1 Introduction
Fed-batch bioprocessing is the most common cultivation method in industrial microbial production of biopharmaceuticals. This batch-wise process essentially includes the repetitive steps of media preparation and reactor setup, fermentation, and subsequent cleaning in place (CIP) and sterilization in place (SIP). In terms of time, the actual fermentation, and particularly the production phase of the recombinant protein, is relatively short. As a result, continuous production becomes more and more interesting due to the greatest possible space time yields and optimal use of the installed assets.[1]In such chemostat cultivations, cells are maintained in a steady-state growth environment by adding fresh medium to the reactor at constant flow. Simultaneously, the cell suspension, and thus the recombinant protein, is removed at the same rate.[2] The growth rate (μ) can be specified depending on the dilution rate (D). Through this process, stable volumetric productivity and high space-time yield can be achieved [3]. In contrast to fed-batch fermentation, the average residence time of a producing cell is always the same, which can be advantageous in terms of product quality. Examples of microbial continuous processes for the production of recombinant proteins have already been described in the literature.[4-6]
For industrial microbial production of recombinant proteins, theEscherichia coli strain BL21(DE3) is often used due to low acetate formation and high production rates resulting from the integrated T7 RNA polymerase (RNAP).[7, 8] In combination with a pET series plasmid, which harbors the gene of interest (GOI) under control of the T7 promoter, extraordinarily high expression rates can be achieved after induction with the non-metabolizable lactose analogue isopropyl-D-1-thiogalactopyranoside (IPTG).[9]
Genetic heterogeneity caused by metabolic burden and toxicity can be problematic at all industrial scales, especially for challenging proteins.[10-12]In bacterial production processes, challenging proteins impose adverse effects on host metabolism, even at low concentrations. Escape variants, which have a growth advantage due to mutations or plasmid loss, can overgrow high-performing producer cells, reducing the overall product yield.[13-15]Therefore, longer production phases, or even continuous production mode, are hardly feasible in such E. coli expression systems.
To obtain stable, high-yield, and predictable E. coli production hosts, engineered producer strains must focus on reducing metabolic load and genetic escape. Attempts to reduce process instability caused by metabolic burden have been made on both the genetic and bioprocessing levels. The metabolic burden and increased selection pressure can be reduced by decoupling growth and production in cascading chemostat cultivations with two bioreactors.[6] Genetic escape can be reduced by removing insertion elements (IS), and by deleting recA or error-prone DNA polymerase genes.[16] Another promising strategy uses the directed evolution approach combined with fluorescence-activated cell sorting to select cells with a lower plasmid mutation rate.[17]
In previous studies, we showed that genomic integration of the GOI under the control of the strong T7 promoter reduces the metabolic burden because plasmid-mediated metabolic load is eliminated, and strong expression has been shown from even a single copy of the GOI. However, after approximately seven doublings under production conditions, mutations in the T7RNAP gene lead to a faster growing non-producing cell population. This phenomenon can be excluded in systems using the host RNAP-specific A1 promoter because full functionality of the host RNAP is required for cell growth.[18]
In combination with a directed evolution approach, this would allow characterization of mutated production hosts, which have adapted themselves to the metabolic load triggered by recombinant protein production and could potentially enable continuous protein production. Moreover, a directed evolution approach can circumvent the complexity of the process of biogenesis and its adverse effects on the host cell.[19-21]
For example, in the study by Walker et al. [22], derivatives of BL21(DE3) were adapted by directed evolution to produce cell membrane proteins that are toxic to host cells. These strains are currently widely used for production of a variety of membrane proteins and toxic proteins.
In the present study, we performed iterated carbon-limited fed-batch-like microbioreactor cultivations under production conditions. The goal was to investigate whether and how long-term metabolic load triggered by the production of recombinant proteins influences the characteristics of mutations occurring in different genome-integratedE. coli production systems.[23, 24] We compared the host RNAP-dependent BL21Q A1 expression system (BQ<A1>) [18] with the T7-based BL21(DE3) expression system (B3<T7>). To study mutation characteristics based on the protein of interest (POI), we used the easy-to-produce protein GFPmut3.1 and the challenging protein Fab fragment dFTN2 as model proteins. To investigate the underlying mutations in the different clones in more detail, we performed comparative whole genome sequencing analyses. We also performed long-term chemostat cultivations in lab-scale bioreactors with the above-mentioned clones and additional robust production strains obtained by the directed evolution approach.