Scale‐up of a Type I secretion system in E. coli using a defined mineral medium

Abstract

Secretion of heterologous proteins into the culture supernatant in laboratory strains of Escherichia coli is possible by utilizing a Type I secretion system (T1SS). One prominent example for a T1SS is based on the hemolysin A toxin. With this system, heterologous protein secretion has already been achieved. However, no cultivations in a defined mineral medium and in stirred tank bioreactors have been described in literature up to now, hampering the broad applicability of the system. In this study, a mineral medium was developed for cultivation under defined conditions. With this medium, the full potential and advantage of a secretion system in E. coli (low secretion of host proteins, no contamination with proteins from complex media compounds) can now be exploited. Additionally, quantification of the protein amount in the supernatant was demonstrated by application of the Bradford assay. In this work, host cell behavior was described in small scale by online monitoring of the oxygen transfer rate. Scalability was demonstrated by stirred tank fermentation yielding 540 mg/L HlyA1 in the supernatant. This work enhances the applicability of a protein secretion system in E. coli and paves the way for an industrial application.

Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using Pseudomonas putida

“…By variation of inducer concentration and time of induction, recombinant protein expression can be tuned and optimized ( Wandrey et al, 2016 ; Mühlmann et al, 2017 ). For Escherichia coli , monitoring of the oxygen transfer rate (OTR) or the scattered light signal was demonstrated to be a useful tool for optimization of recombinant protein production in small scale ( Ladner et al, 2017 ; Ihling et al, 2020 ). In addition, non-invasive online monitoring of the OTR in shake flasks combined with analysis of offline-determined culture parameters can be used for a detailed assessment of cultivation conditions and whole-cell performance.…”

Reproducible Polybutylene Succinate (PBS)-Degrading Artificial Consortia by Introducing the Least Type of PBS-Degrading Strains

“…Microbial pre-culture supernatant samples were mixed with the Bradford reagent in cuvettes, and the absorbance was measured at 595 nm after allowing the reaction to proceed for 5 min at room temperature. The protein concentrations of the samples were determined using a standard curve and calculated using a linear regression equation obtained from the BSA standard curve [ 33 ].…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…It was also revealed that by combining signal peptide and production rate screening, enhanced recombinant protein yields were obtained in the E. coli periplasm (Karyolaimos et al, 2019). One study established scale-up of a type I secretion system in E. coli using a defined mineral medium, paving the way for industrial application (Ihling et al, 2019). The industrially important strain engineering strategies utilized to increase both the quantity and quality of therapeutic products were discussed in another study (Castiñeiras et al, 2018).…”