The production of recombinant proteins plays a vital role in revolutionizing the development of new protein-based drugs. This process has become an integral part of the drug development pipeline. Among the various hosts used for recombinant protein production, bacterial hosts (and more specifically Escherichia coli) stand out as a popular choice, with a significant share of approximately 30% of current biopharmaceuticals on the market. The ease of use, rapid growth rates, and cost-effectiveness of bacteria make them an ideal platform for the large-scale production of recombinant proteins. While E. coli is not a magic bullet for every production application, examples of fermentations using other Gram-negative bacterial hosts are limited. This is largely due to a lack of performant and predictable engineering and/or expression systems that enable the use of these organisms.

In our article, we introduce our phage-based system, ‘pPUT’, specifically designed for recombinant expression in Pseudomonads. By implementing this system in Pseudomonas putida, we're able to radically increase recombinant protein production compared to existing methods. Pseudomonas putida is one of the rising stars in the SynBio field. This bacterium has a versatile metabolism, tolerates endogenous and exogenous stresses, is able to produce toxic, value-added compounds and can grow on diverse carbon sources including waste streams like lignocellulosic biomass, enabling a more circular economy-based production of valuable compounds. With the pPUT system, we expand this potential towards recombinant protein production within the valuable ‘bacterial factory’ P. putida represents.

We were able to streamline the entire cloning process of the pPUT vectors using our previously established, standardized SEVAtile assembly method, allowing easy integration of genes of interest into the expression circuit. As big supporters of the Standard European Vector Architecture platform (SEVA) we made the system SEVA compatible. Indeed, the SEVA vector repository utilizes standardized plasmid assembly & nomenclature and provides accessible functional sequences and user-friendly tools to determine the best vector configuration for specific applications.

Furthermore, the entire pPUT system could be genomically integrated, thus eliminating the need for antibiotic selection and ensuring stable genetic maintenance. Moreover, the phage-based protein expression system is inducible, tuneable and orthogonal to the host metabolism. Our modular setup also allows for optional high-stringency elements and growth decoupling to maximize yields.

During the course of the submission process for this research article, I’ve been fortunate to introduce the pPUT system at various conferences and our team has been pleasantly surprised by the number of research groups interested in adopting the system for their research purposes. While patented and available for commercial applications, I believe it’s very important to make this approach openly accessible for academic researchers.

This work is inspired by one of my scientific heroes, F. William (Bill) Studier, who’s currently a senior biophysicist emeritus at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory. In the 1980s, Studier focused his research on the molecular genetics of bacteriophage T7, particularly the infection process of E. coli. From his research with John Dunn on the T7 RNA polymerase, Studier realized that this polymerase could be harnessed to produce RNA from any gene tagged with a special sequence, called the T7 promoter. He shared these findings in 1986, leading to the establishment of the pET system, which remains to date the seminal approach to recombinant expression. It has easily prompted over 250.000 published studies, and over 100 different versions of the T7 technology available commercially. More importantly, the T7 expression system has had a transformative impact on biomedicine and industry, which, in my humble opinion, would be worthy of a Nobel Prize for Studier and his team.

Historically, bacteriophages in general have been pivotal in the development of molecular biology tools and circuits for E. coli. Many of these discoveries can directly be linked to the 1944 ‘Phage Treaty’, established by Max Delbrück and colleagues, which standardized research on a defined set of phages and E. coli strains (B & K-12) under uniform conditions. This approach resulted in the foundational discoveries in phage biology and bacterial genetics, including defining principles of phage multiplication and mutation, culminating in the 1969 Nobel Prize for Delbrück, Hershey, and Luria. However, this focus on E. coli has led, to some degree, to a biased ‘tunnel vision’ in the mechanisms Nature has in store for us.  Indeed, the immense diversity of phages revealed by sequencing technologies, is significantly expanding the scope of phage research by harnessing their diversity and expanding our understanding on the broader biological and ecological roles that phages have within the environment.

Our lab now harnesses these new phages to broaden the synthetic biology (SynBio) toolbox for non-model microbes. Phages can be seen as nature’s original bioengineers, adeptly preying on their bacterial hosts and commandeering their metabolism to reproduce and create new viral particles. Equipped with an impressive array of genetic building blocks—including promoters, terminators, integrases, and repressors—that operate independently from their bacterial hosts, bacteriophages present a unique opportunity. By mining their genomes, we can now rapidly develop specialized SynBio tools tailored for specific organisms, functioning orthogonally to the native metabolic processes of those microbes. This is especially true for our pPUT system, which, despite pitfalls and problems, was developed within a broader frame of a single PhD (Congratulations Eveline-Marie!). While unique and unexpected in its outcome, our research benefited from the level of conservation between P. putida phi15 and E.coli T7, to which Bill Studier dedicated much of his career. Combined with new high-throughput and efficient methodologies at our fingertips, more groundbreaking advancements in biotechnology are to be expected, especially in non-model microorganisms. Hopefully the implementation of the pPUT system in Pseudomonas can become a part of those advances.