Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains

Abstract

Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7-RNA polymerase-dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315-327, 2018.

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

“…An equivalent behavior is shown here for the first time in a mineral medium using arabinose as inducer. The reason for this observation (more or less constant OTR after induction) remains unclear and was observed for a single amino‐acid exchange, different host strains expressing the same product as well as a single nucleotide exchange . In all cases, biomass formation declined upon induction, which is in agreement with data from literature and a result of a higher ATP demand .…”

“…The phenomenon of a more or less constant OTR after induction is well known from auto‐induction media containing glucose, lactose, and glycerol. Several studies identified a phase of rather constant OTR after induction with IPTG or its analogue lactose as production phase and indication of the strength of metabolic burden . An equivalent behavior is shown here for the first time in a mineral medium using arabinose as inducer.…”

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

“…This increase continues after induction, indicating no strong metabolic burden. Typically, a strong metabolic burden is reflected by visible changes in the OTR after induction (Rahmen et al, 2015a,b;Ihling et al, 2018;Mühlmann et al, 2018). The maximum oxygen transfer capacity (OTRmax) under the chosen cultivation conditions was determined to be 52 mmol/L/h according to Meier et al (2016).…”

Elucidation of auxotrophic deficiencies of Bacillus pumilus DSM 18097 to develop a defined minimal medium

“…As a new and promising tool to clarify auxotrophic deficiencies, the respiration activity monitoring system (RAMOS), based on shake flasks [ 20 , 21 ] and a recently developed system for 48-well microtiter plates (µRAMOS) using the same working principle was applied [ 22 ]. Numerous successful applications of the RAMOS device have already been published such as the detection of protein production [ 23 , 24 ], testing of yeast extract quality [ 5 ], evaluation of cellulose digestibility [ 25 ] and determination of polymer biocompatibility [ 26 ]. The RAMOS technology allows an online measurement of the oxygen transfer rate (OTR) during the entire cultivation time.…”

“…Instead of optical density, OTR offers important information about the metabolic state of the culture [ 27 ]. Moreover, the OTR gives information about carbon source consumption and biomass formation [ 24 ]. In addition, limitations of a second substrate are easily detected.…”