As France's leading biocluster, Genopole is an incubator for cutting-edge projects in biotechnology. Located in the city of Évry, just south of Paris, Genopole provides a unique environment for scientists and entrepreneurs seeking to advance research and innovation.

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Every day, at Genopole, researchers, entrepreneurs and students cross paths, share ideas and unite forces in a veritable melting pot for innovation.

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Biomanufacturing with bacteria. How to tune gene expression in Clostridia bacteria

Researcher Andrew Tolonen and his new team created through the Genopole program Atige* have developed a set of biotech tools to genetically transform and control gene expression in Clostridium genus bacteria.
Andrew Tolonen's team within the unit of Genomics Metabolics (Genoscope - CEA) Andrew Tolonen's team within the unit of Genomics Metabolics (Genoscope - CEA)

Their work creates new vistas in biomanufacturing. Clostridia bacteria are able to ferment plant biomass and especially the lignocellulose fibers present in plant debris. They thus have potential as “factories” for producing high-added-value compounds of interest, including medicines.

Clostridium phytofermentansWithin Genoscope’s Genomics Metabolics mixed research unit, a team led by Andrew Tolonen has developed a method to optimize the genetic engineering of Clostridium genus bacteria for biotech applications. The team used the Clostridium phytofermentans species as a model in the study. The researchers first optimized the genetic transformation of C. phytofermentans, then sought means to modulate the expression of genes of interest.

C. phytofermentans can use lignocellulosic biomass (fibrous plant waste) as a substrate and convert it into a range of compounds. In their work, Tolonen and his colleagues sought to develop the biotechnological tools and methods needed to valorize the potential of Clostridium for biomanufacturing applications.

The “toolbox” developed by the Genopole research team comprises:

  • A simple electroporation (use of an electrical current to make a cell membrane permeable) method to enable the transfer of the gene of interest through the bacterial membrane.
  • Plasmids (non-chromosomal, usually circular DNA), used as vectors, within which a transformation-optimizing DNA fragment can be inserted and delivered to C. phytofermentans.
  • Antibiotic resistance genes that function in C. phytofermentans, used as “markers” to select correctly transformed bacteria via their cultivation in media supplemented with the concerned antibiotic.
  • Variably strong promoters able to increase expression by a factor of 4 to 2000 when inserted upstream of the gene of interest.
  • A promoter that modulates expression under the double influence of a repressor and a repressor inhibitor, the concentrations of each being adjusted as a function of the desired outcome.
  • A dCas12a-mediated CRISPR “interference” system, uniting these regulating elements to experimentally control in a coordinated manner the expression of the genes inserted in the C. phytofermentans and those ensuring its metabolism. The CRISPR-Cas complex known for its role as “genetic scissors” to cut DNA at a precise location was used by Tolonen’s team to specifically repress gene expression.

The complete set of genetic engineering methods and tools developed by the Évry team creates new possibilities for the precise regulation of gene expression in Clostridium, a bacterial genus holding great promise for the transformation of renewable carbon resources, notably plant biomass, into compounds of interest in several sectors. For example, it may be possible to differentially modulate the bacterium’s natural expression to overexpress a particular gene coding for an industrially useful protein, or to insert a gene of interest and to repress the bacterium’s natural metabolic genes in favor of the former

Production of compounds of industrial interest

This work by Tolonen and his team was published on 25 November 2022 in ACS Synthetic Biology. It constitutes a foundation for the development of bioprocesses for the production of compounds of industrial interest, a major element of Genopole’s bioeconomy strategic sector.


Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi

ACS Synthetic Biology, 2022.

Graphical abstract: A biotechnological toolbox for Clostridium genus bacteria. A biotechnological toolbox for Clostridium genus bacteria. UMR Metabolic Genomics - Genoscope
The toolbox contains an electroporation method, the plasmids (grey loop in the image) it makes possible to deliver to the bacteria, and the markers needed to identify those that are correctly transformed.
It also provides a number of genetic tools that can be inserted in the plasmid to refine the expression of the gene of interest (“target” in the image). The image above illustrates an example: a plasmid-delivered “guide” DNA produces a guide RNA capable of binding to the protein coded by a second plasmid-delivered gene, dCas12a. That complex is then able to bind specifically to the promoter of the target gene and suppress its expression.
The expression of dCas12a can be controlled by the Tet repressor (tetR) protein, also included in the plasmid construct. TetR binds to the dCAS12a promoter. The system is modulable experimentally by the addition of anhydrotetracycline (aTc) in the culture medium in greater or lesser quantities.
This schema can be reproduced with different guides to extend it to several target genes, either those belonging to the bacteria or those delivered to it in a plasmid.

A new synthetic biology research team at Genopole, bringing solutions to the major challenge of biological production

  • More on the Atige financial grant program

    The “Genopole Thematic Actions Incentive” or Atige grants give researchers the means they need to join an academic laboratory at Genopole and create a new research team.
    Atige contributes to the emergence of future scientific leaders and new research themes at the biocluster.

    More on the Atige program


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