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Integrating a novel layer of synthetic biology tools in Pseudomonas, inspired by bacterial viruses

Periodic Reporting for period 2 - BIONICbacteria (Integrating a novel layer of synthetic biology tools in Pseudomonas, inspired by bacterial viruses)

Reporting period: 2021-03-01 to 2022-08-31

The goal of BIONICbacteria is 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 aim to i) exploit phage-encoded genetic circuits as synthetic biology biobricks and new biotechnological chassis; ii) build synthetic phage modulators as novel payloads to regulate bacterial metabolism in a targeted manner; and iii) integrate the new circuits to create designer bacteria for applications in industrial fermentations and vaccine design. This innovative approach will allow us to domesticate Pseudomonas strains with the goal of addressing key problems in industrial fermentations and vaccine development for society and industry.
• Our conceptual ideas and the current state of the art have been combined in review articles which explore the potential of bacteriophages for non-model bacteria engineering (Lammens et al., 2020 in Nat. Commun.) and the potential of virulence regulation by phages in biotechnology (Schroven et al., 2020 in Fems. Microbiol. Rev.). Next, to obtain novel phage genetic elements for the design of synthetic biobricks and biotechnological chassis, we defined the transcriptional blueprints of a variety of Pseudomonas phages. For this, we applied differential RNA-seq (Wicke et al., 2021 in RNA Biol.), Grad-seq (Gerovac et al., 2021 in mBio), and ONT-cappable-seq.
• ONT-cappable-seq (Putzeys et al., in subm.) explores the primary prokaryotic transcriptional landscape by combining primary RNA enrichment with Nanopore long-read sequencing technology. This technology has been key to obtain the transcriptional blueprints envisioned in the WP1 and their application in WP2. We have also designed and made available the bioinformatics scripts (available on https://github.com/LoGT-KULeuven/ONT-cappable-seq) to analyze ONT-cappable-seq data.
• SAPPHIRE (Coppens and Lavigne, 2020 in BMC Bioinformatics) and its latest release SAPPHIRE.CNN (in prep.) are promoter prediction software specific for σ70 promoters in Pseudomonas and Salmonella. These tools outperform other promoter predictive software for the mentioned species. This technology has contributed to the identification of novel promoters and the confirmation of TSS identified by ONT-cappable-seq (WP1).
• SEVAtile is a standardized DNA assembly method optimized for Pseudomonas and compatible with the SEVA‐vector backbone (Lammens et al., 2021 in Microb. Biotechnol.). The implementation of this system in Pseudomonas enables the rapid and standardized assembly of genetic parts to create genetic circuits for analysis in non-model hosts.
In the final part of the project, we aim to generate and optimize designer strains as proof-of concept for applications in industrial fermentation and next-generation vaccines. In particular, we expect to generate engineered strains for industrial integrated fermentations with improved production by combining the different circuitry generated so far. In addition, we hope to design a P. aeruginosa strain as a modulated-virulence, self-contained live vaccine by using our specific genetic circuitry and our virulence modulators.
Overview of SynBio applications of phage proteins