Integrated L-phenylalanine separation in an E. coli fed-batch process: from laboratory to pilot scale

Pilot-scale reactive-extraction technology for fully integrated L-phenylalanine (L-Phe) separation in Escherichia coli fed-batch fermentations was investigated in order to prevent an inhibition of microbial L-Phe production by-product accumulation. An optimal reactive-extraction system, consisting of an organic kerosene phase with the cation-selective carrier DEHPA (di-2-ethylhexyl phosphonic acid) and an aqueous stripping phase including sulphuric acid, was found particularly efficient. Using this system with two membrane contactors, mass-transfer coefficients of up to 288 x 10(-7) cm s(-1) for the aqueous/organic and 77 x 10(-7) cm s(-1) for the organic/stripping phase were derived from experimental data using a simple modelling approach. Concentration factors higher than 4 were achieved in the stripping phase as compared to the aqueous donor phase. Reactive extraction enabled a 98% cation portion of L-Phe in the stripping phase, leading to final product purity higher than 99% after L-Phe precipitation. A doubling of L-Phe/glucose yield was observed when kerosene/DEHPA was added to the fermentation solution in the bioreactor to experimentally simulate a fully integrated L-Phe separation process.     

Characterization of stress and protein turnover from protein overexpression in fed-batch E. coli cultures.

A structured kinetic model that accounts for proteolytic degradation due to recombinant protein overexpression is introduced and its performance evaluated by comparison with previously reported fed-batch experimental data. This mathematical model contains an additional pool for a generic key precursor (in our case phenylalanine), an improved IPTG transport term, a phenylalanine transport term, and a variable protein turnover expression that accounts for proteolytic activity. The model predictions concerning proteolytic activity, glucose level, and cell growth are in very good agreement with an amino acid depletion hypothesis. Cultures exposed to greater stress showed higher and/or longer proteolysis, whereas less overall proteolytic activity was observed when the effect of induction was somewhat ameliorated.     

Simple control of fed-batch processes for recombinant protein production with E. coli

A very simple but effective process control technique is proposed that leads to a high batch-to-batch reproducibility with respect to biomass concentration as well as the specific biomass growth rate profiles in E. coli fermentations performed during recombinant protein production. It makes use of the well-established temperature controllers in currently used fermenters, but takes its information from the difference between the controlled culture temperature T (cult) and the temperature T (coolin) of the coolant fed to the fermenter's cooling jacket as adjusted by the fermenter temperature controller. For process control purposes this measured difference is corrected regarding stirrer influences and cumulated before it is used as a new process control variable. As a spin-off of this control, it becomes possible to estimate online the oxygen mass transfer rates and the corresponding k(L)a values during the real cultivation process.     

Open-loop control of specific growth rate in fed-batch cultures of recombinant E.coli

Summary A computer-based algorithm was used for the open-loop control of specific growth rate in fed-batch cultures of recombinant E.coli.. The control of nutrient feed rate to an exponential trajectory resulted in growth of the culture at a constant specific growth rate. Stable specific growth rates between 0.08 and 0.4 h^–1 were achieved.     

Batch and fed-batch cultures of E. coli TB1 at different oxygen transfer rates

Batch cultures of E. coli TB1/pUC13 were carried out at different oxygen transfer rates (OTR) enhanced by the increase of stirring rate and by the increase of air total pressure of the bioreactor. These two variables showed to have little effect on cell growth but a negative effect on cytochrome b5 (recombinant protein) production. However, this effect was more significant of high stirring rates than for values of pressure up to 0.4?MPa. The effects of stirring and pressure were also investigated for fed-batch mode operation. In this type of cell cultivation high cell densities are reached, thus a high capacity of oxygen supply of the system is required. To compare the two ways of improving OTR, cell behaviour was followed in two bioreactors at different operational conditions giving the same maximum OTR value. The first one operated at a high stirring rate (500?rpm) and at atmospheric pressure (0.1?MPa) and the other one at high air pressure (0.48?MPa) and low stirring rate. The increased pressure seemed to be a better way of ensuring an adequate oxygen supply to a culture of E. coli TB1 cells than an increased stirring rate. For the high pressure experiment a higher cellular density was reached, as well as a higher cyt.b5 expression which led to a 4-fold increase in final productivity. These experiments showed that bioreactor pressurization can be successfully used as a means of enhancing oxygen mass transfer to shear sensitive cell cultures.     

Novel approach of high cell density recombinant bioprocess development: Optimisation and scale-up from microlitre to pilot scales while maintaining the fed-batch cultivation mode of E. coli cultures

Background

Bioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale.

Results

Here, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase^® technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor.

Conclusions

Cultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditons from the first screening phase not only shortens the developmental line by guarantying that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all.     

Direct immunological identification of full-length cDNA clones for plant protein without gene fusion to E. coli protein

By immunological screening of a cDNA library constructed from potato tuber poly(A)+ RNA and Escherichia coli expression vector pUC8 by the vector-primer and linker procedure of Okayama and Berg [(1982) Mol. Cell Biol. 2, 161-170], nearly full-length cDNA clones for patatin, a major protein of potato tuber, were identified. The cDNA carrying part of the 5'-noncoding region of the patatin mRNA, in addition to entire coding and 3'-noncoding regions, expressed prepatatin in E. coli cells by translational initiation inside cDNA. These results suggest that nearly full-length cDNA clones with entire coding region can be identified directly by immunological screening without gene fusion to E. coli proteins at least for some plant mRNAs.     

Influences of yeast extract on specific cellular yield of Ovine growth hormone during fed-batch fermentation of E. coli

In most cases of E. coli high cell density fermentation process, maximizing cell concentration helps in increasing the volumetric productivity of recombinant proteins usually at the cost of lower specific cellular protein yield. In this report, we describe a process for maintaining the specific cellular yield of Ovine growth hormone (oGH) from E. coli by optimal feeding of yeast extract during high cell density fermentation process. Recombinant oGH was produced as inclusion bodies in Escherichia coli. Specific cellular yield of recombinant oGH was maintained by feeding yeast extract along with glucose during fed-batch fermentation. Glucose to yeast extract ratio of 0.75 was found to be optimum for maintaining the specific cellular oGH yield of 66 mg/g of E. coli cells. Continuous feeding of yeast extract along with glucose helped in reducing acetic acid secretion and promoted higher cell growth during fed-batch fermentation. High cell growth of E. coli and high specific yield of recombinant oGH thus helped in achieving high volumetric productivity of the expressed protein. A maximum of 2 g/l of ovine growth hormone was expressed as inclusion bodies in 12 h of fed-batch fermentation.     

Tunable recombinant protein expression with E. coli in a mixed-feed environment

Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both d-glucose and l-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of l-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future.     

Dynamics of the redox potential in a fed-batch culture of E. coli

Dynamics of Eh, pH, pO2 and optical density in E. coli cultures under glucose and ammonium exhaustion were studied. It has been shown that changes in the redox potential accompanying the exhaustion of these substances in aerobic cultures are the leaps by their character and reflect the physiological state of cells and changes in the structure of cell surface. A relationship between the changes in the redox potential and in the electrochemical potential of H ions (delta mu H) is suggested.     

Use of disposable reactors to generate inoculum cultures for E. coli production fermentations

Disposable technology is being used more each year in the biotechnology industry. Disposable bioreactors allow one to avoid expenses associated with cleaning, assembly and operations, as well as equipment validation. The WAVE bioreactor is well established for Chinese Hamster Ovary (CHO) production, however, it has not yet been thoroughly tested for E. coli production because of the high oxygen demand and temperature maintenance requirements of that platform. The objective of this study is to establish a robust process to generate inoculum for E. coli production fermentations in a WAVE bioreactor. We opted not to evaluate the WAVE system for production cultures because of the high cell densities required in our current E. coli production processes. Instead, the WAVE bioreactor 20/50 system was evaluated at laboratory scale (10‐L) to generate inoculum with target optical densities (OD₅₅₀) of 15 within 7–9 h (pre‐established target for stainless steel fermentors). The maximum settings for rock rate (40 rpm) and angle (10.5) were used to maximize mass transfer. The gas feed was also supplemented with additional oxygen to meet the high respiratory demand of the culture. The results showed that the growth profiles for the inoculum cultures were similar to those obtained from conventional stainless steel fermentors. These inoculum cultures were subsequently inoculated into 10‐L working volume stainless steel fermentors to evaluate the inocula performance of two different production systems during recombinant protein production. The results of these production cultures using WAVE inocula showed that the growth and recombinant protein production was comparable to the control data set. Furthermore, an economic analysis showed that the WAVE system would require less capital investment for installation and operating expenses would be less than traditional stainless steel systems. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Temperature gradient-based high-cell density fed-batch fermentation for the production of pyruvate oxidase by recombinant E. coli

Pyruvate oxidase (PyOD) is a very powerful enzyme for clinical diagnostic applications and environmental monitoring. Influences of temperature on cell growth, plasmid stability, and PyOD expression during the PyOD fermentation process by recombinant Escherichia coli were investigated. Based on the influences of temperature on the physiological metabolism, a novel high-cell density fed-batch cultivation with gradient temperature decrease strategy for effective PyOD production was achieved, under which the biomass (OD₆₀₀) of recombinant E. coli could reach to 71 and the highest PyOD activity in broth could reach to 3,307 U/L in 26?hr fermentation.     

Industrial control of recombinant E. coli fed-batch culture: new perspectives on traditional controlled variables

Three industrially relevant fed-batch algorithms were applied to the production of recombinant proteins in Escherichia coli BL21 DE3. Starvation dissolved-oxygen (DO)-transient control sustained growth rates greater than those in pH-stat (0.16 h(-1) versus 0.11-0.13 h(-1)) while feed-up DO-transient control better tracked the measured threshold for acetate production (mu approximately 0.2 h(-1)). All controllers supported growth without acetate production, resulting in end concentrations of recombinant protein up to 20 times greater than in batch culture. Both DO-transient control systems were judged superior to pH-stat for their ability to detect and track the acetate threshold. Results also showed that although high cell density at the stationary phase is desirable, this parameter may be dictated by the choice of media and reactor design as opposed to controller type. Controller selection, however, has a great impact on the capacity to track the acetate threshold and therefore to enhance productivity without concomitant acetate production.