Do you ever have to tell your body to sweat when it is too hot or shiver when it is cold? The temperature adjustment of your body happens by itself, just like many other complex processes in nature are autoregulatory, having evolved mechanisms to maintain balance and adapt to changing conditions. This phenomenon can be observed at all scales and domains of life. Even the smallest units of life, cells, are of great complexity and require rigid regulatory processes to survive. If regulation fails this can have detrimental consequences for the growth and survival of the cell. Such interference in internal regulation happens when bacteria are genetically engineered to overexpress proteins for the production of valuable compounds. This introduces a largely unregulated process which, no surprise, often leads to growth inhibition and, in turn, increases the evolutionary pressure to get rid of the “burden” [1]. If externally controlled regulation is so problematic, how do we integrate all the complexity of the living cell and work with (not against) nature? How can we maintain a careful balance of cell metabolism when introducing additional “tasks” - and ensure their execution?

Answers to these questions could significantly improve synthetic biology applicability beyond the lab bench and would be especially relevant for large-scale, continuous microbial bioproduction. Why bioproduction? Because the possibilities for mutations increase with prolonged cultivation time, and both the overexpression of the production genes and the stress factors in bioreactors (e.g. temperature, pH, shear) intensify the rise of non-producing populations [2].

We might have a solution - and it is autoregulatory

It was during the first meeting with Prof. Dr. Annegret Wilde (University of Freiburg) in the weeks of brainstorming project ideas that she proposed an interesting biocontainment concept for biodegradation: couple cell survival to the presence of the degradable compound and your microbes survive only as long as there is something to degrade. Such a solution, to our knowledge, hasn´t been developed yet. What makes it more exciting, we realized that the same novel principle can also be applied to bioproduction by coupling cell survival to the synthesis of the target molecule. Instead of trying to tweak separate parts of metabolic pathways to boost production, you let the cells work in an auto-regulated mode and ensure that only the productive ones survive. Wait, wouldn’t cells just produce what you tell them to? Like, you insert the right genes and as long as the bacteria have them, they will keep producing the target compound, right? Turns out, no: and there are currently no satisfying solutions to ensure phenotypic stability (that is, gene expression) in microbial bioproduction [2, 3] Approaches that focus on genetic stability, like plasmid selection with antibiotic resistance marker genes, fail to secure continuous, long-term gene expression even on the lab scale. That is also what the iGEM Freiburg 2022 team experienced (Figure 1), prompting us to consider one immediate application of the concept Prof. Wilde proposed.

Meet CELLECT, the autoregulatory system!

And so, we were inspired to create CELLECT: the dynamic autoregulatory system that couples cell survival to the presence of your chosen compound. CELLECT consists of three components:

• a target compound-specific riboswitch that detects the compound in the cell
• a toxin-antitoxin system that ensures the induction of cell death in non-productive cells
• your target compound: the molecule that you want to produce or degrade

What is a riboswitch?

A Riboswitch is a natural mRNA gene-regulatory element that changes conformation once the target molecule binds. This conformational change consequently regulates (inhibits or promotes) the expression of the downstream genes. In CELLECT, the riboswitch is located upstream of a toxin gene and inhibits its expression upon binding of the target compound.

How does a toxin-antitoxin system work?

Toxin-antitoxin systems (TAS) are widespread among prokaryotes and are often present in numerous copies of an organism. TAS ensure the survival of the bacteria carrying mobile genetic elements (such as plasmids) by selectively eliminating the cells that have lost it: if the plasmid is missing, the unstable antitoxin is degraded first and the remaining stable toxin eventually kills the cell.

How it actually works: CELLECT proof-of-principle

We illustrate the proof-of-principle of CELLECT by producing the essential vitamin B12 in E. coli (Figure 2). On a plasmid inserted in E. coli, we introduce a gene (bluB) that is necessary for B12 precursor, 5,6-dimethylbenzimidazole (DMB), biosynthesis. Exogenously, we provided another precursor, cobinamide, which together with DMB forms vitamin B12 (why we chose to produce vitamin B12? Read on the Design page).

Additionally, we incorporate a B12-specific riboswitch (what is a riboswitch and how does it work? Read on the Design page) which regulates the expression of a toxin (Figure 3). When B12 is bound to the riboswitch, no toxin is expressed, and therefore only producing cells survive!

Since B12 is not present until we induce the expression of the crucial enzyme, BluB, this could cause unwanted cell death during the preparation of the bacteria cultures. Therefore, our system includes an antitoxin which ensures that the toxin levels remain under control for the initial hours (how does the toxin-antitoxin system work? Read on the Design page). The antitoxin expression is constitutive and therefore does not require induction. However, the antitoxin degrades quicker than the toxin meaning, the toxin can accumulate over time and overcome the inhibition by the antitoxin in the scenario where no B12 is produced.

Considering the parts together, the following 2 scenarios emerge:

1. Produce B12 and survive (Figure 4): the bluB gene is expressed, and as a result, B12 can be produced from its precursors. Next, B12 binds to the B12-specifc riboswitch which in turn inhibits the toxin expression. Thus, the remaining toxin stays under control by the antitoxin; no additional toxin is produced and the productive cell survives.



2. Don’t produce B12 and die (Figure 5): either the bluB gene is not expressed, or something else interferes with the assembly of the complete B12 from the precursors. Either way, if there is no B12 binding to the riboswitch, the toxin is expressed and keeps rising. Eventually, the labile antitoxin can’t deal with the accumulation of the stable toxin, which results in the death of the non-producing cell.

That is, in a nutshell, the proof-of-principle for CELLECT, the novel autoregulatory system that combines elements of sensing the compound to the survival of cellected cells (find a more detailed description of each of the components on the Design page).

The foundational design of CELLECT makes it applicable for:

• various tasks, like biocontainment (during degradation) and microbial bioproduction
• any compound that has a fitting riboswitch
• use in a broad range of chassis organisms

At least, in theory. While we experimentally validated each part of CELLECT by ensuring stable B12 production in E. coli, the rest of the proposed applications remain to be tested. Therefore, we already chose the next challenge: to implement and prove CELLECT in another, industrially relevant chassis. Guess which one?

The next step with CELLECT

Cyanobacteria! We considered working with these guys from the very start of our project. By efficiently converting atmospheric CO₂ into valuable products and utilising light as an energy source, cyanobacteria hold immense potential as more sustainable microbial biofactories [1, 4]. However, as pointed out by our supervisors, they are also relatively challenging (longer doubling time; less researched) compared to the model organisms like E. coli and hence not the optimal choice for the initial testing of CELLECT. Nevertheless, the iGEM UCSC 2017 project on exploring the potential of B12 production in a cyanobacteria model strain, Synechococcus elongatus PCC 7942, served as a major inspiration to continue the work they started and test the theory in practice (see notes from the online meeting with the UCSC 2017 team member McKenna Hicks). Working with another organism besides E. coli fits our objective to test and ensure CELLECT usability in a wide variety of chassis. What’s more, it takes B12 production to the next level: given the complexity of the B12 de novo biosynthesis pathway, finding a chassis that already contains major parts of the pathway is of great industrial relevance. S. elongatus PCC 7942 is one of such organisms and with it, we aim to provide the first proof-of-principle for B12 production in modified cyanobacteria.

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