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