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iGEM Team
2009
Who are we?
Objectives of our Project
•Explore the concept of enzyme channeling
•Identify novel strategies to promote enzyme channeling
•Design a proof of concept system
•Model and Characterized proposed systems
What is it?
•The shuttling of products from one enzymatic reaction directly to a second with minimal diffusion.
Rationale: Why Enzyme Channeling?
Substrate Intermediate Product
Why Should We Care?
•Applications
• Improving Efficiencies of Enzyme Pairs with a Low K1
•Pathway Redirection
Rationale: Why Enzyme Channeling?
•Sequestering of Synthetic Pathways.
Alpha-ketoglutarate Dehydrogenase
So what are ways of optimizing Enzyme Channeling?
•Fusion Proteins
•Protein Scaffold
Optimization of Enzyme Channeling
Microcompartments
So what are Microcompartments?
•Microcompartments are small capsules formed by proteins
•Some have the ability to self assemble
•Contains pores
•Occurs naturally in some species of bacteria
•Channeling Unfavourable reactions (Rubisco in Blue Green)
•Toxic / Highly Reactive Intermediates (T.maritima)
Challenges of using Microcompartments in a System
•Assembly methods unknown
•Targeting
Microcompartment: Problems?
?
Rationale for the Use of Encapsulin
• Targeting Sequence has been elucidated
•On the C-terminal end of protein to be encapsulated
• Self-assembles
•Relatively well characterized
The Encapsulator
Encapsulin Protein from Thermotoga maritima.
250 Ao
Project Overview
Objectives
1) Design, construct and characterize a microcompartment expression system in E. coli.
2) Target a fluorescent marker (eCFP) to the micro-compartment.
3) Identify and prioritize candidate enzyme pairs for channeling.
4) Apply channeling to selected enzyme pairs.
Design Overview
eCFPtgt + Encapsulin
Control Module
Stacy Hung
Control Module
•Constitutive promoter (J23100)•Ribosomal binding sites (B0034) • TetR gene (C0040)•LacI gene (C0012)•Transcriptional terminators (B0010, B0012)
Controlled Expression System
Modeling – Simulations
FarhanRajaAccumulation of Encapsulin at different concentrations of aTc
Enca
psul
in m
onom
er
conc
entra
tion
(mol
/L)
Modeling – Simulations
Accumulation of eCFP monomer at different concentrations of IPTG
eCFP
con
cent
ratio
n (m
ol/L
)
Encapsulin Construct
•TetR repressible promoter (R0040)•Repression inhibited by aTc
•Encapsulin gene (K192000)
eCFPtgt Construct
•LacI repressible promoter (R0010)•Repression inhibited by IPTG
•eCFPtgt (K192001)•Encapsulin target sequence•LVA degradation tag
Proof of Concept
Multiple alignment of extension sequences of DyP and ferritin-like proteins
Sutter et al. (2008)
Proof of Concept
CFP + LVA degradation tag, Encapsulin targeting sequence
Proof of Concept
Degradation occurs, no fluorescence
Encapsulin protects, florescence
“Open source” targeted encapsulation
Protein of choiceTargeting sequence
Ready for localization!
Search for Suitable Enzyme Pairs
Bioinformatics Objective:Identify and prioritize candidate enzyme pairs for channeling
? ?+Meah Gao
1. Specific thermodynamic properties 2. Toxic metabolic intermediates3. Gene fusion products
Search for Suitable Enzyme Pairs
? ?+
Considerations:
1. Biochemically non-adjacent enzymes removed
2. Molecular weight > 100 kDa removed
Further refinements:
Suitable Enzyme Pairs
EC2 Pathway Major Product Applications6.3.4.4 Alanine and aspartate metabolism adenylosuccinate1.2.1.10 Butanoate metabolism Butanoyl-coA2.6.1.52 Glycine, serine and threonine metabolism Phosphoserine4.1.3.1 Glyoxylate and dicarboxylate metabolism Isocitrate3.5.4.9 One carbon pool by folate Formyl-THF3.5.4.9 One carbon pool by folate Formyl-THF5.3.1.12 Pentose and glucuronate interconversions D-Glucuronate5.1.3.1 Pentose phosphate pathway D-ribolose-5P5.3.1.9 Pentose phosphate pathway a-glucose-6P
4.2.1.51Phenylalanine, tyrosine and tryptophan biosynthesis Phenyl-pyruvate
2.7.2.15 Propanoate metabolism Propanoate Food preservatives
2.7.2.1Propanoate metabolism/Pyruvate metabolism Propanoate/acetate Energy
4.1.1.23 Pyrimidine metabolism UMP2.4.2.3 Pyrimidine metabolism Uridine
3.1.3.12 Starch and sucrose metabolism a,a trehalose biotech:overproduction
2.7.2.11 Urea cycle and metabolism of amino groups GlutamateFlavor enhancer, Nutrient, Plant growth
Alternative Microcompartments
1. Gene cluster contains only 7 genes, including 2 for microcompartment proteins
2. Presence of an intact and functional microcompartment enclosing enzymes
3. Targeting sequence?
Clostridium kluyveri
Experimental Results
•Encapsulin construct•Encapsulin gene submitted to 2009 iGEM Parts Registry (K192000)
•eCFPtgt construct •eCFPtgt synthesized•Additional work required
•Control module •Partially assembled•Additional work required
Kenny Zhan
Yen Leung
Conclusions
• Created a conceptual system theoretically capable of engineering metabolic channeling within bacteria cells
• Created a short list of enzymes amenable to targeted encapsulation
• Modeled various components in silico
• Submitted a new part to the registry, Encapsulin (K192000)
Future Directions
• Narrow search for enzyme pair
• Test alternative microcompartments
• Model Encapsulin assembly
• eCFP construct
• Characterization of control module
• Proof of Concept
Thank you!
Left to right: Farhan, James, Graham, John, Yen, Kenny, Meah, Stacy