Heterogeneity of bacterial populations in bioreactors

Production of recombinant proteins by microorganisms such as bacteria (Escherichia coli) or yeasts (Saccharomyces cerevisiae, Pichia 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. Microorganisms used to produce recombinant proteins in these are often seen as a homogeneous population, yet stressed subpopulations may arise in these reactors, resulting in reduced yield and production. According to Müller et al. [1], four factors can induce the emergence of stressed subpopulations :

Heterogeneity in cell cycle phases

The cell cycle can be divided into four phases : Duplication of DNA (S in eucaryotes/C in bacteria), cell division (M/D) and two gaps between these phases (G1/B and G2/pre-D). DNA synthesis and division both have a specific duration for a given organism while gap phases are subject to variation due to stochastic availability of nutrients and environmental conditions, leading to cell cycle phase heterogeneity in populations. As recombinant protein synthesis peaks in gap phases and slows down during DNA synthesis and replication, cell cycle phase heterogeneity directly impacts recombinant protein production.

Heterogeneity in cell age

Microorganisms are often regarded as immortal, having an unlimited and steady potential of division. However, cells can build up unwanted materials over time, especially defective biomolecules. This accumulation leads to a reduction of growth and metabolic activity which can be accounted for aging. Unwanted molecules may be assymmetrically distributed to daughter cells during division, increasing population heterogeneity.

Heterogeneity in environmental conditions

Variation in physico-chemical conditions (temperature, pH, nutrients, ...) induce stress to which cells can respond differently, leading to several subpopulations in different physiological states. Heterogeneity in bioreactors is directly impacted by spatial variations in the medium (imperfect mixing systems, oxygen gradients due to a unilateral injection, ...). During the stationary phase (when the microbial population is the highest), nutrient depletion can induce a physiological state of senescence, where metabolism and recombniant protein production will decrease.

Heterogeneity in genotypes

Mutations and selection can induce heterogeneity in cultures, but occur after relatively long times. Because protein mass production is a stressful process for microorganisms that impact their growth and fitness, mutations relieving engineered organisms from their production are avantageous (e.g. the loss of the plasmid encoding a highly expressed gene of interest), allowing unproductive mutants to be selected and to take over bioreactors.

MightyColi will focus on improving production and yield in bioreactors by reducing population heterogeneity. We will construct a synthetic E. coli that suicides when it enters a stressed physiological state or when its production goes under a threshold. To do so, we will use toxin-antitoxin systems.

Toxin-antitoxin systems

Toxin-antitoxin (TA) systems are couples of linked genes encoding for a toxic protein and an inhibitor of this toxin. To ensure the survival of bacteria expressing one of these toxins, the corresponding antitoxin must also be expressed. TA systems can be contained on mobile genetic elements such as plasmids. When a bacteria possessing such a plasmid divides, the generated daughter cells might not inherit this plasmid due to a stochastic partition. In this case, the antitoxin, which is often unstable, is quickly degraded, allowing the toxin to perform its function and kill the daugther cell in a process known as "post-segregational killing" (Figure 1). This system allows a plasmid to be selected anf maintained in a bacterial population even if it does not confer any advantage. Therefore, TA systems can be seen as selfish entities, virtually making bacteria addicted to them.

TA systems can be divided into five groups, depending on the molecular nature of its components :

We will mainly use type II TAs in which both components are proteins and the antitoxin binds to the toxin, preventing it from performing its function. 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 the DNA gyrase

The ULB-Brussels team aims to work out a system which fights against the appearance of less or none productive microbial sub-populations in bioreactors by repressing :

Our focus will be on E.Coli and S.Cerevisiae. We will maintain the target gene in cells by a mechanism wildly used by the plasmids themselves [4]. Plasmids are closed collections of DNA sequences often find in bacteria and added to the bacterial chromosome. There are actually broad elements for the cell, like virus and transposons, and they have their own existence. They aim to be maximally amplified. They so carry on the exact inverse strategy of the virus: they bring genes that are not essential for the survival of the micro-organism but that give clear evolutionary advantages such as gene of resistance to antibiotics. Despite the metabolic overhead that they represent, the cell can thus have interest to let them breed and to keep them. However some plasmids are greedy and they want to pull the evolutionary phenomenon to their own advantage. When the cell is split, it is important that the plasmid an its copies are present in the both daughter cells but the replication of the plasmids is independent of that of the bacterial chromosome and can't thus use the microtubules. Some plasmids bear genes for a toxin and its antitoxin, the antitoxin being less stable than the toxin, so that the daughter cell which doesn’t inherit at least one copy of the plasmid is sentenced to death: the cell won’t be able to renew its supply of toxin and antitoxin whereas the inherited antitoxin will be quickly degraded, freeing the action of the inherited toxin. That is how some plasmids manipulate the laws of the natural selection…

The fact is that the plasmids are the privileged transforming vectors for bacteria and the yeasts. It is rather easy to insert the target gene on a plasmid and then integrate it inside the micro-organisms. To be ensured that the plasmid, and then the target gene, is maintained into the microbial strains, we could transfer the toxin-antitoxin strategy wildly adopted by numerous plasmids into the process. Moreover we suggest the coupling of the production of the target protein to that of the antitoxin while the toxin is produced independently. So the cells that would have turned off the target gene and thus the antitoxin gene will die. We will actually build what is called a bicistronic gene: the target protein and the antitoxin are set on the same order form (mRNA) for the protein factory (ribosome). A special DNA sequence has to be put between these two genes if one wants that the ribosome produce two distinct proteins with only one mRNA. We will use the gene of the 2A peptide (18 aa.), in both the yeast (S.Cerevisiae) and bacteria model (E.Coli). There is nowadays few literature about the use of 2A peptide in prokaryotes and an important part of our work will aim to improve our knowledge. This peptide is a trick used by some virus to condensate their genome. When the ribosome translates the last amino acid of the 2A peptide, the nascent polypeptide chain is trapped because of steric obstruction inside the ribosomal complex. The translation is momentarily paused. The congestion can be relieved by the hydrolysis of the ester link between the tRNA (linked to the mRNA into the P site of the ribosome) and the last amino acid, which allows the release of the nascent chain, formed by the first target protein in fusion with the 2A peptide. If the second target protein begins by a prolyl residue, the translation can restart. A great advantage of the use of the 2A peptide, unlike other methods, is that allows carrying on a mighty quality control: the antitoxin will be produced only and only if the upstream protein is correctly translated (or punctually muted, which is very rare). Any premature stop codon or “frame-shift” will be detected.

At last, the group of stressed cells because of aging or starvation would be repressed thanks to the exploitation of another wild function of the toxin-antitoxin systems. They are also responsible for the sacrifice of part of the population during an important stress in order to maximize the survival of the remaining cells [6]. The system we will tune will be rather low sensitive so that the micro-organisms won’t be killed for any stress that is inherent to the process (change in the set points, sub-optimal settings, micro-spatial variations, etc.).

The cues we suggest to fight against the appearance of less or none productive sub-populations are universal and would be found in any process using E.Coli and S.Cerevisiae. Here is a great strength of our project: it's a quite simple method that could be easily transferred into many processes!

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