New toolset simplifies building bacterial cell factories

Environment and sustainability 25. feb 2024 2 min Professor and Group Leader Pablo Iván Nikel Written by Kristian Sjøgren

Bacteria can produce many things, including drugs, industrially useful molecules and biofuels. However, this requires considerable genetic engineering, which has now become much easier with a new technology called pAblo·pCasso.

There are many good reasons to engineer the genes in bacteria. One direction can induce the bacteria to make drugs. Taking them in a different direction can make them produce industrially useful molecules or even make antibiotic-resistant bacteria sensitive to antibiotics again.

Changing the genetic structure of bacteria has traditionally been a complex process, but this has now become much simpler after researchers developed a new toolset that can very precisely change the individual building blocks.

Scientists can now practice the art of designing bacteria exactly as required, which explains why the technology has very artistically been dubbed pAblo·pCasso.

“With pAblo·pCasso we have created a new system that can very precisely edit the genetic structure of bacteria. Previously, this could be done in eukaryotic cells such as the cells in animals, but bacteria could not be edited with the same precision,” explains a researcher involved in developing the toolset, Pablo Ivan Nikel, Professor and Group Leader, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby.

The research has been published in Nucleic Acids Research.

Directly changing DNA building blocks

The problem pAblo·pCasso solves is being able to precisely modify a single nucleotide, the building blocks of bacterial DNA.

CRISPR is the genetic scissors that can cut and paste genes, and Pablo Ivan Nikel and colleagues have modified CRISPR to function in a novel system.

Instead of cleaving the DNA and replacing one or more nucleotides, pAblo·pCasso modifies a single nucleotide from a cytosine or an adenine to another nucleotide.

“This makes pAblo·pCasso very useful in our daily laboratory work in designing bacterial cell factories and changing the function of genes by changing one nucleotide. The old techniques and pAblo·pCasso produce the same result, but pAblo·pCasso is much faster,” says Pablo Ivan Nikel.

Borrowing elements of CRISPR

CRISPR-Cas comprises genetic scissors that cleave the DNA at a predetermined site and one sequence to bind the DNA exactly to the cleaved site.

The researchers have retained the element that recognises the DNA in pAblo·pCasso and use this function to precisely identify the site on the DNA where they want to change a single nucleotide – but the scissors have been replaced by another protein that, instead of cutting, modifies the nucleotide.

This can be achieved because nucleotides are quite similar, even though their presence in the DNA can create major changes in how a gene functions.

For this part of pAblo·pCasso, the researchers borrowed techniques to make the same changes in eukaryotic cells – but they had to adapt them to make them work in bacteria.

This involved creating mutations in the specific proteins involved so that they became more flexible in recognising the appropriate site on the DNA to make the switch.

“With pAblo·pCasso, we can now modify bacterial DNA anywhere. We could not do this before,” explains Pablo Ivan Nikel.

Easy to use

According to Pablo Ivan Nikel, the toolset is virtually plug-and-play.

For example, implementing three changes in bacterial DNA to make the bacteria produce a specific protein with a specific function requires entering the information into pAblo·pCasso, which then carries out the changes.

The pAblo part is responsible for base-editing in adenine nucleotides, and pCasso implements cytosine modifications.

pAblo·pCasso enables researchers to modify nucleotides, genes and proteins in bacteria.

“In our experiments, we switched genes on and off by replacing nucleotides. For example, we can switch off genes that make bacteria resistant to antibiotics. pAblo·pCasso can also introduce or change genes in bacteria so that they can make new or better proteins for drugs, industrial useful molecules or even biofuels,” concludes Pablo Ivan Nikel.

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria” has been published in Nucleic Acids Research. The authors are associated with the Novo Nordisk Foundation Center for Biosustainability at the Technical University of Denmark, Kongens Lyngby, which is partly funded by the Novo Nordisk Foundation.

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