Experimental investigations of multiple steady states in aerobic continuous cultivations of Saccharomyces cerevisiae

The steady-state behavior of a glucose-limited, aerobic, continuous cultivation of Saccharomyces cerevisiae CEN.PK113-7D was investigated around the critical dilution rate. Oxido-reductive steady states were obtained at dilution rates up to 0.09 h(-1) lower than the critical dilution rate by operating the bioreactor as a productostat, where the dilution rate was controlled on the basis of an ethanol measurement. Thus, the experimental investigations revealed that multiple steady states exist in a region of dilution rates below the critical dilution rate. The existence of multiple steady states was attributed to two distinct physiological effects occurring when growth changed from oxidative to oxido-reductive: (i) a decrease in the efficiency of ATP production and utilization (at ethanol concentrations below 3 g/L) and (ii) repression of the oxidative metabolism (at higher ethanol concentrations). The first effect was best observed at low ethanol concentrations, where multiple steady states were observed even when no repression of the oxidative metabolism was evident, i.e., the oxidative capacity was constant. However, at higher ethanol concentrations repression of the oxidative metabolism was observed (the oxidative capacity decreased), and this resulted in a broader range of dilution rates where multiple steady states could be found.     

Fluorescence based assay of GAL system in yeast Saccharomyces cerevisiae

The GAL1 promoter is one of the strongest inducible promoters in the yeast Saccharomyces cerevisiae. In order to improve recombinant protein production we have developed a fluorescence based method for screening and evaluating the contribution of various gene deletions to protein expression from the GAL1 promoter. The level of protein synthesis was determined in 28 selected mutant strains simultaneously, by direct measurement of fluorescence in living cells using a microplate reader. The highest, 2.4-fold increase in GFP production was observed in a gal1 mutant strain. Deletion of GAL80 caused a 1.3-fold increase in fluorescence relative to the isogenic strain. GAL3, GAL4 and MTH1 gene deletion completely abrogated GFP synthesis. Growth of gal7, gal10 and gal3 also exhibited reduced fitness in galactose medium. Other genetic perturbations affected the GFP expression level only moderately. The fluorescence based method proved to be useful for screening genes involved in GAL1 promoter regulation and provides insight into more efficient manipulation of the GAL system.     

Two‐compartment processing as a tool to boost recombinant protein production

Pichia pastoris is used extensively as a production platform for many recombinant proteins. The dissolved oxygen (DO) is one of the most important factors influencing protein production. The influence of the DO on productivity has not been studied independent from the feed rate. In this work, various DO levels were investigated independent from the feed rate. The model system was recombinant P. pastoris under the control of methanol‐induced alcohol oxidase promoter, which expressed HRP as the target protein. No significant effect was observed in terms of titer and specific productivity, which is a confirmation of the fact that the DO in a one‐compartment system cannot boost productivity for the model system under study. Hence, a two‐compartment system (a single reactor coupled with a plug flow reactor) was designed and implemented in order to apply oxygen‐related stress in the plug flow reactor and allow the cells to be recovered in the main reactor. Doing so, more than two‐fold increase in the titer and productivity and three‐fold increase in protein‐specific activity were achieved. Hence, partial application of oxygen‐related stress in the two‐compartment system was proposed as a process technology to enhance protein production.     

Advanced photobioreactor LED illumination system: Scale‐down approach to study microalgal growth kinetics

A newly designed and constructed LED illumination device for commercial cylindrical bioreactors is presented for application in microalgal cultivations and investigation of growth kinetics. An ideally illuminated volume is achieved by focusing the light toward the center of the reactor and thereby compensating the mutual shading of the cells. The relevant biomass concentration for homogeneous illumination depending on reactor radius was determined by light distribution measurements for Chlamydomonas to 0.2 g/L (equal 0.435 optical density at 750 nm). It is shown that cultivation experiments with the newly designed illumination device operated in batch mode can be successfully applied for determination of growth rates and photo conversion efficiencies. The exact knowledge of physiological reactions of specific strain(s) and the estimation of relevant parameters for scale‐up can be used for construction of economic pilot plant photobioreactors. The determination of light‐dependent kinetics of growth and product formation is the first necessary step to achieve this. A wide variety of different parameters can be examined like the effect of different illumination conditions (light intensity, frequency of day/night cycles, flashing light, light color…) and thereby for each single application specific, relevant, and interesting parameters will be examined.     

An edeine resistant mRNA-dependent protein synthesis system from a Saccharomyces cerevisiae mutant

A cell-free protein synthesizing system from a mutant of Saccharomyces cerevisiae translated exogenous mRNA in the presence of 2 microM edeine, while a similar system from wild-type strain was completely inhibited by the drug. The mutant ribosomes showed an affinity for [125I]edeine comparable to the wild-type ribosomes, thereby suggesting that these macromolecules alone were not responsible for the edeine-resistant capacity of the mutant.     

Scale‐down simulators for mammalian cell culture as tools to access the impact of inhomogeneities occurring in large‐scale bioreactors

During the scale‐up of a bioprocess, not all characteristics of the process can be kept constant throughout the different scales. This typically results in increased mixing times with increasing reactor volumes. The poor mixing leads in turn to the formation of concentration gradients throughout the reactor and exposes cells to varying external conditions based on their location in the bioreactor. This can affect process performance and complicate process scale‐up. Scale‐down simulators, which aim at replicating the large‐scale environment, expose the cells to changing environmental conditions. This has the potential to reveal adaptation mechanisms, which cells are using to adjust to rapidly fluctuating environmental conditions and can identify possible root causes for difficulties maintaining similar process performance at different scales. This understanding is of utmost importance in process validation. Additionally, these simulators also have the potential to be used for selecting cells, which are most robust when encountering changing extracellular conditions. The aim of this review is to summarize recent work in this interesting and promising area with the focus on mammalian bioprocesses, since microbial processes have been extensively reviewed.     

A global S. cerevisiae small ubiquitin‐related modifier (SUMO) system interactome

The small ubiquitin‐related modifier (SUMO) system has been implicated in a number of biological functions, yet the individual components of the SUMO machinery involved in each of these activities were largely unknown. Here we report the first global SUMO system interactome. Using affinity purification coupled with mass spectrometry, we identify >450 protein–protein interactions surrounding the SUMO E2, Siz type E3s and SUMO‐specific proteases in budding yeast. Exploiting this information‐rich resource, we validate several Siz1‐ and Siz2‐specific substrates, identify a nucleoporin required for proper Ulp1 localization, and uncover important new roles for Ubc9 and Ulp2 in the maintenance of ribosomal DNA.     

Rapid optimization of protein freeze‐drying formulations using ultra scale‐down and factorial design of experiment in microplates

Retaining biopharmaceutical proteins in a stable form is critical to their safety and efficacy, and is a major factor for optimizing the final product. Freeze‐dried formulations offer one route for improved stability. Currently the optimization of formulations for freeze‐drying is an empirical process that requires many time‐consuming experiments and also uses large quantities of product material. Here we describe a generic framework for the rapid identification and optimization of formulation excipients to prevent loss of protein activity during a lyophilization process. Using factorial design of experiment (DOE) methods combined with lyophilization in microplates a range of optimum formulations were rapidly identified that stabilized lactose dehydrogenase (derived from Lactobacillus leichmanii) during freeze‐drying. The procedure outlined herein involves two rounds of factorially designed experiments—an initial screen to identify key excipients and potential interactions followed by a central composite face designed optimization experiment. Polyethylene glycol (PEG) and lactose were shown to have significant effects on maintaining protein stability at the screening stage and optimization resulted in an accurate model that was used to plot a window of operation. The variation of freezing temperatures and rates of sublimation that occur across a microplate during freeze‐drying have been characterized also. The optimum formulation was then freeze‐dried in stoppered vials to verify that the microscale data was relevant to the effects observed at larger pilot scales. This work provides a generic approach to biopharmaceutical formulation screening where possible excipients can be screened for single and interactive effects thereby increasing throughput while reducing costs in terms of time and materials. Biotechnol. Bioeng. 2009; 104: 957–964. © 2009 Wiley Periodicals, Inc.     

Micro‐scale and rapid expression screening of highly expressed and/or stable membrane protein variants in Saccharomyces cerevisiae

Purification of milligram quantities of target proteins is required for structural and biophysical studies. However, mammalian membrane proteins, many of which are important therapeutic targets, are too unstable to be expressed in heterologous hosts and to be solubilized by detergents. One of the most promising ways to overcome these limitations is to stabilize the membrane proteins by generating variants via introduction of truncated flexible regions, fusion partners, and site‐directed mutagenesis. Therefore, an effective screening strategy is a key to obtaining successful protein stabilization. Herein, we report the micro‐scale and high‐throughput screening of stabilized membrane protein variants using Saccharomyces cerevisiae as a host. All steps of the screening, including cultivation and disruption of cells, solubilization of the target protein, and the pretreatment for fluorescence‐detected size exclusion chromatography (FSEC), could be performed in a 96‐well microplate format. We demonstrated that the dispersion among wells was small, enabling detection of a small but important improvement in the protein stability. We also demonstrated that the thermally stable mutants of a human G protein‐coupled receptor could be distinguished based on an increase of the peak height in the FSEC profile, which was well correlated with increased ligand binding activity of the protein. This strategy represents a significant platform for handling numerous mutants, similar to alanine scanning.     

Mimic of a large‐scale diafiltration process by using ultra scale‐down rotating disc filter

Ultra scale‐down (USD) approach is a powerful tool to predict large‐scale process performance by using very small amounts of material. In this article, we present a method to mimic flux and transmission performance in a labscale crossflow operation by an USD rotating disc filter (RDF). The Pellicon 2 labscale system used for evaluation of the mimic can readily be related to small pilot and industrial scale. Adopted from the pulsed sample injection technique by Ghosh and Cui (J Membr Sci. 2000;175:5‐84), the RDF has been modified by building in inserts to allow the flexibility of the chamber volume, so that only 1.5 mL of processing material is required for each diafiltration experiment. The reported method enjoys the simplicity of dead‐end mode operation with accurate control of operation conditions that can mimic well the crossflow operation in large scale. Wall shear rate correlations have been established for both the labscale cassette and the USD device, and a mimic has been developed by operating both scales under conditions with equivalent averaged shear rates. The studies using E. coli lysate show that the flux vs. transmembrane pressure profile follows a first‐order model, and the transmission of antibody fragment (Fab′) is independent of transmembrane pressure. Predicted flux and transmission data agreed well with the experimental results of a labscale diafiltration where the cassette resistance was considered. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Production of S‐lactoylglutathione by high activity whole cell biocatalysts prepared by permeabilization of recombinant Saccharomyces cerevisiae with alcohols

The permeabilization of yeast cells with methanol, ethanol, and isopropyl alcohol under various conditions was studied to develop the preparation method of high activity whole cell biocatalysts. Recombinant Saccharomyces cerevisiae, which intracellularly overexpresses glyoxalase I and catalyzes the conversion of methylglyoxal to S‐lactoylglutathione in the presence of glutathione, was used as the model system. The permeabilization treatments with alcohols significantly enhanced the activities of yeast cells. Especially, the initial S‐lactoylglutathione production rates of cells permeabilized with 40% ethanol and isopropyl alcohol solutions for 10 min at 4°C were high and were 364 and 582 times larger than those of untreated cells, respectively. These permeabilized yeast cells retained high activities during repeated batch reactions. Even in third batch reaction, they showed approximately 70–80% of the activity in the first batch. The plasma membrane of S. cerevisiae cells was damaged by the treatment with alcohol solutions in such a way that leakage of glyoxalase I from the cells is rather small and that both substrate and product show very high permeability. The initial S‐lactoylglutathione production rates of these permeabilized cells were 1.5–2.5 times larger than those of glyoxalase I in cell extracts prepared by ethyl acetate method from the same amount of cells. These results demonstrate that the recombinant S. cerevisiae cells permeabilized with alcohol solutions under the optimum condition are very effective whole cell biocatalysts. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 54–60, 1999.     

An integrated practical implementation of continuous aqueous two‐phase systems for the recovery of human IgG: From the microdevice to a multistage bench‐scale mixer‐settler device

Aqueous two‐phase systems (ATPS) are a liquid‐liquid extraction technology with clear process benefits; however, its lack of industrial embracement is still a challenge to overcome. Antibodies are a potential product to be recovered by ATPS in a commercial context. The objective of this work is to present a more integral approach of the different isolated strategies that have arisen in order to enable a practical, generic implementation of ATPS, using human immunoglobulin G (IgG) as experimental model. A microfluidic device is used for ATPS parameters preselection for product recovery. ATPS were continuously operated in a mixer‐settler device in one stage, multistage and multistage with recirculation configuration. Single‐stage pure IgG extraction with a polyethylene glycol (PEG) 3350‐phophates ATPS within continuous operation allowed a 65% recovery. Further implementation of a multistage platform promoted a higher particle partitioning reaching a 90% recovery. The processing of IgG from a cell supernatant culture harvest in a multistage system with top phase recirculation resulted in 78% IgG recovery in bottom phase. This work conjugates three not widely spread methodologies for ATPS: microfluidics, continuous and multistage operation.