1. ENGINEERING SIGNALSEQUENCES ON
THE 1,2-PROPANEDIOLUTILIZATION
MICROCOMPARTMENT
Kamaria Ashley Kermah
Mentor: Chris Jakobson
Danielle Tullman-Ercek
Department of Chemical and Biomolecular
Engineering
July 28, 2015
Purified
Microcompartment
2. ENGINEERING BACTERIAL CELLS
• Why?
o Ubiquitous
o Easily Replicated
o DNA Easily Manipulated
• How?
o Manipulating the genetic code
4. METABOLIC PATHWAY
• Salmonella enterica
Pathogenicity in
human gut 1,2-propanediol
propionaldehyde
propionyl-CoA
Pdu
CDE
Pdu
P
Pdu
W
propionate
ATP
ADP
5. PDU CDE COMPLEX
• Enzyme localization
• N-terminus of PduD
• Alpha helix
• Hydrophobic and hydrophilic
faces
7. METHODS
7
Growth on 1,2-
propanediol
[G. Cannon 2001. Microcompartments in Prokaryotes doI: 10.1128/AEM.67.12.5351-5361.2001]
Homologous
Recombination
Sequencing
Reaction
TEM
Microscopy
8. RESULTS AND ANALYSIS
Sucrose Sensitivity Confirmation of
gene size
~5kb
Amplification of
pduD gene
1kb
0.5kb
Sucrose Resistance
9. FUTURE
APPLICATIONS
• Engineering microcompartments for industrial
use
o Nanobioreactors
o Antibiotics
Replace PduD N-terminal with PduP
signal peptide
Increasing size of microcompartments
and number of enzymes to increase titer
Localization of co-factors and
heterologous enzymes into MCP
MCP formation in yeast
Editor's Notes
My project was a small step in the larger scheme of engineering bacterial microcompartments for industrial uses
Microcompartments are large organelles inside bacteria that house specific chemical reactions
Now when we picture advancements in the production of biofuels, clean forms of energy or transport of small molecules
We usually imagine a machine created by humans engineered for these different functions
Many people do not think of bacteria as machines however
There are many characteristics of bacteria that make them useful
They are ubiquitous
Therefore they are found in any place and are able to withstand harsh living conditions
Furthermore there are lots of different bacteria naturally found in the human body
Bacteris are easily replicated therefore we can mass produce these bacteria
Most importantly there have been many advances in the last few years that have made it increasingly easier to make mutations at specific points in bacterial DNA
A bacterial cell’s nucleoid is no different than a computers hardware and its DNA is analogous to the code of a computer program
Therefore we can engineer bacteria by making changes in the genome
This summer I worked with organelles inside bacteria called microcompartments
These organelles are made out of a protein shell
Inside are enzymes and co-factors that aide in completion of a specific biochemical pathway
You can think of these microcompartments as minature reaction chambers
The specific microcompartment I worked withwas the pdu mcp
This compartment takes 1-c
This mcp is found in many bacteria and has 2 main functions
It reduces the amount of toxic intermediate chemical compounds in the bcp from entering into the rest of th cell
And it houses a large number of enzymes and co-factors for a more efficient chemical reaction
If we can manipulate the enzymes that go inside the microcompartment
We can produce biofuels inside bacterial cells
Closer look at bcp
Salmonella enterica utilizes pdu MCP for pathogenicity in the human gut
Turns 1,2 propanediol that comes from plant sugars we may eat in our gut into a source of energy to compete with other bacteria
1,2-propanediol is converted into proionaldehyde by the enzyme complex diol dehydratase (removes hydrogen and oxygen in the form of h20)
Propionaldehyde is a toxic intermediate to the cell therefore the
That is why bacteria need microcompartments to sequester this toxic intermediate for other parts of the cell
The enzyme diol dehydratase is essential for formation of microcompartments
We cannot insert enzymes of human interest into mcp until we figure out how enzymes go into the microcompartment in the first place
So to talk about that process there are some pieces of terminology that should be said
Enzymes are proteins that catalyze chemical reactions
When there is an aggregation of an enzyme/protein in a specific area of the cell the protein is said to be localized
The first few amino acids in a cell are called the N-terminus
When that N-terminus binds to another protein and gives a specific direction, it is said to be a signal sequence
How do these enzymes get inside the microcompartment?
The enzymes interact with the proteins that make up the shell
During assembly the shell proteins bring the enzymes along with them
Explain what the N-terminus is
It is formed by the Pdu CDE complex
Previous studies have shown that the N-terminus of the Pdu D protein cause encapsulation of this enzyme complex
The N-terminal is an alpha helix
This alpha helix has a hydrophobic face and a hydrophilic face
There is a specific pattern in the N-terminus that can be manipulated to localize diol dehydratase to lumen of MCP
A big portion of my project was inserting the new gene into the pdu microcompartment genome
This began with a homologous recombination
Homologous recombination is a tool that helps us insert new genes into a genome at specific areas.
In a homologous recombination reaction we take the cells genome and add enzymes that cut it at specific points
Then we apply an electric field which loosens the cells membrane so that the new gene can come inside
If our recombination is successful we end up with the new gene inside the exisisting genome
If you see here the gene in purple is a normal pdu microcompartment gene
At the bottom is the new pduD gene that we have inserted
If you see there gene 21-404 of the pdu gene is still the same. However there is a new portion of the gene that codes for the new n-terminal.
Then we grew on 1,2-propanediol infused media
If you remember from the previous slide, the microcompartment takes 1,2-propanediol and makes energy for itself
If we grow the salmonella cells on only 1,2-propanediol there only choice would be to form the microcompartment and complete the biochemical pathway since there is no other food available.
If the cells grow at an optimum rate, then we know that not only did the microcompartment form, but that the enzyme need to turn 1,2-propanediol into propionaldehyde was inside the compartment.
Then we would sequence the genome of the cells that survived on 1,2-propanediol to see which genes coded for the right n-terminal
From that we studied the genome of surviving cells and computed it into a database
Then we would isolate and observe the surving colonies on a tem microscope to further confirm the formation of microcompartments
