Bacteriophage Inspired Growth-Decoupled Recombinant Protein Production in Escherichia coli

Abstract

Modulating resource allocation in bacteria to redirect metabolic building blocks to the formation of recombinant proteins rather than biomass formation remains a grand challenge in biotechnology. Here, we present a novel approach for improved recombinant protein production (RPP) using Escherichia coli (E. coli) by decoupling recombinant protein synthesis from cell growth. We show that cell division and host mRNA transcription can be successfully inhibited by coexpression of a bacteriophagederived E. coli RNA polymerase (RNAP) inhibitor peptide and that genes overtranscribed by the orthogonal T7 RNAP can finally account to >55% of cell dry mass (CDM). This RNAP inhibitor peptide binds the E. coli RNAP and therefore prevents σ-factor 70 mediated formation of transcriptional qualified open promoter complexes. Thereby, the transcription of σ-factor 70 driven host genes is inhibited, and metabolic resources can be exclusively utilized for synthesis of the protein of interest (POI). Here, we mimic the late phase of bacteriophage infection by coexpressing a phage-derived xenogeneic regulator that reprograms the host cell and thereby are able to significantly improve RPP under industrial relevant fed-batch process conditions at bioreactor scale. We have evaluated production of several different recombinant proteins at different scales (from microscale to 20 L fed-batch scale) and have been able to improve total and soluble proteins yields up to 3.4fold in comparison to the reference expression system E. coli BL21(DE3). This novel approach for growth-decoupled RPP has profound implications for biotechnology and bioengineering and helps to establish more cost-effective and generic manufacturing processes for biologics and biomaterials.

Downregulation of T7 RNA polymerase transcription enhances pET‐based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis

“…Similarly, by inhibiting endogenous RNAP or reducing parts of nonessential proteome production, it is possible to balance cell growth and recombinant protein production on resources allocation (Kim, Darlington, Salvador, Utrilla, & Jimenez, 2019). Recently, the evolved T7 phage RNAP inhibitor Gp2 was used in BL21 (DE3) to decouple recombinant protein production from cell growth, which enhanced protein yields up to 3.4‐fold (Stargardt, Feuchtenhofer, Cserjan‐Puschmann, Striedner, & Mairhofer, 2020). Moreover, the resources can be selectively allocated for transcription or translation of target genes by orthogonal molecular elements (Darlington, Kim, Jimenez, & Bates, 2018; Segall‐Shapiro, Meyer, Ellington, Sontag, & Voigt, 2014), which were beneficial to reduce the metabolic burden of the host cells.…”

Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria

“…Furthermore, bacteriophages secure their energy supply for phage-particle production by shutting down non-essential energy-draining processes such as host transcription, DNA replication, and cell division 116 , 117 . In a similar fashion, these inhibitory proteins could be used to uncouple cellular growth from protein production to maximize substrate-to-product conversion and product yield in bioreactors, as demonstrated successfully in E. coli 118 . In conclusion, phage genomes are an underexploited treasure trove of metabolic modulators and cellular engineering tools, which could allow us to efficiently manipulate cells on multiple levels and revolutionize the SynBio toolbox.…”

Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness

“…This strain is a proprietary expression technology recently developed by enGenes Biotech GmbH. [ 20,21 ] It is derived from BL21(DE3) and carries a genomically integrated sequence coding for the bacteriophage‐derived RNA polymerase inhibitor Gp2 under control of the araB promoter. This protein from the T7 phage inhibits the host RNA polymerase, while the T7 RNA polymerase stays unaffected.…”

“…This approach to decouple growth from recombinant protein production was shown to increase specific yield and product quality. [ 21,22 ] In previous experiments, the X‐press strain showed a high tendency to leak periplasmic protein to the cultivation medium, but strategies to harness this potential were not addressed in more detail. [ 21 ] Therefore, in the present follow‐up study, we further investigated the X‐press strain's response to the process parameters temperature and q S , both known to affect the leakiness of E. coli , in fed‐batch cultivations.…”

“…[ 21,22 ] In previous experiments, the X‐press strain showed a high tendency to leak periplasmic protein to the cultivation medium, but strategies to harness this potential were not addressed in more detail. [ 21 ] Therefore, in the present follow‐up study, we further investigated the X‐press strain's response to the process parameters temperature and q S , both known to affect the leakiness of E. coli , in fed‐batch cultivations. We performed a Design of Experiments (DoE) to find the adequate parameter space for enhancing leakiness while maintaining high productivity and viability.…”