Engineering of In Vivo Nanobioreactors

Modern microbial engineering methods allow the introduction of useful

exogenous metabolic pathways into cells. Metabolism of certain organic

compounds is sometimes limited by the production of toxic intermediates.

Several bacteria have evolved protein based microcompartments capable of

sequestering such reactions, thus protecting cytosolic machinery and processes

from interference by these intermediates. For our iGEM project, we have

cloned (and expressed in Escherichia coli) Salmonella enterica LT2 genes

responsible for the production and assembly of ethanolamine utilization

microcompartments. Additionally, we have demonstrated that a signal

sequence targets an ethanolamine utilization enzyme to the

microcompartment by fusing the sequence to GFP and observing that this

causes the GFP reporter to localize to the compartment. We conclude that

recombinant microcompartments housing targeted enzymes can function as in

vivo bioreactors with high reaction efficiencies.

Abstract

Bacterial Microcompartments (BMCs)

Proposed nanobioreactor model.

(S = substrate, P = product and E1, E2 = enzymes)

• in vivo nanobioreactors can be created by engineering BMCs to encapsulate

targeted enzymes

• Sequestration of enzymes and substrates is expected to increase the overall

efficiency of the reaction

• Prevent dispersion of volatile intermediate

• Protect cell machinery by quarantining toxic intermediate

• Further understand the cell biology and metabolism of prokaryotes

1. Can we form recombinant Eut BMCs within E. coli?

2. Can we target proteins into Eut BMCs?

Challenge

Rationale

Cloning Strategy

Protein Expression

In Memoriam

Ethan, our dear friend, colleague and teacher, died

on September 21st 2010 due to injuries suffered in

a hit-and-run car accident. We are shocked and

devastated by our loss. Ethan joined the Schmidt-

Dannert lab in 2005, and was the go-to guy for all

our questions, scientific and otherwise.

As undergraduates working on the iGEM project in

Claudia’s lab, we feel blessed to call Ethan Johnson

a friend and mentor. Working with and getting to

know Ethan has shown all of us on the iGEM team

how to be better scientists and members of a

community.

Students: Anthony Goering, Ian Windsor, Annie Kathuria, Matt Adams, and Rachel Farr

Instructors: Ethan Johnson, Poonam Srivastava, Jeff Gralnick, Claudia Schmidt-Dannert, and Swati Choudhary

University of Minnesota

• Ethanolamine utilization proteins have been successfully expressed in E. coli.

• Electron Microscopy indicates assembly of a shell-like structure

• EutC signal sequence targets GFP to a distinct region in the cell, suggesting it is being

encapsulated by Eut shell proteins

• EutS is sufficient to co-localize the EutC signal sequence tagged GFP

• Our results indicate that encapsulating enzymes within such shells will create in vivo

nanobioreactors

•To demonstrate the utility of BMCs in improving reaction efficiencies, we will target

genes encoding short catabolic pathways into recombinant BMCs

Conclusions & What’s Next

Acknowledgements

We would like to thank the individuals who helped make our project possible.Kristi Lecy at the BioTechnology InstituteJane Phillips and Aziz Arabkhazaeli at College of Biological Sciences Instructional Labs,University of MinnesotaMark Sanders (Imaging Center, College of Biological Sciences, University of Minnesota)Thank you Brett Couch, Trevor Gould, Michael Jarcho, Bruce Jarvis, Katherine Kirkpatrick,and myriad CBS undergraduates for sharing your lab space this year.

Recombinant BMCs in E. coli

The 17 genes of the Ethanolamine utilization (Eut) operon in Salmonella

enterica LT2. Eut S, M, N, L, and K are believed to be components of the BMC

shell structure.

Model for the metabolism of ethanolamine in the Eut BMC. Ethanolamine enters

the microcompartment. It is then converted into acetaldehyde by EutBC. The

compartment prevents acetaldehyde from diffusing away. EutG converts

acetaldehyde to ethanol. Acetaldehyde is also converted by EutE into acetyl-CoA.

This is then phosphorylated by EutD. Acetyl-phosphate and ethanol can then freely

diffuse out of the compartment

(adapted from Brinsmade et al, Journal of Bacteriology, 2005)

Eut BMC of S. enterica

Imaging by transmission electron microscopy suggests that E. coli transformed with

the pUCBB-EutSMNLK plasmid are able to form a polyhedral shell structure

reminiscent of Eut BMCs.

• SDS-PAGE gels showing that E. coli transformed with Eut BMC plasmids express

recombinant proteins

• Eut shell proteins are present in soluble fraction

It has been suggested that a sequence of 19 amino acids at the N-terminus of the enzyme

EutC may serve as a signal sequence targeting it to the BMC

(Cheng et al, PNAS, 2010). GFP, with or without the predicted EutC N-terminal sequence, is

uniformly spread throughout the cell. However, when co-transformed with the full

complement of Eut shell proteins, it is localized to a distinct site within the cell.

Interestingly, EutS appears to be sufficient to cause this localization.

Microcompartment Targeting

• Proteinaceous polyhedrons

•~100 - 150 nm in width

• Found in several bacterial species including cyanobacteria, chemoautotrophs,

enterobacteria

• Sequester enzymes involved in specific metabolic pathways like CO2 fixation &

utilization of ethanolamine (Eut) & 1,2-propanediol

• Functionally similar to eukaryotic organelles

Model of the Eut Shell structure.Eut BMCs in Salmonella senterica LT2

(Kerfeld et al, Microbe, 2010)

• Eut BMC genes cloned from Salmonellla enterica LT2 genomic DNA

• Inserted in biobrick compatible vector downstream of a constitutive promoter

(* lac promoter)

• Expression cassettes stacked using restriction enzymes EcoRI, PstI, SpeI, and XbaI

pUCBB-EutS pUCBB-EutMN pUCBB-EutLK

pUCBB-EutMNLK

pUCBB-EutSMNLK

The 17 genes of the Eut operon. S, M, N, L, and K are believed to be components

of the shell structure. In Salmonella Enterica.

Figure 4. Model for the metabolism of ethanolamine in the Eut BMC.

Ethanolamine enters the compartment which is believed to be composed of EutS,

M, N, L, and K. It is then converted into acetaldehyde by EutBC. The

compartment prevents acetaldehyde from diffusing away. EutG converts

acetaldehyde to ethanol. EutE also converts trapped acetaldehyde into acetyl-

CoA. This is then phosphorylated by EutD. Acetyl-phosphate and ethanol can

then freely diffuse out of the compartment (adapted from Brinsmade et al. 2005)