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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