Metabolic Engineering

SynBio4ALL Africa

Intermediate Course: Week 4

Instructor: Cholpisit Ice Kiattisewee

May 22^nd, 2024

Final Project Discussion

• An important social problem + a synthetic biology solution
• Form a group now!!! Submit ideas on Week 7
• Presents your ideas, respond to questions, �and provide feedbacks to other groups
• How to present? We will provide asynchronous option, likely video recording. ~To-Be-Determined~
• Have difficulty sharing screen? We can share screen for you if you have sharable materials available (e.g. Google Slide, Google Doc, Notion Page)
• Week 8-10 are dedicated to group projects. We will work together!!!

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Question: Have you form a group yet?

1. Yes!
2. Not yet, still working on it.
3. No idea what that is.

Overview of Today’s Lecture

1. Chemistry vs Metabolism vs Factory
1. Why do we care to Engineer Metabolism

​

• Discover New Chemistries
• Bioprospecting new Enzymes
• Engineering and Screening

​

• Optimize Existing Paths
• Finding the right strategy
• Tuning chemical reactions

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Chemical vs Bio-Production

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BASF plants in Germany (acs.org)

LanzaTech plants with ArcelorMittal Steel (acs.org)

Chemistry vs Metabolism vs Factory

Chemical vs Bio-Production

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Acetone

Clostridium acetobutylicum

Heat and Pressure

~83% of global production came from this process

ABE pathway

Chemistry vs Metabolism vs Factory

Chemical vs Bio-Production

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Clostridium acetobutylicum

Acetone-Butanol-Ethanol

pathway

How many chemical reactions are in this ABE pathway?

1. <10
2. 10-20
3. >20

What about this?

Chemistry vs Metabolism vs Factory

Chemical vs Bio-Production

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Saccharomyces cerevisiae

Ethanol Fermentation

pathway

Heat, Pressure, and Catalyst

Was used a lot in 1960s

Majority of biofuels nowadays are bioethanol

Chemistry vs Metabolism vs Factory

Chemical vs Bio-Production

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Tokyo Train (Japan)

There are lots of unseen connections

Chemistry vs Metabolism vs Factory

Chemical vs Bio-Production

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Tokyo Train (Japan)

Chemistry vs Metabolism vs Factory

Metabolism is very complex!!!

Chemical vs Bio-Production

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Lee et al. Nature Catalysis. 2019

Bio-only industrial chemicals&materials

Bio+Chemical industrial chemicals&materials

Chemistry vs Metabolism vs Factory

Key Challenges

1. New Products
2. More Yields

Finding New Reactions: Known Genes

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Discover New Chemistries

A

B

C

D

E. coli

+ crtEB

= carotenoids

+ crtL

= Lycopene

+ crtY

= beta-Carotene

+ crtZ

= Zeaxanthin

We’re taking crt pathway from plants and put onto E. coli, will it function right away?

1. Yes: E. coli is smart
2. No: E. coli doesn’t know plant language

Finding New Reactions: Known Genes

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Discover New Chemistries

A

B

C

D

X

E. coli

+ crtEB

= carotenoids

+ crtL

= Lycopene

+ crtY

= beta-Carotene

+ crtZ

= Zeaxanthin

+ crtW

= Astaxanthin

Zhu et al. Molecular Plant 2018

Can also be engineered into other organisms

Rice with additional carotenoid nutrients

Finding New Reactions: Unknown?

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Discover New Chemistries

A

B

C

D

X

E. coli

+ crtEB

= carotenoids

+ crtL

= Lycopene

+ crtY

= beta-Carotene

+ crtZ

= Zeaxanthin

+ crtW

= Astaxanthin

Y

+ unknown gene

= blue pigment?

New enzymes are necessary for new chemistries

Finding New Reactions: Unknown?

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Discover New Chemistries

A

B

C

D

X

E. coli

+ crtEB

= carotenoids

+ crtL

= Lycopene

+ crtY

= beta-Carotene

+ crtZ

= Zeaxanthin

+ crtW

= Astaxanthin

Y

+ unknown gene

= blue pigment?

Wongsatit & Srimora et al. Curr. Opin. Syst. Biol. 2024

New enzymes are necessary for new chemistries

Finding New Reactions: Oxygenation

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Discover New Chemistries

King et al. bioRxiv. 2024

Search for enzymes promiscuous activity

De novo design boost enzyme activity

Path Optimization

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Optimize Existing Paths

Where to start?

There are so many ways…

Path Optimization: Wrong Direction

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A

B

C

D

X

E. coli

+ crtEB

= carotenoids

+ crtL

= Lycopene

+ crtY

= beta-Carotene

+ crtZ

= Zeaxanthin

+ crtW

= Astaxanthin

Optimize Existing Paths

Path Optimization: Wrong Direction

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Optimize Existing Paths

A

B

C

D

X

E. coli

+ crtEB

= carotenoids

+ crtL

= Lycopene

+ crtY

= beta-Carotene

+ crtZ

= Zeaxanthin

+ crtW

= Astaxanthin

Z

Enzyme Z’ keep pulling B away.

What should we do?

Undesired product

Enzyme Z’

1. Get rid of enzyme Z’ entirely
2. Reduce the amount of enzyme Z’ to bare minimum
3. Find another route to make C

Path Optimization: Push-Pull-Block

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Optimize Existing Paths

Desired product

Block

Push

Pull

CRISPR/Cas9 🡪 Tuning knob

Fontana, Sparkman-Yager, & Faulkner et al. bioRxiv. 2023

Path Optimization: Push-Pull-Block

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Optimize Existing Paths

CRISPR/Cas9 🡪 Tuning knob

Fontana, Sparkman-Yager, & Faulkner et al. bioRxiv. 2023

Fine-Tune

Path Optimization: Push-Pull-Block

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Optimize Existing Paths

CRISPR/Cas9 🡪 Tuning knob

Fontana, Sparkman-Yager, & Faulkner et al. bioRxiv. 2023

Fine-Tune

Path Optimization: In-and-Out

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Optimize Existing Paths

Incorporation of transporter proteins are usually important

Path Optimization: Growth-vs-Work

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Optimize Existing Paths

Limited Resources

Resources largely allocated to growth/biomass

Resources largely allocated to production

Path Optimization: Choose Start Point

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Optimize Existing Paths

Feedstock

Sugar

Lignin

CO₂

Feedstock

Sugar

Lignin

CO₂

Feedstock

Sugar

Lignin

CO₂

Tolerance

Heat

Salt

Organic

Tolerance

Heat

Salt

Organic

Tolerance

Heat

Salt

Organic

Chassis A

Chassis B

Chassis C

Different cell factories are appropriate for different environments

Will be further discussed on Lecture 7

~~~Microbes for Sustainability~~~

Notable Chassis

• E. coli
• P. putida
• B. subtilis
• C. glutamicum
• C. necator
• R. sphaeroides
• S. elongatus
• S. cerevisiae

Design-Build-Test-Learn Approaches

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Special Topics

Volk, Tran, & Tan et al. Chem. Rev. 2023

AI/ML, Automation, and Self-Driving

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Special Topics

Martin et al. Curr. Opin. Biotechnol. 2023

Set-up the system to operate with minimal set at minimal time spent

Program pathways at DNA level

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Special Topics

Plasmid/

Genomic

DNA

Revisit Week 3 Lecture for strain engineering approaches

Transporter Engineering

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Special Topics

Introduce transporters significantly improve substrate utilization (e.g. C5 sugar)

Volk, Tran, & Tan et al. Chem. Rev. 2023

Artemisinin Biosynthesis

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Special Topics

Volk, Tran, & Tan et al. Chem. Rev. 2023

Complete biosynthesis of the potent vaccine adjuvant QS-21

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Special Topics

Martin et al. Nat. Chem. Biol. 2023 in Tobacco

Liu et al. Nature 2024 in Yeast

Useful Resources

• iBiology Lectures on Synthetic Biology & Metabolic Engineering
• Kris Prather (MIT) Part 1 and Kris Prather (MIT) Part 2
• Reviews
• Volk, Tran, & Tan et al. Metabolic Engineering: Methodologies and Applications
• Intasian & Prakinee et al. Enzymes, In Vivo Biocatalysis, and Metabolic Engineering for Enabling a Circular Economy and Sustainability
• Martin et al. Perspectives for self-driving labs in synthetic biology
• Examples
• Computational stabilization of a non-heme iron enzyme enables efficient evolution of new function
• From Golden Rice to aSTARice: Bioengineering Astaxanthin Biosynthesis in Rice Endosperm
• Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling

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That’s it~~~

Any questions?

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