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Investigating Specificity of Interaction of Signal Sequence and Shell Protein in Bacterial
Microcompartments
Compared to conventional organic synthesis, manufacturing chemicals in a bacterial system
that is chirality-specific, low-cost and environmentally acceptable makes metabolic engineering
a more appealing alternative for chemical synthesis4. Increasing research efforts have been made
to engineer heterologous biosynthetic pathways to produce non-native chemicals in bacteria by
taking advantage of existing metabolic pathways. Yet, major limitations such as diffusion and
toxicity of the intermediates of the intermediates lower the efficiency of the biosynthesis in
bacteria. To overcome the limitations, researchers have tried to utilize engineered Bacterial
Microcompartments (BMCs), a highly-organized bacterial organelle composed entirely of
proteins. Bacteria have evolved BMCs naturally to enhance specific metabolic processes by colocalizing enzymes, substrates, and cytotoxic or volatile intermediates in a delimited
microenvironment1-3, 5, 6. Engineering synthetic BMCs in bacteria may be a promising tool to
solve diffusion or toxicity problems and therefore enhance metabolic efficiency in synthetic
biochemical pathways1. An understanding of the composition and assembly of BMCs is required
in order to engineer recombinant BMCs tailored to specific needs for biosynthetic pathway
designs.
Two major components of BMCs are the shell proteins and the enzymes, or cargo proteins.
Thousands of copies of multimeric shell proteins form polyhedral BMC shells, and other cargo
proteins are targeted to BMCs via N-terminal signal sequences1, 5, 6. Research has suggested that
the N-terminal signal sequence physically interacts with shell proteins to mediate encapsulation
of cargo proteins into the interior of BMCs during shell assembly6. Bobik et al. proposed that the
encapsulation mechanism involves specific interaction of each cargo protein with a specific
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cognate shell protein6; whereas other researchers propose that multiple shell proteins can interact
with multiple cargo proteins using a general targeting mechanism5. So far, there is no direct
evidence to support either hypothesis. It will require more research to determine the mechanisms
of encapsulating cargo proteins into BMCs.
Based on bioinformatic analysis, my hypothesis is that the encapsulation of the cargo protein
involves a general targeting mechanism. In this project, I will work with Professor SchmidtDannert to continue studying ethanolamine utilization (Eut) compartments from Salmonella
enterica as our model system. Even though the exact composition of Eut shells is unknown, it is
believed that five types of shell proteins make up the Eut shells2. The previous studies done in
Schimdt-Dannert labboratory2 have shown that recombinant polyhedral shells can be formed in
Escherichia coli with just the EutS shell protein, and also with a combination of the EutS and
EutM shell proteins. It has been shown that the 19 N-terminal amino acids of EutC serve as a
BMC-targeting signal sequence and are necessary and sufficient to target proteins to recombinant
Eut shells composed of EutS alone or EutS and EutM2. These results and recent studies by Bobik
and colleagues suggest that the cargo proteins are encapsulated through the interaction of their
EutC-derived N-terminal amino acids with EutS and possibly EutM, although interactions have
not been directly shown2, 6, 7.
If targeting cargo proteins into BMCs is a general mechanism, EutC will bind to both EutS
and EutM. To investigate this, I will set up series of experiments utilizing fluorescence
microscopy and heterologous expression of Eut shell and cargo proteins in E. coli2. First, I will
make a EutS mutant that assembles into functional compartments but does not encapsulate cargo
proteins. Selection of residues for mutagenesis will be identified by sequence alignment of EutS
with other BMC shell proteins in different species. The residues identified to be conserved
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among the species will be mutated. A recombinant fusion protein of green fluorescent protein
and first nineteen amino acid of EutC will be made as the cargo protein (EutC1-19-eGFP). We
will examine the co-expression of EutC1-19-eGFP and the EutS mutants in E. coli under the
fluorescence microscope to see if encapsulation of EutC1-19-eGFP occurs. Once the desired EutS
mutant is found, co-expression of the EutS mutant and EutM-red fluorescent protein fusion will
be examined via fluorescence microscopy to show that recombinant Eut shells still form. Then,
we will co-express EutC1-19-eGFP with recombinant shells with the EutS mutant and EutM and
test if colocalization of EutC1-19-eGFP is observed via fluorescence microscopy. If EutS mutant
and EutM do not assemble into compartments as expected, their binding interactions will be
tested using binding affinity assays when time permits. If my hypothesis is correct, we will
observe targeting of EutC1-19-eGFP in the recombinant hybrid Eut shells composed of EutS
mutant and EutM, showing that EutC1-19 also interacts with EutM.
The result of this project will shed some light on the targeting mechanism of encapsulating
enzymes within BMCs. If encapsulation is a specific mechanism, specific pairs of BMC shell
proteins and signal sequences can be used to localize multiple enzymes to engineered BMCs; if
the mechanism is general as I proposed, this suggests that encapsulation of multiple enzymes
should be controlled by other factors such as expression levels of different cargo proteins. The
results will be presented at Undergraduate Symposium as a poster, or be written in a full-length
scientific report. I hope this project will contribute to the development of customized BMCs to
sequester specific sets of heterologous metabolic pathways, and to solve the controversy over the
specificity of the encapsulation signaling mechanism.
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References
(1) Cheng, S.; Liu, Y.; Crowley, C. S.; Yeates, T. O.; Bobik, T. A., Bacterial
microcompartments: their properties and paradoxes. Bioessays 2008, 30 (11-12), 1084-95.
(2) Choudhary, S.; Quin, M. B.; Sanders, M. A.; Johnson, E. T.; Schmidt-Dannert, C.,
Engineered protein nanocompartments for targeted enzyme localization. PloS One 2012, 7 (3),
e33342.
(3) Frank, S., et al., Bacterial microcompartments moving into a synthetic biological world. J.
Biotechnol. 2012,
(4) Keasling, J.D.; Manufacturing Molecules Through Metabolic Engineering. Science 2010,
330,1355-1358.
(5) Kerfeld, C. A.; Heinhorst, S.; Cannon, G. C., Bacterial microcompartments. Annu. Rev.
Microbiol. 2010, 64, 391-408.
(6) Fan, C.; Cheng, S.; Sinha, S.; Bobik, T. A., Interactions between the termini of lumen
enzymes and shell proteins mediate enzyme encapsulation into bacterial microcompartments.
Proc Natl Acad Sci U S A 2012.