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 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
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
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
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
Project overview Lactose cleaving module ? LACTOSE GLUCOSE Chassis used: E. coli Ethanol producing module ? GLUCOSE ETHANOL
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
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
E. coli fermentation pathway Wild type Engineered Theoretical yields: • 0.51 (g EtOH/g glucose) • 0.54 (g EtOH/g lactose)
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.
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
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
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
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)
Characterization results
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
Ethanol tolerance in TOP10 E. coli Toxicity threshold of ethanol: between 3.5 and 4.5% w/v
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
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
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
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