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