Periodic Reporting for period 4 - BIONICbacteria (Integrating a novel layer of synthetic biology tools in Pseudomonas, inspired by bacterial viruses)
Periodo di rendicontazione: 2024-03-01 al 2024-08-31
The goal of BIONICbacteria was to pioneer an unconventional way to perform synthetic biology, inspired by bacteriophages, the natural predators of bacteria. Through billions of years of co-evolution, the molecular biology of phages is fully adapted to its bacterial host, enabling us to tap into an unlimited source of novel phage tools, genetic circuits and phage modulators for synbio applications within Pseudomonas spp. We successfully developed i) phage-encoded genetic circuits as synthetic biology biobricks; ii) introduced novel molecular techniques e.g. ONT-cappable-seq and a CRISPR/Cas3 genome editing tool iii) synthetic phage modulators as novel payloads to regulate bacterial metabolism in a targeted manner; and iv) integrated the new circuits to create designer bacteria for applications in industrial fermentations and towards vaccine design. This research contributed to solutions in industrial fermentations and vaccine development for society and industry.
The genetic circuit designs developed within BIONICbacteria, such as the pPUT expression controller, have been used to create a potent production strain that outperforms the current best in proof-of-concept studies.
At the same time we used these phage-based modulators to effectively attenuate P. aeruginosa virulence in vivo, laying the foundation for live attenuated vaccines.
Together our results demonstrate the potency of phage-based tools to propel synthetic biology applications in non-model bacteria.
The genetic circuit designs developed within BIONICbacteria, such as the pPUT expression controller, have been used to create a potent production strain that outperforms the current best in proof-of-concept studies.
At the same time we used these phage-based modulators to effectively attenuate P. aeruginosa virulence in vivo, laying the foundation for live attenuated vaccines.
Together our results demonstrate the potency of phage-based tools to propel synthetic biology applications in non-model bacteria.
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.
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.
During the reporting period, our research has resulted in the development of novel and advanced high-throughput methodologies for the screening of biological (phage) parts useful in synthetic biology. These methodologies are not only an advance in the synthetic biology field but have also prompted the discovery of several biological breakthrough findings.
In particular, the development of ONT-cappable-seq entails a step further in the field of transcriptomics of bacteria and their viruses. This approach exploits the so far untapped potential of long-read sequencing techniques to determine phage transcriptional landscapes and provide accurate data on transcriptional events. With this method, we acquired vast dataset of TSSs, TTSs and operons across a set of hallmark pseudomonas phages. In addition, this technique has reported, for the first time extensive and time-dependent read-through at phage terminator regions. It thus offers both new synbio tools as well as a unique perspective on phage biology.
Our research on Pseudomonas phage-based anti-virulence factors and its application are an advancement from three different perspectives. 1) We have developed novel methodology to screen phage proteins as virulence attenuators which can be used in P. aeruginosa but also in other pathogens. 2) We have found four new phage-based virulence modulators that are very promising candidates for the creation of attenuated strains of Pseudomonas. 3) The discovery of new specific anti-virulence factors both expands the synbio toolbox and makes those proteins an interesting source for target discovery for small molecule development, thus enabling a more efficient and targeted approach to find anti-virulence compounds.
The developed pPUT system is a breakthrough way of achieving high yield expression in pseudomonads. This makes the pseudomonas chassis a lot more amenable to enzyme overproduction as it has been for decades in E.coli with T7-based expression. This will open up new avenues of utilizing pseudomonads in new ways. Not just in the envisioned fermentations and vaccine systems as done in this project, but also beyond as a key enabling technology.
In particular, the development of ONT-cappable-seq entails a step further in the field of transcriptomics of bacteria and their viruses. This approach exploits the so far untapped potential of long-read sequencing techniques to determine phage transcriptional landscapes and provide accurate data on transcriptional events. With this method, we acquired vast dataset of TSSs, TTSs and operons across a set of hallmark pseudomonas phages. In addition, this technique has reported, for the first time extensive and time-dependent read-through at phage terminator regions. It thus offers both new synbio tools as well as a unique perspective on phage biology.
Our research on Pseudomonas phage-based anti-virulence factors and its application are an advancement from three different perspectives. 1) We have developed novel methodology to screen phage proteins as virulence attenuators which can be used in P. aeruginosa but also in other pathogens. 2) We have found four new phage-based virulence modulators that are very promising candidates for the creation of attenuated strains of Pseudomonas. 3) The discovery of new specific anti-virulence factors both expands the synbio toolbox and makes those proteins an interesting source for target discovery for small molecule development, thus enabling a more efficient and targeted approach to find anti-virulence compounds.
The developed pPUT system is a breakthrough way of achieving high yield expression in pseudomonads. This makes the pseudomonas chassis a lot more amenable to enzyme overproduction as it has been for decades in E.coli with T7-based expression. This will open up new avenues of utilizing pseudomonads in new ways. Not just in the envisioned fermentations and vaccine systems as done in this project, but also beyond as a key enabling technology.