Outline Why whey ? Engineering whey fermentation to ethanol using - - PowerPoint PPT Presentation

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 H

Problem: 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


B0034
 B0010
 B0012
 LacZ


PoPs
input


Ethanol producing module

 Zymomonas mobilis is an

ethanologenic bacterium

• f the soil

 Pyruvate decarboxilase

(pdc)

 Alcohol dehydrogenase

II (adhB)

 Genes were designed

with codon usage bias

• ptimization in E. coli

pyruvate
 acetaldehyde
 ethanol
 pdc adhB

B0030
 B0010
 B0012


pdc


adhB
 B0030


PoPs
input


• 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
 B0012


pdc


adhB
 B0030


PoPs
input


B0034
 B0010
 B0012


lacZ


PoPs
input


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
 lux
pR
 B0010
 B0012


PLac
 J23100


B0034
 tetR
 Ptet
 B0010
 B0012
 J23100/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 = dF

φ

dt ⋅ 1 OD600,φ dFJ 23101 dt ⋅ 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


Local
evapora4on
 the
“frame
effect”
 GFP
vs
O.D.600
 serial
diluKons
of
fluorescent
bacteria
 O.D.600
vs
culture
concentra4on
 Serial
diluKons
of
bacteria
 Bacterial
growth
in
microplate
 vs
falcon
tube/flask


Device characterization steps: aTc sensor driven by BBa_J23118 promoter

0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 100 200 300 400

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‐gal
generator
 expressed
by
Ptet
 (TOP10)
 PosiKve
control
 (BW20767

strain)
 NegaKve
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 (phenotype)

Weak
expression
of
the
operon:
 normal
colonies
 Strong
expression
of
the


• peron:
small
colonies


High Copy Number plasmid with different promoters

Ethanol production results (quantitative)

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


Experimental
condiKons:


• 24h
of
fermentaKon
in
10%
glucose

• homoserine
lactone
sensing
promoter


(Plux)


• HC/LC
induced

• HC/LC
not
induced
(exploiKng
Plux
leakage)


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

• ptimal 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