Synthetic Biology/Metabolic Engineering
Transcript of Synthetic Biology/Metabolic Engineering
SYNTHETIC BIOCHEMISTRY
Making Chemicals the Cell-Free Way
James BowieUCLA/Invizyne Technologies
B C D ENADPH NADP+
CoA
ATP ADP
The Problem(s) with Cells
P
Solution: Synthetic Biochemistry
B C D ENADPH NADP+
CoA
ATP ADP
Engineered Microbe
Plasmid A
P
A B C Z product
Synthetic Biochemistry
1. Design Biochemical Transformation
2. Clone/Express Enzymes
3. Purify/Isolate Activities
4. Mix Enzymes
ATPCoA
NADPH
Input:Glucose
Output:Product
(Commodity Chemicals, Drug Intermediates,
Biofuels)
5. Run Bioreactor
Synthetic Biochemistry
• High yields
• Easy optimization/Total Control
• Rapid Design-Build-Test Cycles
• Great flexibility in pathway design
• No toxicity headaches
• Easier product purification
• Potential for much higher productivity
Main Advantages
Main Challenges
• Enzyme cost
• Enzyme stability
• Cofactor Recycling and
Maintenance
Synthetic Biochemistry
The GoalSystems that function continuously
for long periods of time without
intervention
Synthetic Biochemistry System Design
Perfect cofactor balance is not ideal
Sugar
Fuel
nNAD+
nNADH
nNADH
nNAD+
Spontaneous oxidation
nADP
nATP
nATP
nADP
Spontaneous hydrolysis
Breakdown
Phase
Build
Phase
Example: Molecular Rheostat for Maintaining ATP levels
Glucose
Top Glycolysis
Bottom Glycolysis
Pyruvate
G-3P
1,3-BPG
3-PG
PiNADP+
NADPH
ADP
mGap
Pgk
NADP+
NADPH
GapN
Nature Chem. Biol., 2017
ATP
ADP + Pi
Example: Molecular Rheostat for Maintaining ATP levels
Glucose
Top Glycolysis
Bottom Glycolysis
Pyruvate
G-3P
1,3-BPG
3-PG
PiNADP+
NADPH
ADP
mGap
Pgk
NADP+
NADPH
GapN
Nature Chem. Biol., 2017
ATP
High Pi/Low ATP
Example: Molecular Rheostat for Maintaining ATP levels
Glucose
Top Glycolysis
Bottom Glycolysis
Pyruvate
G-3P
1,3-BPG
3-PG
Pi
NADP+
NADPH
ADP
mGap
Pgk
NADP+
NADPH
GapN
Nature Chem. Biol., 2017
ATP
Low Pi/High ATP
Valliere M et al., Nature Comm, 2019
Opgenorth PH, et. al. Nat Chem Biol 2017
Korman TP, et. al. Nat Comm 2017
Opgenorth PH, et. al. Nat Chem Biol 2016
Opgenorth PH, et. al. Nat Comm 2014
Developed Flexible “Purge Valve” and “Rheostat” Systems
to Effectively Manage Cofactor Levels
Rheostat
(ATP)
Purge Valve
(NAD(P)H)
Cell Free should be better at making toxic chemicalsLet’ try!
Toxic to cells
Limit is ~20 g/L, but productivity falls off much sooner.
Isobutanol: Commodity chemical and biofuel
First Try at Isobutanol Production
Cell-Free Isobutanol Pathway Design
Opgenorth PH, et. al. Nat Chem Biol 2017
• Max Productivity: 1.8 g/L/h• Final titer: 24.1 ± 1.8 g/L• Yield > 91%
Stoichiometrically balanced
“Managed” ATP excess
The Cell-Free Approach Works
Valliere M et al., Nature Comm, 2019
Opgenorth PH, et. al. Nat Chem Biol 2017
Korman TP, et. al. Nat Comm 2017
Opgenorth PH, et. al. Nat Chem Biol 2016
Opgenorth PH, et. al. Nat Comm 2014
Some results so far
Isobutanol (Commodity Chemical): 275 g/L (>10X better than cell-based)
4 g/l/hr, >90% yields (UNPUBLISHED)
Monoterpenes (Diverse uses): 16 g/L (10x toxicity limit)
0.1 g/L/hr, >90% yields
Cannabinoids (Pharmaceutical): >1.5 g/L (>100-fold better than cells based)
0.1 g/L/hr, >90% yields
Results obtained rapidly with small teams and limited
resources—the power of the cell-free approach!
The Potential of Synthetic Biochemistry
Fermentation
Ethanol Production
Thousands of years of optimization
Productivity: ~2 g/L/hr
Yield: 85-95 %
Titer: ~100 g/L
Time: 1-3 days
Synthetic Biochemistry
Isobutanol Now
~4 person year
Prod.: 4 g/L/hr
Yield: ~95%
Titer: 275 g/L
Time: 4 days
Isobutanol Future?
Prod.: 20 g/L/hr?
Yield: ~95%
Titer: 1000 g/L?
Time: 5 days?
?
Goal: carbon neutral or negative production of chemicals
We don’t need to be tied to one approach
Carbon Feedstock CO, CO2/H2
Acetate
PyruvateLactate
Ethanol
Succinate
Cells do some things really well
CO, H20, CO2
Acetate
Electrochemistry does some things really well
Formate Ethanol
Ethylene
Breakdown ModuleMostly Glycolysis
Build ModuleMevalonate Pathway
Possible combined approach to zero carbon, electrically powered cell-free carbon upgrading
1.5
Glucose
1.5 G6P
1.5 F6P
1.5 FBP
3xG3P
3xBPG
3x3PG
3x2PG
3xPEP
P
P
3xPYR
3 Acetyl-CoA
0-6 NADPH
3 ATP
1.5 DHAP
ACAC-CoA
3 Acetyl-CoA
HMG-CoA
MEV
MEV-P
MEV-PP
IPPDMAPP
GPP
Monoterpenes
Build ModuleMevalonate Pathway Acetate
CO, CO2/H2
Power Converter
System
e-NAD(P)H
ATP
Possible combined approach to zero carbon, electrically powered cell-free carbon upgrading
?
ACAC-CoA
3 Acetyl-CoA
HMG-CoA
MEV
MEV-P
MEV-PP
IPPDMAPP
GPP
Monoterpenes
Acknowledgements
Tyler Korman
Paul Opgenorth
Salem Faham
Meaghan Valliere
Sum ChanSakenSherkhanov
Leo Liu
Chong Liu