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

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 levels.

Problem Statement

LUX SYSTEM Alpha Subunit Beta Subunit RNA Polymerase α β Reductase C Synthetase Transferase E D luxE luxD luxA luxB luxC

LUX SYSTEM Alpha Subunit Luciferase Beta Subunit α β α β Reductase C Synthetase Transferase E D luxE luxD luxA luxB luxC

LUX SYSTEM Luciferase α β Reductase C Synthetase Transferase E D luxE luxD luxA luxB luxC

LUX SYSTEM FATTY ACID RCOOH D

LUX SYSTEM E E C C FATTY ACID REDUCTASE C C COMPLEX E E FATTY ACID RCOOH D

LUX SYSTEM RCHO ALDEHYDE E E C C FATTY ACID REDUCTASE C C COMPLEX E E FATTY ACID RCOOH RCOOH D

REDUCED FLAVIN RCHO RCHO FMNH 2 FMNH 2 ALDEHYDE O 2 O 2 E E C C FATTY ACID α β LUCIFERASE REDUCTASE C C COMPLEX E E FATTY ACID RCOOH D

REDUCED FLAVIN RCHO FMNH 2 ALDEHYDE O 2 E E C C FATTY ACID RCOOH α β LUCIFERASE REDUCTASE C C COMPLEX E E OXIDIZED FLAVIN FMN FATTY ACID RCOOH H 2 O LIGHT D

STOICHIOMETRIC PROTEIN PRODUCTION C D E C C D E E C D E D E C D C D E E

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

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

Problem Statement

Problem Statement

Chemical Reaction Network

Chemical Reaction Network

Chemical Reaction Network

Chemical Reaction Network

Chemical Reaction Network

Reaction Equations

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 [unpublished]

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 [unpublished]

Model Assumptions ν LuxD = 2 ν LuxD 1 D C D E 2 Light LuxCDE = Light LuxCDE_E = Light LuxCDE_C 3 [O 2 ], [NADPH], [ATP], [H + ], [H 2 O] : constant Isolated system 4

Simulation Results

Simulation Results

Simulation Results B

Simulation Results C

Simulation Results

Problem Statement

Problem Statement

Reaction 1 A + B -> C Reaction 2 C + D -> E Flux Balance Analysis Reaction 3 A + D -> F . . . Reaction N 1 2 3 … V1 A -1 0 -1 … V2 B -1 0 0 … V3 0 C 1 -1 0 … V4 D 0 -1 -1 … V5 E 0 1 0 … V6 F 0 0 1 … V7 S v b

Constraint-Based Modeling Physical Constraints Infinite Solution Space Compartments: mitochondria, cytoplasm, extracellular, … Flux Boundaries: Lower Bound < v reaction < Upper Bound

Metabolic Burden (mmol/gDW*hr) (mmol/gDW*hr) Light and growth are strongly coupled!

Reactions Coupled to Light

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

FUTURE DIRECTIONS Fitting model to experimentally validated parameters

Team THANK YOU Bart Borek Phillip Kyriakakis Jahir Gutierrez Todd Coleman