Ethanol, whey not?

iGEM Jamboree 2009

UNIPV-Pavia

Outline• Why whey?• Engineering whey fermentation to

ethanol• Promoters characterization• Ethanol production and

conclusions

Motivation: why whey?

Cheese whey composition after extraction

Components % w/v

Proteins 0,75

Fat 0,40

Lactose 4,6

Ash 0,012

Cheese Whey is a residue of cheese curdling in dairy industries

• Its high nutrition load contributes to the proliferation of water microorganisms that steal oxygen in their environment• It causes water asphyxia, when

introduced into river systems• It is considered as a special waste for

Italian law (B.O.D.5 2000 times higher than legal limit)• Many industries extract high value

substances in order to decrease its pollutant power and to valorize it

Cheese whey valorization• Substances of interest:

- whey proteins- purified fatty acids- dry whey

• The residual liquid of these treatments is still a special waste for its high lactose content (~4.5%)• Complete lactose extraction

and purification is not convenient• New valorization techniques

should be developed

Solution: fermentation of lactose into ethanol• Ethanol is an important alternative and renewable source of energy• It is already used as a fuel in some countries such as Brazil and Australia• It is produced from feedstock such as sugar cane by fermentation

• Lactose can be easily converted into glucose by some microorganisms (such as E. coli) Idea: • whey can be considered as a

free feedstock• design a new biological

system able to convert lactose into ethanol with high

efficiency

Problem: no wild type organism is able to perform both functions efficiently

• Glucose can be fermented into ethanol by many microbiota (such as S. cerevisiae)

Project overview• Lactose cleaving module

• Ethanol producing module

PoPs input

Chassis used: E.coli

PoPs input

Lactose cleaving module

• E. coli β-galactosidase breaks lactose with high efficiency

• β-galactosidase overexpression to increase lactose cleaving capability

Alfa-D-glucose

D-galactoseLactose

B0034B0010 B0010

LacZ

• Zymomonas mobilis is an ethanologenic bacterium of the soil.

• Pyruvate decarboxilase (pdc)• Alcohol dehydrogenase II

(adhB)• Genes were designed with

codon usage bias optimization.

Ethanol-producing module

B0030 B0010 B0010pdc adhBB0030

pyruvate

acetaldehyde

ethanol

pdc

adhB

E. coli fermentation pathwaysWild type EngineeredVS

Theoretical yields:• 0.51 (g EtOH g glucose-1)• 0.54 (g EtOH g lactose-1)

Inducible promoters used

• aTc inducible devices:

• 3OC6HSL sensor: BBa_F2620

pTet B0034 LuxR lux pRB0010 B0012

PLac R23100 R23101 R23118

• Lac promoter (BBa_R0011), BBa_J231xx

B0034 tetR PtetB0010 B0012

Relative Promoter Units• It quantifies a promoter activity:

• The studied promoter expresses a GFP gene• Negative control is a non fluorescent culture• Benchmark: BBa_J23101 which leads GFP expression

R.P.U. calculation:

Hard model to study. We approximated it to the steady state:

Simplified in: Definition:

O.D.O.D.m O.D.LB

FlFlm Flcell

C.F .U.nO.D. p

G.F .P.mFl q

Scell d[G.F .P.]

dt

1

C.F .U.StotalC.F .U.

][

])[(][][

][)(][

IaS

IaMdt

Id

MtPoPsdt

Md

cell

I

M

PoPSSS M (aI )an

ScellSS

R.P.U. PoPS

SS

PoPSJ 23101SS

R.P.U. Scell,SS

Scell,J 23101SS

Measurement system• Tecan Infinite F200 Microplate reader

– bacterial incubation in multiwell plates– fluorescence and absorbance kinetics

• Experimental setup– optimized for promoter characterization– standard growth conditions

μl

Local evaporationthe “frame effect”

GFP vs O.D.600serial dilutions of fluorescent bacteria

O.D.600 vs culture concentrationSerial dilutions of bacteria

Bacterial growth in microplatevs falcon tube/flask

Device characterization steps:aTc sensor driven by J23118 promoter

Characterization results

β-galactosidase activity results

• X-Gal plates confirmed the cleaving capability of the Registry’s β-galactosidase

• Dynamic tests will be done to check if our system cleaves lactose more rapidly than the wild type one

Beta-gal generator expressed by Ptet

(TOP10)

Positive control (BW20767 strain)

Negative control (TOP10 with BBa_B0032)

Ethanol tolerance in TOP10 E. coli

• Toxicity threshold of ethanol: between 3.5 and 4.5 %w/v

Ethanol production results

Mean of three growth curves (96-well microplate) in LB+10% glucose: our engineered strains reach higher ODs than the negative control

Weak expression of the operon: normal

colonies

Strong expression of the operon: small

colonies

Conclusions• A complete ethanol producing operon has been assembled

and submitted using BioBrick Standard Parts.• The feasibility of conversion from lactose to ethanol, by a

synthetic biological device has been demonstrated.• The capability of engineered E. coli in ethanol production was

tested, still best fermentation conditions haven’t been found yet.

• A set of inducible devices and promoters has been tested, in order to fine regulate gene expression, like a genetic knob.

A lot of work has still to be done for:• Optimizing gene expression of our ethanol-producing operon• Speeding up the lactose cleaving process in E. coli.

Acknowledgements