Scientists have invented a new method that allows for the flexible engineering of essential and non-essential enzymes without additional engineering. They describe a method based on CRISPR/Cas9, which enables flexible engineering of essential and non-essential enzymes without additional engineering.

This could be of great importance for various aspects including the development of bio-based production of pharmaceuticals, food additives, fuels, and cosmetics. The study published in the journal Metabolic Engineering.

When having a production strain, this will make it easier for one to engineer certain limiting enzymes in the biosynthetic pathway and increase efficiency, specificity or diversity. People would be able to discover the best trade-off enzyme variants in the pathway and increase production of valuable compounds.

The newly developed method is named CasPER and is building on existing technologies, such as CRISPR/Cas9, that has been used for genome engineering and re-programming in yeast during the last years.

Nucleotide

However, the new tool enables scientists to engineer enzymes or their active domains by integrating much longer diversified fragments providing the opportunity to target every single nucleotide in a specific region.

Discovery of enzymes variants

In-depth characterization of the new method concludes that the main difference between already existing CRISPR/Cas9 methods is that CasPER allows very efficient integration and in a multiplex manner of large DNA fragments bearing various mutations to generate pools of cells with hundreds of thousands of enzymes variants.

While other CRISPR methods rely mostly on the integration of shorter sequences to diversify DNA and require multiple rounds of engineering, CasPER significantly broadens the length of engineered DNA fragments.

Enzymes

Furthermore, it does not require any additional steps making it faster and more effective to diversify enzymes to produce higher yields of desired chemicals.

Screening platform

Before the introduction of CRISPR/Cas9, it was a rather slow process to engineer essential enzymes in, e.g., yeast. Today it is much more flexible regarding what you can target, and that makes it more viable to engineer enzymes to be more efficient and specific allowing them to transform more substrate into a product.

As a proof-of-concept in this study, the scientists targeted several essential enzymes in the mevalonate pathway. This biosynthetic route is responsible for the production of sterols and is essential in most living organisms. 

To prove the applicability and efficiency of CasPER scientists targeted two essential enzymes in the mevalonate pathway and were able to select cell factories with up to 11-fold increased production of carotenoids.

Great potential in industry and academia

In the future, CasPER can be widely used both in academia and industry for various purposes. Although the main application of the method was to speed up and lower the costs for engineering and optimizing cell factories, the method can also be applied to any experiment where diversification of DNA is needed.

You can study protein functions to develop protein structure prediction tools, and study protein interactions with DNA, substrates, and other molecules to diversify regulatory elements such as promoters, terminators, and enhancers. The method was validated in yeast, but it can also be applied in other organisms with efficient homologous recombination machinery.