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iGEM Freiburg 2022 presents
chAMBER
chAMBER – Tidying up bacterial cells with natural and engineered nanocompartments
Let’s start with a little exercise … and some background information
We’d like you to think for a moment about the place you live in. Go around the different rooms and spaces in your mind. What do you see? You probably easily recognise a certain pattern: each room contains mostly objects/furniture/equipment needed to perform a certain task, for instance prepare, consume and store food, or relax and entertain yourself. Things that are unrelated to the task or could negatively impact on its realisation are kept out of the room. You won’t find a toilet in the kitchen, or a bed in the hallway!
Well, the cells in your body – and in all other living organisms – follow this very principle of 
Figure 1: Schematic representation of an animal cell. A: Membrane-bound organelles in an animal cell. B: Membraneless organelles in an animal cell. Figure was adapted from [2].
You might be surprised to read that, despite their small sise, also bacteria have sophisticated and dynamic internal organization, featuring distinct microenvironments optimised for a given task. Compartments in bacteria are essentially membraneless, proteinaceous microenvironments (with the notable exception of the nucleus of the newly identified bacterium, Thiomargarita magnifica, which is a membrane-bound sac, similar to the eukaryotic nucleus [3]). Bacterial microcompartments (BMCs) have been so far exclusively associated with the catabolism of nutrients in heterotrophic organisms (that is, organisms that cannot produce their own food and must obtain nutrients from outside sources. In this case, we speak of metabolosomes), and the fixation of carbon dioxide (CO2) in autotrophic organisms (that is, organisms that can produce their own food using, in fact, CO2, but also light, water or other chemicals. In this case, we speak of carboxysomes) [4]. BMCs bring several advantages to the cells producing them: 1. they protect the cytosol from toxic intermediates, which are quickly channelled to downstream enzymes in the same BMC before diffusing out of the BMC; 2. they prevent unwanted side reactions, which would happen if the enzymes were freely diffusing in the cytoplasm; 3. they improve metabolic fluxes because they enhance reaction kinetics due to the high concentrations of enzymes within the BMCs; 4. they protect enzymes from proteolytic degradation or misfolding Figure 2 [5].
Figure 2: Simplified representation of a bacterium equipped with bacterial microcompartments (BMCs).The enzymes of a metabolic pathway are included in BMCs by different mechanisms. Substrates enter the BMCs and are converted into products by the enzymes via intermediate steps.
Beyond BMCs, which possess a rigid shell, cells feature many compartments of a different kind called liquid droplets. They form from the process of liquid-liquid phase separation [6]. Liquid droplets are also found in bacteria [7][8] and, in some cases, are formed in response to stress and help the cells survive [9]. Liquid droplets are very dynamic structures, unlike BMCs.Why care?
Synthetic biologists often look for inspiration in nature to find elegant solutions to certain problems. Since some bacterial species naturally use BMCs to improve metabolic pathways, synthetic biologists thought: why not transplant the genes coding for the proteins responsible for BMC formation into other bacterial species, better suited to be chassis (e.g. Escherichia coli) and incorporate other, desired enzymes into them? This could help improve various metabolic pathways, which are naturally not encapsulated into BMCs (please see below “The motivation behind chAMBER” to learn the reason why it is so important to optimise metabolic pathways in bacteria). As a matter of fact, the carboxysome and the 1,2-propanediol utilization microcompartment (PDU) have been successfully assembled in E. coli cells when the necessary genes were brought into this organism [10]. Studies have first shown that the shells can be formed even in the absence of the natural cargo (as empty structures) [11], then have further proven that other proteins can be brought into the BMCs for instance by fusion with enzymes that are naturally recruited into them [12]. The idea of creating plug-and-play compartments has been conceived about a decade ago [13]. Because of the power of compartmentalisation for metabolic engineering applications, it is not surprising that many iGEM teams in the past have selected to work with BMCs (LMU 2010, UW-Madison 2011, UZurich 2019, UANL 2019, Virginia 2020 – please note this is not a comprehensive list).
Now that you know why it is worth working with BMCs, it’s time for you to learn what sets our project apart from all previous ones…
Our project chAMBER in a nutshell
Our aim was to engineer a compartmentalisation toolbox for future iGEM teams and researchers worldwide. We set ourselves the goal to compare different compartmentalisation systems for their ability to locase enzymes and promote metabolic processes in E. coli. We chose two compartments with rigid shells and one liquid droplet. We aimed to further manipulate these compartments via the incorporation of non-canonical amino acids (ncAAs) at positions we determined based on 3D structures. With ncAAs, BMCs as well as enzymes can be engineered, improved and studied [14][15][16]. Therefore, another major part of our project was the establishment of ncAA incorporation in E. coli, which led to the creation of another toolbox, a series of plasmids with GFP carrying the AMBER stop codon at various positions, which is very useful to test the incorporation of various ncAAs and the corresponding tRNA/synthetase pair quickly and easily. Moreover, we developed a database, INCLUSIVE , collecting the important information from the literature, and a software to predict the monoisotopic mass of the peptides containing the ncAA of choice, that will make working with ncAAs much easier for future iGEM teams and researchers worldwide. Aware of the burden imposed on the cells by the production of many heterologous proteins, we additionally thought of testing genome-reduced E. coli strains, which might offer an elegant solution to the problem of burden [17]. Thus, we aimed to assess ncAA incorporation, compartment formation and both processes simultaneously in genome-reduced strains.
Despite our major objective being the introduction of a foundational advance in synthetic biology in the form of a toolbox of natural and engineered compartments, we wished to have an application with a strong societal impact: the sustainable bioproduction of the natural pigment indigo, the antileukemia agent indirubin and the autophagy-inducing, obesity-suppressing sugar trehalose . The concept can be quickly applied to any pathway through a single cloning step per enzyme, thanks to the SpyTag/Catcher and SnoopTag/Catcher system. With chAMBER we want to create a versatile platform with which it is possible to enable or improve pathways.
The motivation behind chAMBER
We are absolute supporters of basic science and foundational advances, because we are convinced that they are the basis of any application. Nonetheless, the reason for us to join the iGEM competition was to contribute to the fight against climate change. Extreme weather conditions worldwide have become more and more frequent over the last years, bringing to our attention the alarming state of the earth’s climate. Sadly, it is clear that we humans are the major cause of it, mostly due to the combustion and processing of fossil fuels, albeit many different factors also play a role. Modelling the effects of greenhouse gas sources and sinks on future global temperatures demands the expertise of atmospheric researchers, oceanographers, ecological scientists, economists, even policy analysts and others [18]. It is becoming clearer and clearer that the best option to stop global warming is to leave fossil fuels in the ground [19]. The use of fossil fuels as we know them has been the basis of human progress for decades. One can state that fossil fuels have been the driving force behind the industrial revolution. Fossil energy has grown from a vanishingly small significance at the beginning of the 19th century to an annual output of almost 10,000 million tonnes of oil equivalents [20].
In order to stop using fossil fuels, we need to find sustainable and economically feasible alternatives. Here synthetic biology comes into play! Indeed, the production of valuable compounds such as pigments or drugs in bacteria is a more ecologically friendly and sustainable approach compared with chemical synthesis, which requires fossil fuels.
Usually, the metabolic pathway required for the biosynthesis of the compound is transferred from the source organism into a model bacterium such as E. coli. A long optimization process follows to tune the expression levels of the heterologous enzymes; however, the right stoichiometry between enzymes in the pathway must be achieved, and this can be tedious and laborious, being mostly based on trial-and-error.
Moreover, another common problem is the production of toxic intermediates, which hinder the growth of the host organism, eventually negatively impacting on the final product yield.
There are already many possibilities to produce various products from so-called microbial factories [21][22][23][24][25].
However, so far few have made it to the industrial level because the yields are typically too low, and the production would simply be too expensive. Something has to change, and new approaches must be found to make bioproduction of goods more effective, cheaper and overall attractive for the use in industrial production. We decided to exploit and combine several strategies such as compartmentalisation, ncAA incorporation and genome reduction to increase the yield of three very valuable compounds: indigo, indirubin and trehalose.
