Gen enes t s to J Jea eans A A Green een S Solution t to - - PowerPoint PPT Presentation

UC Berkeley iGEM

Gen enes t s to J Jea eans

A A Green een S Solution t to Blue e Den enim

Bernardo Cervantes Hojae Lee Roy Park Ramya Prathuri Thomas Rich

3,000,000,000

jeans per year tons of indigo per year

40,000 66,000,000,000

dollar denim industry

petroleum indigo

Indi digo i is synt nthes esized zed from

• m o
• il

KOH NaOH H2SO4 HNO3 ClCH2COOH

Air (O2)

Jeans m must b t be dyed with a a soluble f form o

• f ind

ndigo

Na2S2O4

We w want nt t to d dye e jean ans s sustainab nably

• 1. Make soluble indigo
• 2. Dye using our soluble form of indigo

Consulted experts on design principles

Key Design Considerations:

ABPDU

• Consumers like green

products

• Most consumers won’t

pay (much) more for green products

• Anaerobic conditions can

lower fermentation cost

• Secretion reduces

process cost

Jesse Genet

• Dr. Julio Baez

Polygonum tinctorium

indole indoxyl p450 GT indoxyl GLU O2 Healthy Leaf Stressed Leaf indican (soluble) indican (soluble) indigo (insoluble)

Plan ants m mak ake a e a soluble e form o

• f i

indi digo

indole indoxyl indigo (insoluble) indican (soluble) FMO GT GLU O2 FMO from M. aminisulfidivorans – BBa_K1131000 GT from S. antibioticus – BBa_K1131006 GLU from B. circulans – BBa_K1131002

The he en enzymatic ic dyein eing meth ethod

indole indoxyl indigo dye

O2

Mak Making ng indox

• xyl

yl wi with F FMO

Flask of bio-indigo

FMO

Best Titer of indigo: 230 mg/L

Char aracter erizat zation

• n o
• f F

FMO MO Kinet netics cs

Char aracter erizat zation

• n o
• f F

FMO MO Kinet netics cs

Char aracter erizat zation

• n o
• f F

FMO MO Kinet netics cs

Char aracter erizat zation

• n o
• f F

FMO MO Kinet netics cs

Char aracter erizat zation

• n o
• f F

FMO MO Kinet netics cs

Km = 0.87 mM

indole indoxyl indigo (insoluble) indican (soluble) FMO GT GLU O2

The he en enzymatic ic dyein eing meth ethod

The he indican production p pathway

Paper describing activity

Accession ID

Protein Database

MATRIAILGAGPSGMAQLRAQS AQEKGAEIPELVCFEKQADWG QWNYTWRTGLDENGEPVHSS MYRYLWSNGPKECLEFADYTF DEHFGKPIASYPPREVLWDYIK GRVEKAGVRKYIRFNTAVRHVE FNEDSQTFTVTVQDHTTDTIY SEEFDYVVCCTGHFSTPYVPEFE GFEKFGGRILHAHDFRDALEFK DKTVLLVGSSYSAEDIGSQCYK YGAKKLISCYRTAPMGYKWPE NWDERPNLVRVDTENAYFAD

Protein Sequence indole indoxyl FMO indican (soluble) GT

GT sequence data does not exist. 

The he indican production p pathway

indole indoxyl FMO indican (soluble)

Finding a missing activity:

• 1. Screening cDNA libraries
• 2. T

esting GTs that act on similar substrates

GT

Sc Screening cDNA NA libraries

mRNA cDNA

B-glucosyl transferases

Multiple Sequence Alignment Indigo producing plant

Char aracter erizi zing ng previou

• usly u

unk nkno nown G n GTs

New Glucosyl Transferases are being characterized and submitted to NCBI

Tes esting ng OleD eD: : a pr promiscuous G GT T

OleD

No indican

Indoxyl (Acceptor) UDP-Glucose (Donor)

16 hrs Aerobic reaction

OleD (Purified Enzyme)

Two h hyp ypothese ses

OleD-ASP

Active site modifications

Thorson et.al, 2010

OleD

Hypothesis 1: Aerobic conditions disfavor indican production Hypothesis 2: Native OleD does not recognize indoxyl

Tes esting ng o

• ur h

hypothes eses

OleD or OleD ASP (Purified Enzymes)

?

Indoxyl (Acceptor) UDP-Glucose (Donor)

16 hrs Anaerobic reaction

Indican i is pr prod

• duced

ed un under er a anaer aerob

• bic c

condition

• ns

OleD + Indoxyl OleD + Indoxyl + UDPG

Indican i is pr prod

• duced

ed un under er a anaer aerob

• bic c

condition

• ns

OleD + Indoxyl + UDPG

First demonstration of indican production with a recombinant GT! 

B-gluco cosidas dase to dye j e jeans ans

indole indoxyl indigo (insoluble) indican (soluble) FMO GT GLU O2

B-Glucosidase produces i indigo from i indican

50mM Indican + GLU lysate Indigo

0 min 30 min 10 min

=

GLU KanR ColE1

Char aracter erizi zing ng B B-glucos cosida dase kinet netics cs

Km = 1.78 mM

Can w n we d dye e cloth w with B-gl glucos

• sidase?

100% Cotton Cloth 10 g/L Indican 3.4 g/L B-Glucosidase Dark jeans 100% Cotton Cloth 1 g/L Indican 3.4 g/L B-Glucosidase Light jeans

Thes ese c e color

• rs don
• n’t

’t ru run!

H2O 1% SDS 70% EtOH Acetone

From t tiny y be bears t rs to t thousa sands o s of j jeans

Dip fabric in indican Dip/spray fabric with GLU, dry in air

Engineer neering ng a a cost-effect ective s e system em

• Anaerobic production of

indican

• Overproduction of

tryptophan

• Overproduction of UDPG
• Secretion of GLU

Accom complishmen ents

Added 5 well-characterized parts to the registry and a novel protein to NCBI Produced indican for the first time using a recombinant protein Dyed fabric using indican and GLU Developed a novel, green, scalable dyeing process that is both green

Ack ckno nowled edgem emen ents

Ack ckno nowled edgem emen ents!

Thanks to the Dueber Lab and EBB for hosting us! Special thanks to: John Dueber T erry Johnson Zach Russ Chris Eiben

Qu Questions?