their ability to fully capture in vivo enzyme activity. Goal - - PowerPoint PPT Presentation

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their ability to fully capture in vivo
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their ability to fully capture in vivo enzyme activity. Goal - - PowerPoint PPT Presentation

Oct 31, 2023 228 likes •658 views

Problem Current approaches are limited in their ability to fully capture in vivo enzyme activity. Goal Express and optimize the bacterial lux system in Saccharomyces Cerevisiae to find rate-limiting steps via stoichiometric control of enzyme

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Problem

Current approaches are limited in their ability to fully capture in vivo enzyme activity.

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Goal

Express and optimize the bacterial lux system in Saccharomyces Cerevisiae to find rate-limiting steps via stoichiometric control of enzyme levels.

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Problem Statement

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D

Reductase Alpha Subunit Beta Subunit Synthetase

luxC luxD luxA luxB luxE

β α C E

RNA Polymerase

Transferase

LUX SYSTEM

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luxC luxD luxA luxB luxE

β α C D E β α

Reductase Alpha Subunit Beta Subunit Synthetase Transferase Luciferase

LUX SYSTEM

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C D E β α

Reductase Synthetase Transferase Luciferase

luxC luxD luxA luxB luxE

LUX SYSTEM

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D

FATTY ACID

RCOOH

LUX SYSTEM

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D E E E E C C C C

FATTY ACID REDUCTASE COMPLEX FATTY ACID

RCOOH

LUX SYSTEM

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RCOOH

D E E E E C C C C

FATTY ACID REDUCTASE COMPLEX FATTY ACID

RCOOH RCHO

ALDEHYDE

LUX SYSTEM

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O2

RCHO

D E E E E C C C C

FATTY ACID REDUCTASE COMPLEX

FMNH2

FATTY ACID

RCOOH

ALDEHYDE REDUCED FLAVIN

FMNH2

β α

LUCIFERASE

RCHO

O2

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SLIDE 12

RCHO

D β α E E E E C C C C

FATTY ACID REDUCTASE COMPLEX LUCIFERASE

LIGHT

FMNH2 FMN

O2

H2O

FATTY ACID

RCOOH

ALDEHYDE REDUCED FLAVIN OXIDIZED FLAVIN

RCOOH

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STOICHIOMETRIC PROTEIN PRODUCTION

C D E C D E C D E C D E C C D D E E E

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PLASMID DESIGN

Feature Purpose Bidrectional Promoter Modular plasmid construction Enhanced LuxAB Autonomous production of luminescence frp FMNH2 regeneration P2A Linkers Stoichiometric protein expression C D E AB frp Dup

P2A Linker

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Benefits

+ Optimize lux system in a eukaryotic organism + Improve flexibility and strength as reporter system + Develop modular platform to optimize other metabolic pathways

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Problem Statement

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Problem Statement

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Chemical Reaction Network

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Chemical Reaction Network

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Chemical Reaction Network

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Chemical Reaction Network

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Chemical Reaction Network

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Reaction Equations

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Reaction Equations

WelhamP, Stekel D (2009) Mathematical model of the lux luminescence system in the terrestrial bacterium Photorhabdus luminescens. Mol Biosyst 5(1):68–76. Iqbal M. , Stekel D. (2015). An extended mathematical model of Lux bioluminescence in bacteria. [unpublished]

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System of Differential Equations

WelhamP, Stekel D (2009) Mathematical model of the lux luminescence system in the terrestrial bacterium Photorhabdus luminescens. Mol Biosyst 5(1):68–76. Iqbal M. , Stekel D. (2015). An extended mathematical model of Lux bioluminescence in bacteria. [unpublished]

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Model Assumptions

C D E D

νLuxD = 2νLuxD LightLuxCDE = LightLuxCDE_E = LightLuxCDE_C [O2], [NADPH], [ATP], [H+], [H2O] : constant

1 2 3 4

Isolated system

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Simulation Results

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Simulation Results

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Simulation Results

B

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Simulation Results

C

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Simulation Results

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Problem Statement

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Problem Statement

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Reaction 1 A + B -> C Reaction 2 C + D -> E Reaction 3 A + D -> F . . . Reaction N 1 2 3 … A

  • 1
  • 1

… B

  • 1

… C 1

  • 1

… D

  • 1
  • 1

… E 1 … F 1 … V1 V2 V3 V4 V5 V6 V7 S v b

Flux Balance Analysis

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Infinite Solution Space Physical Constraints Flux Boundaries: Lower Bound < vreaction < Upper Bound

Constraint-Based Modeling

Compartments: mitochondria, cytoplasm, extracellular, …

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Light and growth are strongly coupled!

Metabolic Burden

(mmol/gDW*hr) (mmol/gDW*hr)

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Reactions Coupled to Light

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Pentose Phosphate Pathway Glycolysis TCA Cycle Fatty Acid Biosynthesis Lux System Light

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FUTURE DIRECTIONS

Fitting model to experimentally validated parameters

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THANK YOU

Team Phillip Kyriakakis Bart Borek Jahir Gutierrez Todd Coleman