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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


  1. Outline  Why whey ?  Engineering whey fermentation to ethanol using BioBrick parts  Promoters characterization  Ethanol production and conclusions

  2. Motivation: why whey?  Residue of cheese curdling in dairy industries  High nutritional load  proliferation of water microorganisms  water Cheese whey composition asphyxia after extraction Components % w/v  Special waste for Italian law Proteins 0,75 (B.O.D.5 2000 times higher Fat 0,40 than legal limit) Lactose 4,6 Ash 0,012

  3. Cheese whey valorization  Substances of interest: WHEY DRY WHEY  Whey proteins  Purified fatty acids  Dry whey  The residual liquid of these RESIDUAL treatments is still a special ULTRA-FILTRATION / LIQUID CRYSTALLIZATION waste for its high lactose (rich in lactose) content (~4.5%)  Complete lactose extraction and purification is not convenient.  New valorization techniques WHEY FATTY ACIDS should be developed. PROTEINS

  4. 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 can be fermented into glucose by some microorganisms ethanol by many microbiota (such (such as E. coli ) as S. cerevisae ) GLUCOSE LACTOSE O O PYRUVATE O CH 3
 
H +
 
CO 2
 GLUCOSE H O ACETALDEHYDE CH 3
 NADH
+
H +
 
NAD +
 H OH
 Problem: no wild type organism is able ETHANOL H CH 3
 to perform both functions efficiently

  5. Engineering lactose fermentation pathway  Whey can be LACTOSE considered as a free feedstock GLUCOSE  Design a new O synthetic O PYRUVATE O biological system CH 3
 
H +
 
CO 2
 able to convert H O ACETALDEHYDE lactose into CH 3
 NADH
+
H +
 ethanol with high 
NAD +
 H OH
 ETHANOL efficiency H CH 3


  6. Project overview  Lactose cleaving module ? LACTOSE GLUCOSE Chassis used: E. coli  Ethanol producing module ? GLUCOSE ETHANOL

  7. Lactose cleaving module Alpha‐D‐ glucose
 D‐galactose
 Lactose
  E. coli β -galactosidase breaks lactose with high efficiency  β -galactosidase overexpression to increase lactose cleaving capability B0012
 B0010
 B0034
 LacZ
 PoPs
input


  8. Ethanol producing module pyruvate
  Zymomonas mobilis is an ethanologenic bacterium of the soil pdc  Pyruvate decarboxilase (pdc) acetaldehyde
  Alcohol dehydrogenase II (adhB)  Genes were designed adhB with codon usage bias optimization in E. coli ethanol
 B0030
 B0010
 B0012
 adhB
 pdc 
 B0030
 PoPs
input


  9. E. coli fermentation pathway Wild type Engineered Theoretical yields: • 0.51 (g EtOH/g glucose) • 0.54 (g EtOH/g lactose)

  10. Quantitative characterization: why? B0034
 B0010
 B0012
 lacZ 
 PoPs
input
 B0030
 B0010
 B0012
 pdc 
 adhB
 B0030
 PoPs
input
  Inducible systems: well characterized gene expression knobs to choose best promoter for our actuator.

  11. Inducible promoters used  Lac promoter (BBa_R0011), BBa_J231xx PLac
 J23100
  aTc inducible devices (BBa_K173007, BBa_K173011) tetR
 B0010
 B0012
 Ptet
 J23100/J23118
 B0034
  3OC6-HSL receiver device: BBa_F2620 LuxR
 lux
pR
 B0034
 B0010
 B0012
 pTet


  12. 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 dF 1 φ dt ⋅  R.P.U. estimation OD 600, φ R . P . U . φ = dF J 23101 1 ⋅ dt OD 600, J 23101  Blank subtraction

  13. Measurement system  TECAN Infinite F200 Microplate reader Local
evapora4on
 the
“frame
effect”
  Bacterial incubation in multi-well plates  Fluorescence and absorbance kinetics μl
  Experimental setup  Optimized for promoter characterization  Standard growth conditions GFP
vs
O.D.600
 O.D.600
vs
culture
concentra4on
 Bacterial
growth
in
microplate
 serial
diluKons
of
fluorescent
bacteria
 Serial
diluKons
of
bacteria
 vs
falcon
tube/flask


  14. Device characterization steps: aTc sensor driven by BBa_J23118 promoter 1,8 1,6 1,4 1,2 R.P.U. 1 0,8 0,6 0,4 0,2 0 0 100 200 300 400 aTc induction (ng/ml)

  15. Characterization results

  16. β -galactosidase activity results Beta‐gal
generator
 NegaKve
control
(TOP10
 PosiKve
control
 expressed
by
Ptet
 with
BBa_B0032)
 (BW20767

strain)
 (TOP10)
  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

  17. Ethanol tolerance in TOP10 E. coli  Toxicity threshold of ethanol: between 3.5 and 4.5% w/v

  18. Ethanol production results (phenotype) High Copy Number plasmid with different promoters Strong
expression
of
the
 Weak
expression
of
the
operon:
 operon:
 small
colonies
 normal
colonies


  19. Ethanol production results (quantitative) Experimental
condiKons:
 • 
24h
of
fermentaKon
in
10%
glucose
 • 
homoserine
lactone
sensing
promoter
 (Plux)
 • 
HC/LC
induced
 • 
HC/LC
not
induced
(exploiKng
Plux
leakage)
 Mean
of
three
growth
curves
(96‐well
microplate)
in
LB+10%
 glucose:
our
engineered
strains
reach
higher
ODs
than
the
 negaKve
control


  20. 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

  21. 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

  22. Acknowledgements Università degli Studi di Pavia

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