Engineering of In Vivo Nanobioreactors
Transcript of Engineering of In Vivo Nanobioreactors
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)