Phage-mediated just-in-time circuit amplification and delivery for recombinant expression of toxic proteins

In order to address outstanding challenges in bacterial bioreactor bioprocessing, we have developed an approach that alleviates the need for chemical inducers and selective agents (such as antibiotics), and mitigates leaky expression of the production circuit. More specifically, temperate bacteriophage Lambda ({lambda}) was engineered as a chassis to just-in-time amplify and deliver a production circuit to a wild-type Escherichia coli population. Since the expression of the production pathway is engineered to be dependent on {lambda}s central regulatory switch towards the lysogenic state, {lambda} will first lytically amplify in the background without significantly altering overall bioreactor growth dynamics or gene expression. Only when {lambda} eventually starts outnumbering the host population, its natural switch to lysogeny will actively install and trigger the production circuit. As such, the {lambda} chassis serves as an intrinsic self-amplifying inducer that meanwhile rules out any leaky expression. The impact of the initial phage-to-host ratio on population dynamics and production efficiency was assessed experimentally and through ODE modeling, revealing that it can be used to fine-tune the trade-off between gene dosage and biomass conversion into virions. We demonstrate that this leakproof approach is particularly promising for the expression of toxic proteins such as Benzonase, a highly potent DNase and RNase.