Pastoris Culture Research Articles

Monitoring of sorbitol in Pichia pastoris cultivation applying sequential injection analysis

A simple and robust analyser system based on sequential injection analysis for the determination of sorbitol in Pichia pastoris Mut S phenotype batch fermentation is described. Applicability for the bioprocess monitoring of sorbitol in batch culture medium in a concentration range of 8–1.5 g l −1 with satisfying sorbitol recovery was demonstrated during 60 h. Optimization of physical and chemical parameters is further described. The method proved to be reproducible (R.S.D. < 3%), robust, and fast (17 injections per hour). The SIA sorbitol analyzer showed to be applicable for the monitoring of sorbitol concentration in a mixed substrate P. pastoris fermentation strategy. No interference of methanol was observed. The method is suited for the implementation of sorbitol concentration closed-loop control strategies and for the study of the effect of sorbitol concentration in heterologous protein productivity.

Recombinant truncated tilapia growth hormone enhances growth and innate immunity in tilapia fry ( Oreochromis sp.)

Pichia pastoris cells transformed with a plasmid engineered for the expression of tilapia growth hormone as a secreted product produced a proteolytically cleaved form of the recombinant protein. The sequence of this truncated variant was obtained by mass spectrometry analysis. The cleavage site was determined to be between residues Tyr 158 and Tyr 159. The resulting truncated tilapia growth hormone was a single chain protein lacking 46 amino acids of the C-terminal portion. In this study, we showed that the truncated growth hormone produced in the P. pastoris culture supernatant has growth promoting effects and stimulates innate immune parameters (lysozyme and lectins) in tilapia larvae. These results suggest that the C-terminal portion of growth hormone is not required for its growth promoting activity and the innate immune functions studied herein in fish. In addition, we found that the culture supernatant containing truncated tilapia growth hormone has a stronger effect over growth and immune system than cells lysate containing intact tilapia growth hormone expressed in P. pastoris.

Preparation of High-Purity Fructo-oligosaccharides by Aspergillus japonicus β-Fructofuranosidase and Successive Cultivation with Yeast

The short-chain FOSs with high purity were prepared using a two-step strategy: Aspergillus japonicus extracellular beta-fructofuranosidase-catalyzed synthesis of FOSs followed by cultivation with Pichia pastoris (P. pastoris). The higher FOSs content was obtained after 8 h under the catalysis of beta-fructofuranosidase at pH 5.5 and 55 degrees C. Successive P. pastoris cultivation exhausts almost all monosugars in 12 h at 30 degrees C, which increases the purity of FOSs, and also recovers beta-fructofuranosidase activity by ceasing the inhibition of glucose from catalysis of the enzyme, yielding more FOSs. Finally, the FOSs purity was increased from 56.55 to 84.45% (26.47% 1-kestose and 57.98% nystose).

Overexpression of the Neocallimastix frontalis xylanase gene in the methylotrophic yeasts Pichia pastoris and Pichia methanolica

The xylanase gene from the rumen fungus Neocallimastix frontalis was expressed in Pichia pastoris and Pichia methanolica. Using a complex medium with medium replacement before induction and the maintenance of the methanol induction level at 0.5%, P. pastoris was able to produce about 5400 U/ml of xylanase after 10 days of induction. With P. methanolica, on the other hand, about 6200 U/ml of xylanase was reached after 10 days of induction using synthetic medium as first culture medium and then direct induction by continuous methanol feed at 1.8 ml l −1 h −1. In general, the advantages of using P. methanolica to produce the xylanase included higher protein production, the lack of medium replacement, and an ease of scale up. However, because the P. pastoris culture supernatant contained fewer secreted non-target proteins compared to P. methanolica, xylanase purification would be easier with the P. pastoris system. In addition, experiments involving methanol pulses suggested that a relationship exists among base feeding, methanol consumption and xylanase activity.

On-line parallel factor analysis. A step forward in the monitoring of bioprocesses in real time

There is a widely growing interest to obtain robust and rapid methodologies capable of monitoring bioprocesses in real time. Different analytical methods have been adapted to measure cell density evolution throughout a culture, and fluorescence spectroscopy is becoming promising technique due to its sensitivity, selectivity towards important chemical analytes and its easy implementation as a non-invasive procedure. This work is focused on showing the advantages of coupling the trilinear algorithm Parallel Factor Analysis (PARAFAC) to the Multivariate Statistical Process Control (MSPC) as a monitoring and real-time control tool for bioprocesses. In this context, both induced (growing on methanol) and non induced (growing on glycerol) Pichia pastoris cultures were monitored by multiwavelength-fluorescence. In the first one methanol was used as substrate; whereas glycerol was used in the second one. Taking advantages of the mathematical properties of PARAFAC, batches of a bioprocess measured under normal operating conditions (NOC) were used to develop a calibration models. Residuals of the model in combination with MSPC were used to establish two control limits. The control limits were used for new batches in real time. Fluorescence spectroscopy combined with PARAFAC and MSPC is a feasible approach for controlling and performing fault diagnosis of bioprocesses offering the opportunity of performing real-time process surveillance based on relevant quality measurements.

Construction, production, and characterization of recombinant scFv antibodies against methamidophos expressed in Pichia pastoris

Pichia pastoris was used to express a recombinant scFv antibody against methamidophos derived from a recombinant phage-display library. The specific scFv gene was amplified from a positive clone and then subcloned into the expression vector pPICZα C. The resulting plasmid, pPICZα C–scFv, was linearized and transformed into P. pastoris (X-33). A transformant named X-33-Pp-Met-28D4, which showed strong expression of antibodies, was isolated, and the culture conditions were optimized. Under optimal conditions, P. pastoris cultures yielded much higher levels of scFv product than the Escherichia coli expression system. Immunochemical characterization of the scFv antibodies produced in P. pastoris indicated that the affinity and specificity of scFv against methamidophos are comparable to those of scFv antibodies produced in E. coli. Recoveries of methamidophos-fortified samples demonstrated that the P. pastoris-derived scFv antibodies can be used to determine the content of methamidophos residue in environmental and agricultural samples. For our purposes, expression in Pichia proved to be an efficient and economical method for the large-scale production of functional scFv antibodies against methamidophos for downstream applications.

Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures

In the methylotrophic yeast Pichia pastoris, alcohol oxidase (AOX) is a key enzyme involved in the dissimilation of methanol. Heterologous proteins are usually expressed under the control of the AOX1 promoter, which drives the expression of alcohol oxidase 1 in the wild-type strain. This study investigates the regulation of the alcohol oxidase enzyme of a recombinant P. pastoris Mut+ strain in cultures on glycerol and methanol as sole carbon sources and in mixed substrate cultures on both substrates. The aim was to have a better insight in the transition from growth on glycerol to growth on methanol, which is a key step in standard high cell density P. pastoris cultures for the production of foreign proteins. Nutrient shifts in chemostat cultures showed that after growth on glycerol use of mixed feeds of glycerol and methanol allowed faster induction of alcohol oxidase and faster adaptation of cellular metabolism than with a feed containing methanol as sole carbon source. The results of this study showed also how critical it is to avoid transient methanol accumulation during P. pastoris cultures operated at low residual methanol concentrations. Indeed, pulse experiments during chemostat cultures showed that sudden increase in methanol concentrations in cultures performed under methanol-limited or dual methanol and glycerol-limited growth conditions leads to wash-out of the culture because of too high consumption rate of methanol, which leads to excretion of toxic intermediates. High rate of methanol consumption was due to high specific AOX activities observed at low residual methanol concentrations.

Expression of the nucleocytoplasmic tobacco lectin in the yeast Pichia pastoris

The Nicotiana tabacum lectin, also called Nictaba, is a nucleocytoplasmic plant lectin expressed in tobacco leaves after exposure to jasmonates. Purification of the lectin from raw material is a time-consuming process, demanding large amounts of induced plant material. In addition, the lectin yield is low and purified lectin fractions are always contaminated with low molecular weight compounds such as phenols. In a way to improve and facilitate the purification of the tobacco lectin, we cloned the Nictaba gene in a vector optimized for protein expression in the methylotrophic yeast Pichia pastoris. In this report, we present data of the expression profile of recombinant Nictaba in the P. pastoris culture medium and in P. pastoris cells together with the purification strategy using ion exchange chromatography and affinity chromatography on a column with immobilized ovomucoid. Pichia transformants were estimated to express approximately 6mg of recombinant lectin per liter medium after a 72h culture. SDS–PAGE and Western blot analysis revealed that the recombinant lectin expressed in Pichia exists in two molecular forms. Edman degradation and mass spectrometry analysis confirmed the presence of at least two forms of recombinant lectin with molecular weights of 19,060 and 20,100Da, corresponding to lectin polypeptides similar to the fully processed Nictaba which is N-terminally blocked, and Nictaba extended at the N-terminus with the amino acids residues EAEAYVEFT due to incomplete processing of the α-factor mating sequence. Further characterisation of the recombinant lectin revealed agglutination and carbohydrate-binding properties similar to the native tobacco lectin.

Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures: A quantitative study based on concentration gradients in transient continuous cultures

Transient continuous cultures constitute a means to speed up strain characterization, by avoiding the need for many time-consuming steady-state experiments. In this study, mixed substrate growth on glycerol and methanol of a Pichia pastoris strain expressing and secreting recombinant avidin was characterized quantitatively by performing a nutrient gradient with linear increase of the methanol fraction in the feed medium from 0.5 to 0.93 C-mol C-mol(-1) at a dilution rate of 0.06 h(-1). The influence of the methanol fraction in the feed medium on recombinant avidin productivity and on specific alcohol oxidase activity were also examined. Results showed that, compared with cultures on methanol as sole carbon source, the specific recombinant avidin production rate was the same provided the methanol fraction in the feed medium was higher than 0.6 C-mol C-mol(-1). The volumetric avidin production rate was even 1.1-fold higher with a methanol fraction in the feed medium of 0.62 C-mol C-mol(-1) as a result of the higher biomass yield on mixed substrate growth compared with methanol alone. Moreover, since heat production and oxygen uptake rates are lower during mixed substrate growth on glycerol and methanol, mixed substrate cultures present technical advantages for the performance of high cell density P. pastoris cultures. Results obtained in a high cell density fed-batch culture with a mixed feed of 0.65 C-mol C-mol(-1) methanol and 0.35 C-mol C-mol(-1) glycerol were in agreement with results obtained during the transient nutrient gradient.

Screen-less expanded bed column: new approach for the recovery and purification of a malaria transmission blocking vaccine candidate from Pichia pastoris

An experimental malaria transmission blocking vaccine antigen, Pfs25H, expressed and secreted from Pichia pastoris was recovered and purified using a screenless expanded bed column equipped with a rotating fluid distribution system. This column was able to accommodate feed stock, containing 30% biomass, at a flow rate of 300-400 cm/h without affecting column stability. This capability is three times higher than the capability of the expanded bed column currently in use, which is equipped with a perforated plate fluid distribution system; this design could accommodate biomass concentrations of only up to 10%. The screen-less design did not affect the binding capacity, purification level or process yield and, therefore, shorten the process. Purified Pfs25H of 6.4 g were recovered from 37 l of Pichia pastoris culture in one step.

Parallel factor analysis combined with PLS regression applied to the on-line monitoring of Pichia pastoris cultures

Various key variables (biomass, substrate and product) of bioprocesses should be monitored in order to retrieve useful information on the system, with the biomass (the cell density) the principal target. Although several analytical methods have been adapted and used to monitor the evolution of cell density evolution in cultures, a general method for performing this determination has not yet been established, as each technique has its own advantages and drawbacks. In the present work, noninduced glycerol batch cultures (for which biomass and substrate are the key variables) were monitored using multiwavelength fluorescence spectroscopy. The data gathered were modelled via PARAFAC-PLS chemometric methodologies, resulting in important qualitative and quantitative information about the behaviours of different biogenic fluorophors in batch cultures of the yeast Pichia pastoris. This information was used to predict the target process variables in such cultures; this permitted the applicability of this combined technique to bioprocess monitoring to be assessed.

Interfacing Pichia pastoris cultivation with expanded bed adsorption

For improved interfacing of the Pichia pastoris fed-batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density. The concept was applied to a one-step recovery and purification procedure for a fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A fused to lipase B from Candida antarctica (CALB). The modified cultivation technique resulted in lower cell death and consequently lower concentration of proteases and other contaminating proteins in the culture broth. Flow cytometry analysis showed 1% dead (propidium-stained) cells compared to 3.5% in the reference process. During the whole process of cultivation and recovery, no proteolysis was detected and in the end of the cultivation, the product constituted 87% of the total supernatant protein. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 2.2 g/L of CBM–CALB. In the EBA process, no cell-adsorbent interaction was detected but the cell density had to be reduced by a two-times dilution to keep a proper bed expansion. At flow velocity of 400 cm/h, the breakthrough capacity was 12.4 g/L, the product yield 98%, the concentration factor 3.6 times, the purity about 90%, and the productivity 2.1 g/L · h. © 2006 Wiley Periodicals, Inc.

A methodology for centrifuge selection for the separation of high solids density cell broths by visualisation of performance using windows of operation

Expression systems capable of growing to high cell densities are now readily available and are popular due to the benefits of increased product concentration. However, such high solids density cultures pose a major challenge for bioprocess engineers as choosing the right separation equipment and operating it at optimal conditions is crucial for efficient recovery. This study proposes a methodology for the rapid determination of suitable operating conditions for the centrifugal recovery of high cell density fermentation broths. An ultra scale-down (USD) approach for the prediction of clarification and dewatering levels achieved in a range of typical high-speed centrifuges is presented. Together with a visualisation tool, a Window of Operation, this provides for the rapid analysis of separation performance and evaluation of the available operating conditions, as an aid in the selection of the centrifuge equipment most appropriate for a given process duty. A case study examining centrifuge selection for the processing of a high cell density Pichia pastoris culture demonstrates the method. The study examines semi-continuous disc-stack centrifuges and batch-operated machines such as multi-chamber bowls and Carr Powerfuges. Performance is assessed based on the variables of clarification, dewatering and product yield. Inclusion of limits imposed by the centrifuge type and design, and operation itself, serve to constrain the process and to define the Windows of Operation. The insight gained from the case study provides a useful indication of the utility of the methodology presented and illustrates the challenges of centrifuge selection for the demanding case of high solids concentration feed streams.

Production of pediocin PA-1 in the methylotrophic yeast Pichia pastoris reveals unexpected inhibition of its biological activity due to the presence of collagen-like material

Expression of the pedA gene from Pediococcus acidilactici, coding for mature bacteriocin Pediocin PA-1, was investigated using the yeast Pichia pastoris to obtain larger quantities of pediocin to support additional studies, including structure–function research. Following various cloning strategies, a KM71H (Mut s) strain was selected. A significant concentration (74 μg/ml) of extracellular recombinant pediocin was obtained but the pediocin showed no biological activity. Supernatant fluids from P. pastoris cultures, harboring or not pedA, inhibited the biological activity of natural pediocin PA-1. The recombinant pediocin appeared as a mixture of three main fractions (7–8, 11, 20 kDa vs. 4.6 kDa for natural pediocin PA-1). The recombinant pediocin was also less hydrophobic and behaved differently when subjected to isoelectric focusing. Strong evidence indicated that some “collagen-like” material was tightly associated, most probably via covalent binding, to the recombinant pediocin. The “collagen-like” material was most probably responsible for the lack of biological activity of the recombinant pediocin and for the differences observed regarding some of the physico-chemical properties. Both the recombinant pediocin and natural pediocin were sensitive to collagenase, suggesting that pediocin PA-1 may possess a somewhat “collagen-like” nature. Interestingly, recombinant pediocin preparations showed the ability to assemble into fibrils.

Adaptive dissolved oxygen control through the glycerol feeding in a recombinant Pichia pastoris cultivation in conditions of oxygen transfer limitation

In high cell density cultivation processes the productivity is frequently constrained by the bioreactor maximum oxygen transfer capacity. The productivity can often be increased by operating the process at low dissolved oxygen concentrations close to the limitation level. This may be accomplished with a closed-loop controller that regulates the dissolved oxygen concentration by manipulating the dominant carbon source feeding rate. In this work we study this control problem in a pilot 50 l bioreactor with a high cell density recombinant P. pastoris cultivation in complex media. The study focuses on the design of accurate stable adaptive controllers, with guaranteed exponential convergence and its relation with the calibration of controller parameters. Two adaptive control strategies were tested in the pilot bioreactor: a model reference adaptive controller with a linear reference model and an integral feedback controller with adaptive gain. The latter alternative proved to be more robust to errors in the measurements of the off-gas composition. Concerning the instrumentation, algorithms were derived assuming that both the dissolved oxygen tension and off-gas composition are measured on-line, but also the case of only dissolved oxygen being measured is addressed. It was verified that the measurement of off-gas composition might not improve the controller performance due to measurement and process time delays.

Bioreactor Retrofitting to Avoid Aeration with Oxygen in Pichia Pastoris Cultivation Processes for Recombinant Protein Production

It is demonstrated that bioreactor construction is of practical importance to some recombinant protein production processes. Here we focus on a case where the oxygen mass transfer capability of the bioreactor depicts a limitation to productivity that must be overcome by technical measures. Choosing different impellers can significantly enhance the mass transfer capability of the bioreactor. This is shown through the concrete example of cultivation of Pichia pastoris on methanol. In order to operate the bioreactor at biomass concentrations larger than 150 g kg −1 and specific growth rates larger than 0.03 h −1 , it is usually necessary to supplement the aeration with molecular oxygen. When, however, as is shown here, the standard Rushton turbines in the bioreactor are replaced by a set of lower power number impellers with improved air handling capabilities it is possible to operate the reactor at even more demanding growth and biomass conditions without additional oxygen while achieving even higher productivities. Here we took a combination of two hollow blades and a single hydrofoil impeller in order to enhance the mass transfer rates. Its advantage is shown at the 10-litre laboratory-scale with a Pichia pastoris culture that produces the recombinant protein dipeptidylpeptidase IV. The enzyme serves as a target for the development of specific inhibitors to control blood glucose homeostasis. Optimization of the fermentation process and higher enzyme yields allow inhibitor evaluation and co-crystallization studies.

High-level expression, purification, and characterization of recombinant wheat xylanase inhibitor TAXI-I secreted by the yeast Pichia pastoris

Triticum aestivum xylanase inhibitor I (TAXI-I) is a wheat protein that inhibits microbial xylanases belonging to glycoside hydrolase family 11. In the present study, recombinant TAXI-I (rTAXI-I) was successfully produced by the methylotrophic yeast Pichia pastoris at high expression levels (∼75 mg/L). The rTAXI-I protein was purified from the P. pastoris culture medium using cation exchange and gel filtration chromatographic steps. rTAXI-I has an iso-electric point of at least 9.3 and a mass spectrometry molecular mass of 42,013 Da indicative of one N-linked glycosylation. The recombinant protein fold was confirmed by circular dichroism spectroscopy. Xylanase inhibition by rTAXI-I was optimal at 20–30 °C and at pH 5.0. rTAXI-I still showed xylanase inhibition activity at 30 °C after a 40 min pre-incubation step at temperatures between 4 and 70 °C and after 2 h pre-incubation at room temperature at a pH ranging from 3.0 to 12.0, respectively. All tested glycoside hydrolase family 11 xylanases were inhibited by rTAXI-I whereas those belonging to family 10 were not. Specific inhibition activities against family 11 Aspergillus niger and Bacillus subtilis xylanases were 3570 and 2940 IU/mg protein, respectively. The obtained biochemical characteristics of rTAXI-I produced by P. pastoris (no proteolytical cleft) were similar to those of natural TAXI-I (mixture of proteolytically processed and non-processed forms) and non-glycosylated rTAXI-I expressed in Escherichia coli. The present results show that xylanase inhibition activity of TAXI-I is only affected to a limited degree by its glycosylation or proteolytic processing.

Detection of non-host viable contaminants in Pichia pastoris cultures and fermentation broths

The ability to detect viable contaminants in cultures propagated from the original host-expression system ensures that the integrity and purity of seed banks, fermentation broths, and ultimately the final product are continually controlled and maintained. The method developed to detect such agents must be selective for a broad spectrum of microbes, which may be present at very low levels, while discriminating from the host organisms. Although Pichia pastoris strains are frequently used as cell lines for the expression of heterologous proteins, a method that is specific for monitoring culture purity has yet to be reported for this type of organism. An assay that is capable of recovering contaminating bacteria, fungi, and closely related yeast from cultures of P. pastoris at parts per million detection limits is described here.

Advances in understanding and modeling the gas–liquid mass transfer in shake flasks

The gas–liquid mass transfer in 250 ml shake flasks has previously been sucessfully modelled on basis of Higbie’s penetration theory. The current contribution presents advances in understanding and modelling the gas–liquid mass transfer in shake flasks at waterlike liquid viscosity in flask sizes between 50 and 1000 ml. An experimental investigation of the maximum gas–liquid mass transfer capacity OTR max using the sodium sulphite system was extended to relative filling volumes of 4–16%, shaking diameters of 1.25, 2.5, 5, 7, 10 cm and shaking frequencies of 50–500 rpm for the above flask sizes. Simultaneously, the previous model of the gas–liquid mass transfer was extended to a “two sub-reactor model” to account for different mechanisms of mass transfer in the liquid film on the flask wall and the bulk of the liquid rotating within the flask. The shake flask is for the first time considered to be a two-reactor system consisting of a stirred tank reactor (bulk liquid) and a film reactor (film on flask wall and base). The mass transfer into the film on the flask wall and base at “in-phase” operating conditions is described by Higbie’s penetration theory. Two different mass transfer theories were applied to successfully describe the mass transfer into the bulk liquid: a model by Kawase and Moo-Young and a model by Gnielinski. The agreement between the new modelling approach, which requires absolutely no fitting parameters and the experimental is within ±30%. The applicability of the models to a biological system was shown using a Pichia pastoris culture. This is particularly notable since geometrically non-similar liquid distributions in very different sizes of shaking flasks are covered. A comparable description of the gas–liquid mass transfer in bubble aerated reactors like stirred tanks is absolutely out of reach. A spatially- and time-resolved consideration of the mass transfer in the liquid film on the flask wall and base has shown that the validity of Higbie’s theory sensitively depends on the film thickness and contact time.

Endostatin capture from Pichia pastoris culture in a fluidized bed : From on-chip process optimization to application

One of the characteristics of the methylothrophic yeast Pichia pastoris is its ability to grow to a very high cell density. Biomass concentrations of 300–400 g wet mass/l are common. It is therefore obvious that the recovery processes of extracellular proteins from this microorganism should take into account the effect of high biomass content. Separation by filtration and/or centrifugation is possible but these steps are cumbersome and can affect the protein recovery. The use of fluidized beds is attractive proteins capture option since it eliminates the biomass while capturing the desired protein. Zirconia-based resins possess unique properties which make them appropriate for processing high biomass concentrations in an expanded bed mode. The beads are particularly heavy (density is 3.2 g/ml) and small (75 μm) and therefore can accommodate high fluidization velocity and high mass transport. Specific operating conditions for effective capture of expressed protein have to be determined. This determination is generally time consuming and requires relatively large amount of feedstock for the lab trials. To avoid multiple chromatographic trials in columns, optimal conditions of adsorption and elution were determined by ProteinChip technology coupled with mass spectrometry. This technology involves flat chip surfaces functionalized as chromatographic beads where it is possible to adsorb and desorb proteins. Four different functional groups (strong anion-exchange, weak cation-exchange, hydrophobic and metal chelate) were tested and the retained proteins were analyzed directly by mass spectrometry. The weak cation-exchange group was chosen for further work. The Zirconia-based weak cation-exchange sorbent (CM HyperZ) was evaluated for binding capacity in a packed column and then for capturing endostatin from crude feed stock. Based on the previously determined conditions; 45 l of culture containing approximately 15 kg of biomass (wet mass) and 3 g endostatin were applied on an expanded bed at a flow-rate of 535 cm/h, yielding 80% of the endostatin and removing approximately 80% of foreign proteins.