For the future one could think of much more radical strategies for peroxisomal engineering with a final goal of a “minimal peroxisome” by redirecting metabolic pathways through changing the protein-localization-signal in the yeast genome. Additionally, endogenous metabolic pathways could be redirected to our novel artificial compartment for establishing a customized metabolism specifically tailored for the user's application.
Introduction
In order to achieve a fully controllable artificial compartment, the first step was to design a completely orthogonal import system. Next was the knockout of endogenous import systems. However, a few proteins are imported neither by the Pex5 nor the Pex7 import machinery. Therefore, specific genome engineering designs, such as knockouts, deleting or redirecting the protein localization could be utilized for the ultimate goal of creating a synthetic organelle.
Additionally, knockouts or genome integrations enable customization of the peroxisomal properties, such as membrane permeability, size/number, decoupling of peroxisomes from cytoskeleton and the peroxisomal metabolism.
All these strategies allow a rational design of an artificial compartment, which is fully engineerable regarding the proteome, metabolome and the entire peroxisomal environment.
Design of yeast multi -knockout strains
The Crispr Cas9 System
The demands on yeast engineering have significantly increased with the design of more complex systems and extensive metabolic pathways. Genetic techniques that have historically relied on marker recycling are unable to keep up with the ambitions of synthetic biologists. In recent years the Crispr Cas9 system has been used for several strain-engineering purposes, including:
- Markerless integration of multiple genetic cassettes into selected genomic loci
- Multiplexed and iterative gene knockouts without the need to recycle a marker
- Precise genome editing – nucleotide substitutions, etc.
We utilized the Cas9 system as a tool for peroxisomal engineering and have adopted the existing toolbox from (Lee et al. 2015) and the complete cloning system which also provides the possibilities for genome integration and gene editing by Cas9. For this, two oligonucleotides have to be designed for targeting the Cas protein to the gene of interest.
Two oligos, containing the targeting sequence of the gRNA, have to be annealed and can then be integrated in the gRNA entry Vector by a Golden Gate reaction. Adapted from (Lee et al. 2015)
Several gRNA vectors can subsequently be assembled into one vector with a Cas9 expression cassette and then be transformed into yeast. The expression of Cas9 together with gene specific gRNA´s leads to double strand breakage followed by non-homologous end joining repair or homologous recombination, in case of added repair DNA (figure 3).
Vector for Cas9 and gRNA expression, assembled by a Golden Gate reaction, containing a URA marker, Cen6 yeast origin and a kanamycin resistance. Adapted from (Lee et al. 2015)
The combination of the Cas9 system with DNA repair sequences enables not only knockouts of peroxisomal proteins, but also allows redirecting protein localization by changing protein targeting signals or integration of linear DNA into yeast chromosomes. Genome engineering facilitates yeast strain development for customized peroxisomes.
