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

Abstract

With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X‐press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (qS) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy‐chain single‐domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X‐press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and qS are used to control productivity and leakiness. In the X‐press strain, extracellular SpA and VHH titer reach up to 349 and 19.6 mg g−1, respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X‐press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.

Economic and ecological benefits of a leaky E. coli strain for downstream processing: a case study for staphylococcal protein A

“…Several leaky E. coli strains for extracellular protein production have been reported, but they often suffer from impaired growth or viability which limits their applicability 9–12 . The recently developed strain E. coli X‐press was shown to enhance expression of various recombinant proteins and we demonstrated that this strain leaks ≤90% of soluble recombinant product to the extracellular space while maintaining viability 13,14 . In this case study, we show the potential of E. coli X‐press to reduce downstream costs and resource consumption in direct comparison to the industrial standard strain E. coli BL21(DE3).…”

“…The theoretical pI of SpA is 5.0, calculated with the ExPASy ProtParam tool (http://web.expasy.org). Details about the strains and the protein sequence are described elsewhere 13,14 . The processes using X‐press for extracellular and BL21(DE3) for intracellular protein production are hereafter referred to as ‘process EX’ and ‘process IN’, respectively.…”

“…The upstream processes for production of SpA were conducted equally for both strains. The process parameters temperature and specific substrate uptake rate were screened in our preceding study and the parameters yielding the highest productivity of SpA in both strains were chosen for the present work 14 . The semisynthetic media used for the preculture and the defined bioreactor medium were prepared as described previously 14 .…”

“…The process parameters temperature and specific substrate uptake rate were screened in our preceding study and the parameters yielding the highest productivity of SpA in both strains were chosen for the present work 14 . The semisynthetic media used for the preculture and the defined bioreactor medium were prepared as described previously 14 . The preparation of precultures and the 20 L bioreactor setup are described elsewhere 13 .…”

“…The limit of detection was 0.005 mg L −1 . Cell lysis in the cultivation was estimated with the DNA data according to a previous study 14 …”