Our conceptual ideas and the initial state of the art have been combined in high-impact review articles which explore the potential of bacteriophages for non-model bacteria engineering (Lammens et al., Nat. Commun., 2020), the potential of virulence regulation by phages in biotechnology (Schroven et al., Fems. Microbiol. Rev., 2020), and on high-throughput approaches for understanding phage biology through transcriptomics (Putzeys et al., Curr. Opin. Microbiol., 2024).
To obtain novel phage genetic elements for the design of synthetic biobricks and biotechnological chassis, we have developed several dedicated tools and methods. These include the development of ONT-cappable-seq to define the transcriptional blueprints of Pseudomonas phages to unprecedented accuracy, SAPPHIRE.CNN to predict promoter elements with improved accuracy, and tailored assays to discover specific phage modulators. These methods enabled the discovery of hundreds of phage-derived DNA parts and different protein- and RNA-based modulators impacting diverse metabolic processes in the host cell. These include virulence, replication, translation, post-translation and RNA stability. In addition, new repressors and orthogonal RNA polymerase-promoter pairs have been identified, enabling precise control of Pseudomonas transcription. With the creation of a standardized and efficient cloning method ‘SEVAtile’, we were able to test these different parts in high-throughput and combine them into genetic circuitry that can be readily controlled.
To introduce novel genetic modulators and circuits into Pseudomonas, we had to develop and implement a Cas3-based editing strategy as classical CRISPR-Cas9 editing was inefficient for Pseudomonas and ineffective on its phages. It is compatible with our SEVAtile method and includes an efficient plasmid curing strategy that significantly reduced time to edit. This method became core to edit our proof-of-concept strains.
Multiple phage modulators were combined into a final proof-of-concept pseudomonas strain to demonstrate their performance in an industrially relevant example. For this, the genetic circuits ‘pPUT’ encompassing a performant phage RNAP-promoter pair as well as a specific phage RNAP modulator, were integrated in the genome at optimised loci. This enabled high yield expression in pseudomonas while maintaining a very stringent (non-leaky) expression control. The strain was improved with a modulator that decouples the cell growth phase and production phase to maximise cellular resources towards the product of interest. These adaptations enabled up to 5-fold increased protein yields compared to the current best expression systems in pseudomonas (publication in review).
To lay the foundation for a live-attenuated P. aeruginosa vaccine strain, P. aeruginosa strains with several virulence modulators were tested in both a wax moth larvae & human cell line model. This demonstrated effective reduction of pseudomonas virulence in live examples. Additionally, we have identified triggers that can activate virulence modulators upon reaching clinically relevant infection sites (e.g. human airway epithelia). When combined with our phage-based kill switches for bio-containment, this strain forms a new step in the generation of a new vaccine for Pseudomonas infections.
The different results of our work were presented at 13 (inter)national conferences and led to 22 accepted publications. This project has directly led to four PhDs and 16 MSc’s graduating in this research area and has sprouted new collaborations to further explore phage-based Synbio opportunities in the future.
