$\Ogo$ur $\MyColi$ project is based on several biological concepts, like production mechanisms of proteins & toxin-antitoxin systems, and bioengineering topics, like bioreactor optimization & effect of an additional peptide.
Recombinant Proteins
Production of recombinant proteins by microorganisms such as bacteria ($\small Escherichia$ $\small Coli$) or yeasts ($\small Saccharomyces$ $\SCere$, $\small Pichia$ $\small Pastoris$) is a key process in pharmacy (vaccines, insulin) and biotechnology (enzymes, antibodies). On industrial scale, proteins and other biological molecules are produced in bioreactors.
Heterogeneity in bioreactors
Microorganisms used to produce recombinant proteins in bioreactors are often seen as a homogeneous population. However, stressed subpopulations may appear, resulting in reduced quantity and quality of the production. Indeed,stressed cells consume nutrients and space but have a reduced productivity (Davey et al., 1996). Several factors may induce heterogeneity in a population : desynchronisation in cell cycle phases, emergence of mutants and new genotypes or variation in physico-chemical conditions within the reactor (Muller et al., 2010). Getting rid of these stressed subpopulations might be an effective way to increase both quality and quantity of production in bioreactors.
The Mighty Coli solution
To reduce inefficient production due to population heterogeneity in bioreactors, we designed $\MyColi$, a synthetic $\EColi$ that dies when it enters a stressed physiological state or when its production decreases.
Toxin-Antitoxin Systems
$\small Toxin$-$\small Antitoxin$ (TA) systems are
two linked genes encoding respectively for a stable toxic protein and an unstable inhibitor of this toxin.
Thus, to ensure the survival of bacteria expressing one of these toxins, the corresponding antitoxin must be continuously expressed in order to compensate its unstability.
TA systems naturally occur in mobile genetic elements such as plasmids, and are used to maintain the plasmid in the microbial populattion. When a bacterium possessing such a plasmid divides, the generated daughter cells might not inherit a copy of the plasmid due to a stochastic partition. In this case, the antitoxin, unstable, is quickly degraded, allowing the toxin to perform its function and kill the daugther cell in a process known as $\small Post$-$\small Segregational$ $\small Killing$ (PSK) [Fig. 1]. This system allows a plasmid to be selected and maintained in a bacterial population even if it doesn't confer any advantage for the host. Therefore, TA systems can be seen as selfish entities, virtually making bacteria addicted to them (Hayes & Van Melderen, 2011).
Figure 1 $:\hspace{0.16cm}$ PSK and plasmid addiction. Cells that inherit a plasmid encoding a TA system can grow normally. As there is no system to part plasmids equivalently in daughter cells, some cells might not receive a plasmid during division. Such cells would still have toxins and antitoxins but these will not be renewed due to the loss of TA genes. Antitoxins are often unstable and quickly degraded by a protease under these conditions, leaving the toxins free and able to kill the cell, effectively eliminating cells losing plasmids that encode TA systems.
TA systems can be divided into five groups, depending on the molecular nature of its components (Hayes & Van Melderen, 2011). We will mainly use II TAs in which both components are proteins and the antitoxin binds to the toxin, preventing it from performing its function [Fig. 2]. Toxin functions and structures in type II TA systems are diversified, allowing us to chose how cells will die when stressed. Two systems will be used to illustrate our project : CcdBA and Kid/Kis.
Figure 2 : Type II TA systems. In such systems, toxins and antitoxins are translated from the same polycistronic mRNA. The toxin is a protein that inhibits a vital function (translation, replication, peptidoglycan synthesis, etc.) and the antitoxin is another protein that binds to this toxin, preventing it from causing harm to the cell. This antitoxin is often unstable and subject to proteolytic degradation. Should the transcription of the TA operon stops, the antitoxin would swiftly be degraded, allowing the toxin to kill or damage the cell. Type II TA operons are negatively regulated.
CcdBA and the DNA gyrase
CcdBA is the most studied type II TA system where CcdB is the toxin and CcdA the antitoxin. CcdB is an inhibitor of the DNA gyrase, an enzyme that supercoils circular DNA, such as bacterial chromosomes and plasmids, making it more compact. DNA gyrase complexes must bind to DNA to perform supercoiling. CcdB binds the DNA gyrase subunit that binds to DNA and inhibits its activity when it is bound to DNA, resulting in DNA double strand breaks, activation of emergency signals and possibly death of the cell [Fig. 3] (Dao-Thi et al., 2005).
Figure 3 $:\hspace{0.16cm}$ DNA gyrase mechanism & CcdB poisoning. $\hspace{0.2cm}a)$ The DNA gyrase is a tetramer made of two GyrA and two GyrB subunits. $\hspace{0.2cm}b)$ An undefined DNAsegment (G-segment) can bind between two GyrB subunits. $\hspace{0.2cm}c)$ Another segment (T-segment) binds, situated between both GyrA subunits of the complex. $\hspace{0.2cm}d)$ The gyrase catalyses a double strand break in the G-segment. $\hspace{0.2cm}e)$ The T-segment is translocated from GyrA to GyrB subunits, above the G-segment. $\hspace{0.2cm}f)$ Both ends of the G-segment are joined and supercoiled DNA is released from the complex. $\hspace{0.2cm}g)$ CcdB binds to GyrA subunits. $\hspace{0.2cm}h)$ CcdB prevents the gyrase from joining G-segment ends, resulting in DNA double strand breaks.
We want to construct a biological system selecting only highly productive cells. By coupling the production of a $\small Protein$ $\small of$ $\small Interest$ (PI) with an antitoxin (A-Tox) in a strain expressing its corresponding toxin, we hope that stressed cells experiencing a drop in PI productivity will be killed by the ensuing drop in A-Tox production. There are many ways to couple the production of two proteins, f.e. polycistronic mRNAs in procaryotes or Internal Ribosome Entry Sites. We chose to use a 2A peptide to pair the PI and the A-Tox.
The 2A Peptide
The 2A peptide sequence (19 nucleotides) allows the post-transcriptional cleavage of 1 ARN sequence into 2 amino-acid sequences: one upstream and one downstream of p2A. This cleavage is done by ribosome skipping within the sequence of p2A. The construction of the peptidic bound between the two last amino-acids of p2A is interrupted, resulting in the termination of the translation of the upstream mRNA sequence. The ribosome can then either stop the translation of the mRNA or continue to translate the downstream sequence into a second, separated protein. The C-terminal extremity of the upstream protein is thus fused with the N-terminal extremity of p2A, and N-terminal extremity of the downstream protein is fused with the last amino-acid of p2A (a prolin).
Bibliography
- H.M. Davey & D.B. Kell, (1996). Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiological reviews, 60(4), 641-696.
- F. Hayes & L. Van Melderen, (2011). Toxins-antitoxins: diversity, evolution and function. Critical Reviews in Biochemistry and Molecular Biology, 46(5), 386-408.
- S. Müller, H. Harms & T. Bley, (2010). Origin and analysis of microbial population heterogeneity in bioprocesses. Current opinion in biotechnology, 21(1), 100-113.
- M.H. Dao-Thi, L. Van Melderen, E. De Genst, H. Afif, L. Buts, L. Wyns & R. Loris, (2005). Molecular basis of gyrase poisoning by the addiction toxin CcdB. Journal of molecular biology, 348(5), 1091-1102.
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