iGEM Team 2009 Who are we? Objectives of our Project Explore the - - PowerPoint PPT Presentation

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 A

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

Farhan Raja

Accumulation of Encapsulin at different concentrations of aTc

Encapsulin monomer concentration (mol/L)

Modeling – Simulations

Accumulation of eCFP monomer at different concentrations of IPTG eCFP concentration (mol/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 choice Targeting 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 intermediates
• 3. 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 Applications 6.3.4.4

Alanine and aspartate metabolism

adenylosuccinate 1.2.1.10

Butanoate metabolism

Butanoyl-coA 2.6.1.52

Glycine, serine and threonine metabolism

Phosphoserine 4.1.3.1

Glyoxylate and dicarboxylate metabolism

Isocitrate 3.5.4.9

One carbon pool by folate

Formyl-THF 3.5.4.9

One carbon pool by folate

Formyl-THF 5.3.1.12

Pentose and glucuronate interconversions

D-Glucuronate 5.1.3.1

Pentose phosphate pathway

D-ribolose-5P 5.3.1.9

Pentose phosphate pathway

a-glucose-6P 4.2.1.51

Phenylalanine, tyrosine and tryptophan biosynthesis

Phenyl-pyruvate 2.7.2.15

Propanoate metabolism

Propanoate Food preservatives 2.7.2.1

Propanoate metabolism/Pyruvate metabolism

Propanoate/acetate Energy 4.1.1.23

Pyrimidine metabolism

UMP 2.4.2.3

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 Glutamate

Flavor 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