYC1 K122003 pGAL1 + ZeoR K122001 tADH1 K122003 pADH1 K122002 TAL K122005 tPGK1 K122013

• allows for integration selection

Zeo ZeoR

+G +GAL ALACT CTOSE OSE +GL +GLUCOSE UCOSE

ZeoR ZeoR

Our strategy was to create a circuit that is comprised of three expression cassettes

pPGK1 K122000 4CL::STS K122010 tCYC1 K122003 pGAL1 + ZeoR K122001 tADH1 K122003 pADH1 K122002 TAL K122005 tPGK1 K122013 pPGK1 tPGK1

Brewer’s Yeast Genomic

PGK1

Our genetic circuit Genomic of Desired Strain

New yeast parts submitted to registry

tCYC1 K122003 4CL::STS K122010 pADH1 K122002 pGAL1 + ZeoR K122001 pPGK1 K122000 tADH1 K122003 tPGK1 K122013 pPGK1 K122000 4CL::STS K122010 tCYC1 K122003 pGAL1 + ZeoR K122001 tADH1 K122003 pGAL1 + ZeoR K122001 tADH1 K122003 pPGK1 K122000 4CL::STS K122010 tCYC1 K122003

pPGK1 K122000 4CL::STS K122010 tCYC1 K122003 pGAL1 + ZeoR K122001 tADH1 K122003 pGAL1 + ZeoR K122001 tADH1 K122003 pPGK1 K122000 4CL::STS K122010 tCYC1 K122003

New yeast parts submitted to registry

tCYC1 K122003 4CL::STS K122010 pADH1 K122002 pGAL1 + ZeoR K122001 pPGK1 K122000 tADH1 K122003 tPGK1 K122013

Our strategy was to create a circuit that is comprised of three expression cassettes

pPGK1 tPGK1

Brewer’s Yeast Genomic

PGK1

Our feeding circuit Incorporation of feeding circuit

The feeding circuit has been transformed into Hefeweizen Strain

YPG media with 0.2 µg/mL Bleocin

Transformed Hefe strain WT Hefe Lab Strain

The feeding circuit has been transformed into Hefeweizen strain

YPG media with 0.2 µg/mL bleocin

Transformed Hefe strain WT Hefe Lab Strain

PCR screen results confirm genomic integration

1.0 kb –– 0.5 kb ––

C1 C1 C2 C2 C3 C3 C4 C4 P1 P1 G1 G1 C1 C1 C2 C2 C3 C3 C4 C4

900 bp

PCR G1 = Genomic DNA from parent strain

700 bp

PCR P1 = plasmid containing integration circuit

––1.0 kb ––0.5 kb

HPLC can be used to quantify resveratrol concentrations

50 100 150 200 250 300 30 30.5 31 31.5 32 32.5 33

mAU (290nm) Retention Time (min)

20 ug/ml 10 ug/mL 5 ug/mL 2.5 ug/mL

HPLC can be used to quantify resveratrol concentrations

20 40 60 80 100 120 140 160 180 200 21 23 25 27 29 31 33 mAU (290nm) Retention Time (min)

We have performed analytical HPLC on several wines

20 40 60 80 100 120 140 160 180 200 30 30.5 31 31.5 32 32.5 33 mAU (290nm) Retention Time (min)

Chardonnay Merlot Primitivo Suavignon Blanc 10 ug/mL STD Shiraz

Sauvignon Blanc Merlot Primitivo Shiraz Chardonnay 10 ug/mL Standard

Our current HPLC assay can quantify low levels of resveratrol in wine

Note: all wine sample handling was performed by Rice team members who are over 21.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Primitivo (Italy) Merlot (Australia) Shiraz (Australia) Savignon Blanc (France) Chardonnay (N. California)

*

Mer Merlot lot

Austr Australia alia

Shir Shiraz az

Austr Australia alia

Sauvignon Sauvignon Blanc Blanc

France ance

tr trans ans-r

• resv

esver eratr trol (

• l (µg/

g/mL mL) )

Primitiv Primitivo

• Italy

Italy

Char Chardonna donnay

• N. Calif

. California

• rnia

Mer Merlot lot

Austr Australia alia

Shir Shiraz az

Austr Australia alia

Sauvignon Sauvignon Blanc Blanc

France ance

Char Chardonna donnay y

• N. Car

. Carolina

• lina

Cur Currently perf ently performing

• rming p-coumaric

coumaric acid acid feeding trials on r eeding trials on recombinants ecombinants

50 100 150 200 250 300 27 28 29 30 31 32 33

mA mAU (290nm) U (290nm) Retention T etention Time (min) ime (min) standards WT strain 1 strain 2

HPLC analysis shows no trans-resveratrol after 24 hours of fermentation

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…

BIOENGINEERING BIOENGINEERING

Ole Oleg Ig g Igoshin

• shin

Justin Judd Justin Judd Junghae Suh Junghae Suh

BIOCHEMISTR BIOCHEMISTRY AND CELL BIOL Y AND CELL BIOLOGY OGY

Beth Beason Beth Beason Geor George Bennett e Bennett P Peter Nguy eter Nguyen en Jof Joff Silber Silberg g

CHEMICAL AND BIOMOLECUL CHEMICAL AND BIOMOLECULAR ENGINEERING AR ENGINEERING

K Ken Co en Cox x

Sar Sarah Duk ah Duke e Arielle La Arielle Layman yman Da David Ouy vid Ouyang ang Thomas-Se homas-Segall Sha all Shapir piro

• Selim Sheikh

Selim Sheikh Taylor Ste ylor Stevenson enson

Special T Special Thank hanks to Br s to Broc

• ck

k Wagner (Rice Univ gner (Rice Univer ersity sity Class of Class of 1987) of 1987) of St. St. Arnold Arnold’s Br s Brewery in ery in Houston, TX Houston, TX

BIOENGINEERING BIOENGINEERING

Ole Oleg Ig g Igoshin

• shin

Justin Judd Justin Judd Junghae Suh Junghae Suh

BIOCHEMISTR BIOCHEMISTRY AND CELL BIOL Y AND CELL BIOLOGY OGY

Beth Beason Beth Beason Geor George Bennett e Bennett P Peter Nguy eter Nguyen en Jof Joff Silber Silberg g

CHEMICAL AND BIOMOLECUL CHEMICAL AND BIOMOLECULAR ENGINEERING AR ENGINEERING

K Ken Co en Cox x

Sar Sarah Duk ah Duke e Arielle La Arielle Layman yman Da David Ouy vid Ouyang ang Thomas-Se homas-Segall Sha all Shapir piro

• Selim Sheikh

Selim Sheikh Taylor Ste ylor Stevenson enson

Special T Special Thank hanks to Br s to Broc

• ck

k Wagner (Rice Univ gner (Rice Univer ersity sity Class of Class of 1987) of 1987) of St. St. Arnold Arnold’s Br s Brewery in ery in Houston, TX Houston, TX