Our MightyColi 2014 Project

Heterogeneity of bacterial populations in Bioreactors and Productivity

The production of complex biomolecules by micro-organisms in bioreactors is nowadays a widely spread process in the pharmaceutical (synthesis of antibodies, vaccines and other therapeutic proteins) or food (emulsifiers, flavors, food conservatives and enzymes) industries. However, the productivity is compromised by the appearance of sub-populations which are less or none productive. According to Müller et al. (1), the origin of this heterogeneity is linked to 5 key points.

1) Cell Cycle

There are variations of the production levels of the target molecule during the life cycle and the micro-organisms are generally unsynchronized. The heterogeneity in the phase of the cycle so explains a part of the heterogeneity in the level of productions.

2) Aging

The prolonged use of a microbial culture can lead to a decrease of the growth rate, the fitness and the metabolic efficiency of the micro-organisms. Aging can very appear because of the old biomolecules rafts build-up. This problem can affect with more or less gravity the different strains. The dysfunctional biomolecules can indeed be asymmetrically allocated between the two daughters cells during the division. It can progressively lead to the appearance of more or less productive strains.

3) Spatial and temporal variations of conditions in Bioreactors

The physic-chemical parameters in bioreactors can vary during the process. This variations can be purposely carried on (change in the pH or T° set points, …) or can happen because of the microbial growth itself (supply depletion,…) but they both induce stress. The micro-organisms react in different physiological ways so as sub-populations in different physiological states appear. This phenomenon is enforced by the micro-spatial condition variations in the bioreactors (imperfect stir, vertical O2 concentration gradient because of injection by the bottom, etc.)

The problematic of the starvation during the stationary phase takes an important part in the productivity of a bioreactor. Some micro-organisms are stressed and can become senescent. Their main protection systems against material damages are turned off and their metabolism is minimal (and so is the production of the target molecule). Without protections, their proteins are oxidized until the cell dies.

4)Mutations

Mutations inside the target gene can also bring heterogeneous sub-populations in bioreactors. The types “non-sense” (appearance of a premature stop codon) and “frame-shift” are quite frequent. They can have an important economic impact in so far as the presence of muted target proteins, even if the concentration is low, can turn unsellable the entire production.

Another important genetic phenomenon can happen when the transforming vector is a plasmid. The plasmid has actually a big weight on the metabolism (energy surcharge) and it can be against-selected leading to the proliferation of the wild strain (without the vector).

5)Regulation of the expression of the target gene

The last but not the least key point regards the mechanisms of regulation of the gene expression. In general ways, there are several mechanisms allowing the cell to be protected against the introduction of broad genetic material (plasmid, virus, transposon, etc.) and against anormally high levels of production. Molecular sensors are actually able to turn of the expression of the dubious gene. Micro-organisms in bioreactors are so able to refuse its work defined by the humans.

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 the cells by a mechanism wildly used by the plasmids themselves (4). Plasmids are little circular molecule of DNA that we often find in bacteria and that are 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 cańt 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-organism. 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 would 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 little 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 that 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 that 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 is a quite simple method that could be easily transferred into many processes!