Bacteria are just bags of freely diffusing molecules.
This is what you might be thinking,
but is it really like that?
Well, bacteria actually do have fine and dynamic internal organization!
Some proteins form rigid 3D structures...
while others form liquid droplets!
We have established four types of compartments for you to use:
- The full wiffleball,
- the minimal wiffleball,
- the encapsulin
- and the SPD-5 lquid droplet.
Compartmentalisation is naturally employed by cells to
and thereby improving an organisms stress tolerance.
In compartments, the concentration of substrates and/or enzymes is increased, leading to a variety of positive effects!
- boost reaction rates,
- protect molecules from the surrounding environment,
In bacteria, microcompartments are known to boost metabolic pathways:
One example for this is the activity of RuBISCO, a carbon fixating enzyme that is inhibited by oxygen. Microcompartments have been dicovered to shield RuBISCO from oxygen, thereby improving its activity!
Compartments can fulfill different functions depending on their radius, stability, structure or pore size.
How about modifying the proteins forming these compartments to influence any of these properties?
The incorporation of non-canonical amino acids (ncAAs) into proteins may change their properties.
We have tested this concept by incorporating pAzF and pBpA into the T1 protein of the wiffleball!
Bacteria are known to be burdened when expressing heterologous proteins...
Fortunately, special strains have been engineered whose genome has been reduced to a minimum.
Such genome-reduced strains can act as an improved chassis for expressing engineered compartments to produce useful products.
We asked ourselves:
> Can we produce higher amounts of useful compounds with bacteria if we encapsulate some of the enzymes into compartments?
> Are engineered compartments even better?
> And what influence have genome-reduced strains on all of this?
We chose to apply these various strategies of three different concepts on the production of three industrially relevant molecules:
- Indigo - the pigment for our jeans
- Indirubin - a potential drug against leukemia
- Threhalose - much more than just a sugar.
Our project in a nutshell
Despite their small size, bacteria have sophisticated and dynamic internal organisation, featuring distinct microenvironments optimized for a given task, such as carboxysomes that perform CO2 fixation. We aimed to engineer and compare different compartmentalisation systems (rigid shells and liquid droplets) for their ability to localise enzymes and promote metabolic processes due to reduced formation of toxic side-products and more efficient interplay between pathway components thanks to physical proximity. Using computational structural biology, we predicted sites at which the incorporation of a non-canonical amino acid (ncAA) could influence pore size. To facilitate the use of ncAAs for future iGEM teams, we developed INCLUSIVE, a database collecting the important information from the literature. Furthermore, we investigated the impact of genome reduction on compartment formation and metabolic pathway output. In conclusion, our project lays the foundations for using compartmentalisation as a tool to exploit the full power of bacterial cells.