Limiting factors in Escherichia coli fed‐batch production of recombinant proteins

Abstract

Fed-batch production of recombinant beta-galactosidase in E. coli was studied with respect to the specific growth rate at induction. The cultivations were designed to induce protein production by IPTG at a glucose feed rate corresponding to high mu = 0.5 h(-1)) or low (mu = 0.1 h(-1)) specific growth rate. Protein production rate was approximately 100% higher at the higher specific growth rate, resulting in the accumulation of beta-galactosidase up to 30% of the total cell protein. Transcription analysis showed that beta-galactosidase-specific messenger RNA was immediately formed after induction (<5 min), but the amount was the same in both cases and was thus not the initial limiting factor. The content of ribosomes, as represented by rRNA, rapidly decreased with specific growth rate from a relative level of 100%, at the high specific growth rate, to 20% at the low specific growth rate. At high specific growth rate, ribosomes were additionally degraded upon induction due to the high production level. Translation therefore seemed to be the initial limiting factor of the protein synthesis capacity. The alarmone guanosine tetraphosphate increased at both high and low feed level inductions, indicating an induction-forced starvation of charged tRNA and/or glucose. The altered physiological status was also detected by the formation of acetic acid. However, the higher production rate resulted in high-level accumulation of acetic acid, which was absent at low feed rate production. Acetic acid production is thus coupled to the high product formation rate and is proposed to be due either to a precursor drain of Krebs cycle intermediates and a time lag before induction of the glyoxalate shunt, or to single amino acid overflow, since the model product is relatively poor in glycin and alanin. In conclusion, it is proposed that production at high specific growth rate becomes precursor-limited, while production at low specific growth rate is carbon- and/or energy-limited.

Heterologous protein production using the Pichia pastoris expression system

“…However, increased copy number might reasonably be expected to exert a knock-on effect on transcription and translation, both of which may become rate-limiting due to a lack of resources, such as precursors and energy [47], as has been previously reported for recombinant protein production in E. coli [62,93]. However, in Pichia it has been proposed that it is more likely that any limitations are due to post-translational events, such as folding within the endoplasmic reticulum (ER), membrane translocation and signal sequence processing [47].…”

Recombinant protein production in an Escherichia coli reduced genome strain

“…Fed-batch fermentations were used to assess E. coli MDS40 at three different the fed-batch growth rates: 0.15 h −1 (low), 0.25 h −1 (intermediate), and 0.5 hr −1 (high), where the batch phase growth rates were not different. The parent strain, E. coli MG1655, was only assessed at 0.25 h −1 , as the behavior of E. coli MG1655 and other related strains has been well investigated with respect to recombinant protein productivity at these controlled growth rates (Curless et al, 1990;Hellmuth et al, 1994;Sanden et al, 2003;Turner et al, 1994). The growth curves for E. coli MG1655 and MDS40 are shown in Figure 2.…”

“…Some expression systems have extremely powerful promoters which have been observed to stress the cells or cause a metabolic burden (Glick, 1995;Haddadin and Harcum, 2005;Hoffman et al, 2002;Sanden et al, 2003;Wood and Peretti, 1990). To assess the effect of strong promoters on E. coli MDS40, the effect of changing the induction strength was investigated.…”

“…Although E. coli is a well studied and a commonly used recombinant host, the behavior of E. coli can still be unpredictable at times, especially while expressing recombinant protein (Andersson et al, 1996;Baneyx, 1999;Baneyx and Mujacic, 2004;Gill et al, 2000;Oh and Liao, 2000;Sanden et al, 2003;Swartz, 1996). One of the overall goals of the multiple deletion strain project is to improve the growth/yield properties and robustness of E. coli as a recombinant host by eliminating potentially nonessential genes.…”

Proteomic Profiling of Recombinant Escherichia coli in High-Cell-Density Fermentations for Improved Production of an Antibody Fragment Biopharmaceutical

“…Previous work has shown that synthesis rates (35,38) and steady-state levels (17) of ribosomal proteins, as well as rRNA levels (36), decrease during recombinant protein synthesis in fed-batch fermentations. In the present study, the ribosomal protein decrease seemed to occur in both control and production runs.…”