Outline

 Why whey?  Engineering whey fermentation to

ethanol using BioBrick parts  Promoters characterization  Ethanol production and conclusions

Motivation: why whey?

  Residue of cheese curdling in dairy industries

  High nutritional load proliferation of water microorganisms water asphyxia

  Special waste for Italian law (B.O.D.5 2000 times higher than legal limit)

Cheese whey composition after extraction

Components % w/v

Proteins 0,75

Fat 0,40

Lactose 4,6

Ash 0,012

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.

WHEY

ULTRA-FILTRATION / CRYSTALLIZATION

RESIDUAL LIQUID

(rich in lactose)

FATTY ACIDS WHEY

PROTEINS

DRY WHEY

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   It is produced from feedstocks such as sugar cane by fermentation

  Lactose can be easily converted into glucose by some microorganisms (such as E. coli)

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

GLUCOSE

LACTOSE

GLUCOSE

PYRUVATE

ACETALDEHYDE

ETHANOL

O

CH3

O

O

O

CH3

H

H+

CO2

NADH+H+

NAD+

OH

CH3

H

HProblem: no wild type organism is able

to perform both functions efficiently

Engineering lactose fermentation pathway

  Whey can be considered as a free feedstock

  Design a new synthetic biological system able to convert lactose into ethanol with high efficiency

GLUCOSE

PYRUVATE

ACETALDEHYDE

ETHANOL

O

CH3

O

O

O

CH3

H

H+

CO2

NADH+H+

NAD+

OH

CH3

H

H

LACTOSE

Project overview

  Lactose cleaving module

  Ethanol producing module

? LACTOSE GLUCOSE

GLUCOSE ETHANOL

Chassis used: E. coli

?

Lactose cleaving module

  E. coli β-galactosidase breaks lactose with high efficiency

  β-galactosidase overexpression to increase lactose cleaving capability

Alpha‐D‐glucose

D‐galactose

Lactose

B0034B0010 B0012

LacZ

PoPsinput

Ethanol producing module

  Zymomonas mobilis is an ethanologenic bacterium of the soil

  Pyruvate decarboxilase (pdc)

  Alcohol dehydrogenase II (adhB)

  Genes were designed with codon usage bias optimization in E. coli

pyruvate

acetaldehyde

ethanol

pdc

adhB

B0030 B0010 B0012pdc adhBB0030

PoPsinput

E. coli fermentation pathway

Wild type

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

Engineered

Quantitative characterization: why?

  Inducible systems: well characterized gene expression knobs to choose best promoter for our actuator.

B0030 B0010 B0012pdc adhBB0030

PoPsinput

B0034 B0010 B0012lacZ

PoPsinput

Inducible promoters used

 Lac promoter (BBa_R0011), BBa_J231xx

 aTc inducible devices (BBa_K173007, BBa_K173011)

 3OC6-HSL receiver device: BBa_F2620

pTet B0034 LuxR luxpRB0010 B0012

PLac J23100

B0034 tetR PtetB0010 B0012J23100/J23118

Relative Promoter Units

  Approach for promoter strength quantitative measurement (Kelly J. et al., 2008)

  Standard approach: reproducibility across labs   Relative units: use of a reference standard promoter   R.P.U. computation steps:

 Hypothesis:  Steady state for gene expression and proteins synthesis

 R.P.U. estimation

 Blank subtraction

€

R.P.U.φ =

dFφdt

⋅1

OD600,φ

dFJ 23101dt

⋅1

OD600,J 23101

Measurement system

  TECAN Infinite F200 Microplate reader   Bacterial incubation in multi-well plates   Fluorescence and absorbance kinetics

  Experimental setup   Optimized for promoter characterization   Standard growth conditions

μl

Localevapora4on

the“frameeffect”

GFPvsO.D.600

serialdiluKonsoffluorescentbacteriaO.D.600vscultureconcentra4on

SerialdiluKonsofbacteria

Bacterialgrowthinmicroplate

vsfalcontube/flask

Device characterization steps: aTc sensor driven by BBa_J23118 promoter

0 0,2 0,4 0,6 0,8

1 1,2 1,4 1,6 1,8

0 100 200 300 400

aTc induction (ng/ml)

R.P.U

.

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‐galgeneratorexpressedbyPtet

(TOP10)

PosiKvecontrol(BW20767strain)

NegaKvecontrol(TOP10withBBa_B0032)

Ethanol tolerance in TOP10 E. coli

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

Ethanol production results (phenotype)

Weakexpressionoftheoperon:normalcolonies

Strongexpressionoftheoperon:smallcolonies

High Copy Number plasmid with different promoters

Ethanol production results (quantitative)

Meanofthreegrowthcurves(96‐wellmicroplate)inLB+10%glucose:ourengineeredstrainsreachhigherODsthanthenegaKvecontrol

ExperimentalcondiKons:• 24hoffermentaKonin10%glucose• homoserinelactonesensingpromoter(Plux)• HC/LCinduced• HC/LCnotinduced(exploiKngPluxleakage)

Conclusions 1/2

 The ethanol producing operon was tested and promising working conditions were found.

 Lactose conversion to ethanol is feasible and we have shown that our machine is suitable for biofuel production

Conclusions 2/2

  27 new parts have been submitted to the Registry.   A standard measurement method (R.P.U.) was validated and

used to characterize the activity of several promoters and devices.

  10 standard parts and devices have been characterized, including tunable gene expression knobs in order to choose the optimal promoter for our actuators.

  Additional results:   PnhaA promoter has been tested as a pH/Na+ sensor   Enterobacteria Phage T4 Lysis actuator has been characterized

  Sequence debugging of 12 existing parts, including BBa_T9002 and BBa_F2620

  A software of composite parts sequence alignment has been developed

Acknowledgements

Università degli Studi di Pavia