Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield

Streptavidin is widely used as an analytical tool and affinity tag together with biotinylated surfaces or molecules. We report for the first time a simple strategy that yields high biomass of a Pichia pastoris strain containing a methanol induced core streptavidin (cStp) gene. Three factors were evaluated for biomass production: glycerol concentration, aeration, and feed flow rates in a bioreactor. Recycling of recombinant cells, either free or immobilized, was investigated during induction. Concentration of 2.0 M glycerol, feeding flow rate of 0.11 mL min^?1, and aeration by air injection dispersed with a porous stone combined with agitation at 500 rpm were the set of conditions resulting into maximum biomass yield (150 g L^?1). These parameters yielded 4.0 g L^?1 of cStp, after 96 h of induction. Recombinant biomass was recycled twice before being discarded, which can reduce production costs and simplify the process. Immobilized P. pastoris biomass produced 2.94 and 1.70 g L^?1 of cStp in the first and second induction cycle, respectively. Immobilization and recycling of recombinant P. pastoris biomass opens new possibilities as a potential strategy to improve volumetric productivity for heterologous protein expression. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Polymer characterization and optimization of conditions for the enhanced bioproduction of benzaldehyde by Pichia pastoris in a two‐ ;phase partitioning bioreactor

Benzaldehyde, with its apricot and almond‐like aroma, is the second most abundantly used molecule in the flavor industry, and is most commonly produced via chemical routes, such as by the oxidation of toluene. Biologically produced benzaldehyde, whether by extraction of plant material or via microbial biotransformation, commands a substantial price advantage, and greater consumer acceptance. Methylotrophic yeast, such as Pichia pastoris, contain the enzyme alcohol oxidase (AOX), which, in the presence of alcohols other than methanol, are able to yield aldehydes as dead‐end products, for example, benzaldehyde from benzyl alcohol. In this work, we have determined that benzaldehyde, and not benzyl alcohol, is inhibitory to the transformation reaction by P. pastoris, prompting the development of a selection strategy for identifying sequestering polymers for use in a partitioning bioreactor that was based on the ratio of partition coefficients (PCs) for the two target molecules. Additionally, we have now confirmed for the first time, that the mechanism of solute uptake by amorphous polymers is via absorption, not adsorption. Finally, we have adopted a common strategy used for the production of heterologous proteins by P. pastoris, namely the use of a mixed methanol/glycerol feed for inducing the required AOX enzyme, while reducing the time required for high density biomass generation. All of these components were combined in a final experiment in which 10% of the polymer Kraton D1102K, whose PC ratio of benzaldehyde to benzyl alcohol was 14.9, was used to detoxify the biotransformation in a 5 L partitioning bioreactor, resulting in a 3.4‐fold increase in benzaldehyde produced (14.4 g vs. 4.2 g) relative to single phase operation, at more than double the volumetric productivity (97 mg L^?1 h^?1 vs. 41 mg L^?1 h^?1). Biotechnol. Bioeng. 2013; 110: 1098–1105. © 2012 Wiley Periodicals, Inc.     

Effects of temperature and glycerol and methanol‐feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris

Optimization of protein production from methanol‐induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol‐feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut⁺ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5‐fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. © 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728–735, 2014     

Quantitative physiology of Pichia pastoris during glucose‐limited high‐cell density fed‐batch cultivation for recombinant protein production

Pichia pastoris has become one of the major microorganisms for the production of proteins in recent years. This development was mainly driven by the readily available genetic tools and the ease of high‐cell density cultivations using methanol (or methanol/glycerol mixtures) as inducer and carbon source. To overcome the observed limitations of methanol use such as high heat development, cell lysis, and explosion hazard, we here revisited the possibility to produce proteins with P. pastoris using glucose as sole carbon source. Using a recombinant P. pastoris strain in glucose limited fed‐batch cultivations, very high‐cell densities were reached (more than 200 g_CDW L^?1) resulting in a recombinant protein titer of about 6.5 g L^?1. To investigate the impact of recombinant protein production and high‐cell density fermentation on the metabolism of P. pastoris, we used ¹³C‐tracer‐based metabolic flux analysis in batch and fed‐batch experiments. At a controlled growth rate of 0.12 h^?1 in fed‐batch experiments an increased TCA cycle flux of 1.1 mmol g^?1 h^?1 compared to 0.7 mmol g^?1 h^?1 for the recombinant and reference strains, respectively, suggest a limited but significant flux rerouting of carbon and energy resources. This change in flux is most likely causal to protein synthesis. In summary, the results highlight the potential of glucose as carbon and energy source, enabling high biomass concentrations and protein titers. The insights into the operation of metabolism during recombinant protein production might guide strain design and fermentation development. Biotechnol. Bioeng. 2010;107: 357–368. © 2010 Wiley Periodicals, Inc.     

Enhanced truncated-t-PA (CT-b) expression in high-cell-density fed-batch cultures of <Emphasis Type="Italic">Pichia pastoris</Emphasis> through optimization of a mixed feeding strategy by response surface methodology

Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µ _set glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µ _set glycerol and that of methanol to be 0.05 and 0.03 h^?1, respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained.     

Optimization of norovirus virus‐like particle production in Pichia pastoris using a real‐time near‐infrared bioprocess monitor

The production of norovirus virus‐like particles (NoV VLPs) displaying NY‐ESO‐1 cancer testis antigen in Pichia pastoris BG11 Mut⁺ has been enhanced through feed‐strategy optimization using a near‐infrared bioprocess monitor (RTBio^® Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real‐time. The production of NoV VLPs displaying NY‐ESO‐1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two‐fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP‐NY‐ESO‐1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L^?1 during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1‐NY‐ESO‐1 yield of 0.85 g L^?1. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518–526, 2016     

Improving intracellular production of recombinant protein in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using an optimized preinduction glycerol-feeding scheme

High-cell-density production of recombinant growth hormone of Lateolabrax japonicus (rljGH) expressed intracellularly in Pichia pastoris was investigated. In the regular strategy of induction at a cell density of 160 g l⁻¹, short duration of intracellular rljGH accumulation (17 h) resulted in a low final cell density of 226 g l⁻¹. Thus, a novel strategy of induction at a cell density of 320 g l⁻¹ was investigated. In this strategy, the preinduction glycerol-feeding scheme had a significant effect on the post-induction production. Constant glycerol feeding led to a decrease of the specific rljGH production and specific production rate because of low preinduction specific growth rate. This decrease was avoided by exponential glycerol feeding to maintain a preinduction specific growth rate of 0.16 h⁻¹. The results from exponential glycerol feeding indicated that the rljGH production depended on the preinduction specific growth rate. Moreover, mixed feeding of methanol and glycerol during induction improved the specific production rate to 0.07 mg g⁻¹ h⁻¹ from 0.043 mg g⁻¹ h⁻¹. Consequently, both high cell density (428 g l⁻¹) and high rljGH production could be achieved by the novel strategy: growing the cells at the specific growth rate of 0.16 h⁻¹ to the cell density of 320 g l⁻¹ and inducing the expression by mixed feeding.     

GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin‐auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol‐based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol‐based processes lead to large proportions of biotin‐blocked binding sites of streptavidin due to biotin‐supplemented media. Targeting these problems, this paper provides strategies for the methanol‐free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin‐blocked streptavidin. The optimized, easily scalable fed‐batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin‐free streptavidin and a productivity of 57.8 nM h^?1 based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un‐optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h^?1), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855–864, 2016     

Continuous culture of Candida boidinii variant 60 growing on methanol

Summary A new variant, Candida boidinii variant 60, which is less sensitive to methanol and formaldehyde shocks was grown in continuous cultures with methanol as sole carbon source. The substrate concentration in the feeding medium was either 1% methanol or 3% methanol. Biomass production, methanol consumption, the formation of formaldehyde and gas exchange were measured at different dilution rates. With low methanol feeding (10 g/l) maximal productivity of 0.44 g biomass/l·h is obtained at a dilution rate of 0.14 h^–1. Maximal specific growth rate is 0.18 h^–1. A yield of 0.32 g biomass/g methanol was obtained and the respiration quotient was determined as 0.55. Independently of initial substrate concentration, biomass decreases if methanol and formaldehyde are accumulating in the culture broth.In the culture with high methanol feeding (30 g/l) cell concentratioon increases up to 9 g/l at D=0.04 h^–1. At higher dilution rates methanol and form-aldehyde appear in the medium. Formaldehyde is then preferably oxidized without energy advantages for the cells. It seems that this enables the cells to overcome toxic effects caused by methanol and formaldehyde.     

Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rate

The intracellular metabolic fluxes through the central carbon pathways in the bioprocess for recombinant human erythropoietin (rHuEPO) production by Pichia pastoris (Mut⁺) were calculated to investigate the metabolic effects of dual carbon sources (methanol/sorbitol) and the methanol feed rate, and to obtain a deeper understanding of the regulatory circuitry of P. pastoris, using the established stoichiometry‐based model containing 102 metabolites and 141 reaction fluxes. Four fed‐batch operations with (MS‐) and without (M‐) sorbitol were performed at three different constant specific growth rates (h^?1), and denoted as M‐0.03, MS‐0.02, MS‐0.03, and MS‐0.04. Considering the methanol consumption pathway, the M‐0.03 and MS‐0.02 conditions produced similar effects and had >85% of formaldehyde flux towards the assimilatory pathway. In contrast, the use of the dual carbon source condition generated a shift in metabolism towards the dissimilatory pathway that corresponded to the shift in dilution rate from MS‐0.03 to MS‐0.04, indicating that the methanol feed exceeded the metabolic requirements at the higher µ₀. Comparing M‐0.03 and MS‐0.03 conditions, which had the same methanol feeding rates, sorbitol addition increased the rHuEPO synthetic flux 4.4‐fold. The glycolysis, gluconeogenesis, and PPP pathways worked uninterruptedly only at MS‐0.02 condition. PPP and TCA cycles worked with the highest disturbances at MS‐0.04 condition, which shows the stress of increased feeding rates of methanol on cell metabolism. Biotechnol. Bioeng. 2010; 105: 317–329. © 2009 Wiley Periodicals, Inc.     

<Emphasis Type="Italic">Pichia pastoris</Emphasis> fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement

Fed-batch fermentation of a methanol utilization plus (Mut⁺) Pichia pastoris strain typically has a growth phase followed by a production phase (induction phase). In the growth phase glycerol is usually used as carbon for cell growth while in the production phase methanol serves as both inducer and carbon source for recombinant protein expression. Some researchers employed a mixed glycerol-methanol feeding strategy during the induction phase to improve production, but growth kinetics on glycerol and methanol and the interaction between them were not reported. The objective of this paper is to optimize the mixed feeding strategy based on growth kinetic studies using a Mut⁺ Pichia strain, which expresses the heavy-chain fragment C of botulinum neurotoxin serotype C [BoNT/C(Hc)] intracellularly, as a model system. Growth models on glycerol and methanol that describe the relationship between specific growth rate (μ) and specific glycerol/methanol consumption rate (ν _gly, ν _MeOH) were established. A mixed feeding strategy with desired μ _gly/μ _MeOH =1, 2, 3, 4 (desired μ _MeOH set at 0.015 h⁻¹) was employed to study growth interactions and their effect on production. The results show that the optimal desired μ _gly/μ _MeOH is around 2 for obtaining the highest BoNT/C(Hc) protein content in cells: about 3 mg/g wet cells. Electronic Publication     

Productivity of Chlorella sorokiniana in a short light‐path (SLP) panel photobioreactor under high irradiance

Maximal productivity of a 14 mm light‐path panel photobioreactor under high irradiance was determined. Under continuous illumination of 2,100 µmol photons m^?2 s^?1 with red light emitting diodes (LEDs) the effect of dilution rate on photobioreactor productivity was studied. The light intensity used in this work is similar to the maximal irradiance on a horizontal surface at latitudes lower than 37°. Chlorella sorokiniana, a fast‐growing green microalga, was used as a reference strain in this study. The dilution rate was varied from 0.06 to 0.26 h^?1. The maximal productivity was reached at a dilution rate of 0.24 h^?1, with a value of 7.7 g dw m^?2 h^?1 (m² of illuminated photobioreactor surface) and a volumetric productivity of 0.5 g dw L^?1 h^?1. At this dilution rate the biomass concentration inside the reactor was 2.1 g L^?1 and the photosynthetic efficiency was 1.0 g dw mol photons. This biomass yield on light energy is high but still lower than the theoretical maximal yield of 1.8 g mol photons^?1 which must be related to photosaturation and thermal dissipation of absorbed light energy. Biotechnol. Bioeng. 2009; 104: 352–359 © 2009 Wiley Periodicals, Inc.     

Production of a sterol esterase from Ophiostoma piceae in batch and fed‐batch bioprocesses using different Pichia pastoris phenotypes as cell factory

The potential biotechnological applications for the Ophiostoma piceae sterol esterase (OPE) are conditioned to the availability of high enzyme amounts at low prices. This enzyme is a versatile biocatalyst with different biotechnological applications. In this work a systematic study on its heterologous production in different Pichia pastoris strains and operational strategies is presented. The best results were obtained using an AOX1 defective yeast strain in a fed‐batch bioprocess using methanol as inducer substrate at a set point of 2.5 g L^?1 and sorbitol as cosubstrate by means of a preprogramed exponential feeding rate at a μ = 0.02 h^?1, reaching 30 U mL^?1 of enzyme and a volumetric productivity of 403.5 U L^?1 h^?1. These values are twofold higher than those obtained with a Mut⁺ phenotype using methanol a sole carbon source. OPE was the main protein secreted by the yeast, 55% for Mut^s versus 25% for Mut^+. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1012–1020, 2014     

Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose‐Limited Cultivation of Penicillium chrysogenum

During glucose‐limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of β‐lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady‐state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose‐limited chemostat cultures at the dilution rates (D) of both 0.03 h^?1 and 0.05 h^?1 are determined to evaluate the quantitative impact on the physiology of a high‐yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h^?1, while this is not the case at D = 0.05 h^?1. In addition, a higher cytosolic redox status (NADH/NAD⁺), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h^?1, which are virtually absent at D = 0.03 h^?1. In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose‐limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial‐scale scenario.     

Benchmarking recombinant Pichia pastoris for 3-hydroxypropionic acid production from glycerol

The use of the methylotrophic yeast Pichia pastoris (Komagataella phaffi) to produce heterologous proteins has been largely reported. However, investigations addressing the potential of this yeast to produce bulk chemicals are still scarce. In this study, we have studied the use of P. pastoris as a cell factory to produce the commodity chemical 3-hydroxypropionic acid (3-HP) from glycerol. 3-HP is a chemical platform which can be converted into acrylic acid and to other alternatives to petroleum-based products. To this end, the mcr gene from Chloroflexus aurantiacus was introduced into P. pastoris. This single modification allowed the production of 3-HP from glycerol through the malonyl-CoA pathway. Further enzyme and metabolic engineering modifications aimed at increasing cofactor and metabolic precursors availability allowed a 14-fold increase in the production of 3-HP compared to the initial strain. The best strain (PpHP6) was tested in a fed-batch culture, achieving a final concentration of 3-HP of 24.75 g l⁻¹, a product yield of 0.13 g g⁻¹ and a volumetric productivity of 0.54 g l⁻¹ h⁻¹, which, to our knowledge, is the highest volumetric productivity reported in yeast. These results benchmark P. pastoris as a promising platform to produce bulk chemicals for the revalorization of crude glycerol and, in particular, to produce 3-HP.     

Mixed-feed exponential feeding for fed-batch culture of recombinant methylotrophic yeast

A simple, accurate model capable of predicting cell growth and methanol utilization during the mixed substrate fed-batch fermentation of Mut^S recombinant Pichia pastoris was developed and was used to design an exponential feeding strategy for mixed substrate fed-batch fermentation at a constant specific growth rate. Mixed substrate feeding has been shown to boost productivity in recombinant fed-batch culture of P. pastoris, while fixed growth rate exponential feeding during fed-batch culture is a useful tool in process optimization and control.     

Antibody expression kinetics in glycoengineered Pichia pastoris

Growth of the antibody market has fueled the development of alternative expression systems such as glycoengineered yeast. Although intact antibody expression levels in excess of 1 g L^?1 have been demonstrated in glycoengineered yeast, this is still significantly below the titers reported for antibody fragments in fungal expression systems. This study presents a simplified approach to estimate antibody secretion kinetics and oxygen uptake rate requirements as a function of growth‐rate controlled by a limiting methanol feed rate in glycoengineered Pichia pastoris. The yield of biomass from methanol and the specific oxygen requirements predicted in this study compare well with values reported in the literature for wild‐type P. pastoris, indicating the intrinsic nature of these yields independent of glycoengineering or the heterologous protein expressed. Specific productivity was found to be a non‐linear function of specific growth rate. Based on comparison with relationships between specific growth rate and specific productivity reported in the literature this correlation seems empirical in nature and cannot be established a priori. These correlations were then used in a simple mass balance based model to predict the cultivation performance of carbon limited cultivations under oxygen transfer limited conditions to indicate the usefulness of this approach to predict large scale performance and aid in process development. Biotechnol. Bioeng. 2010;106: 918–927. © 2010 Wiley Periodicals, Inc.     

In situ cell retention of a CHO culture by a reverse‐flow diafiltration membrane bioreactor

Heterogeneities occur in various bioreactor designs including cell retention devices. Whereas in external devices changing environmental conditions cannot be prevented, cells are retained in their optimal environment in internal devices. Conventional reverse‐flow diafiltration utilizes an internal membrane device, but pulsed feeding causes temporal heterogeneities. In this study, the influence of conventional reverse‐flow diafiltration on the yeast Hansenula polymorpha is investigated. Alternating 180 s of feeding with 360 s of non‐feeding at a dilution rate of 0.2 h^?1 results in an oscillating DOT signal with an amplitude of 60%. Thereby, induced short‐term oxygen limitations result in the formation of ethanol and a reduced product concentration of 25%. This effect is enforced at increased dilution rate. To overcome this cyclic problem, sequential operation of three membranes is introduced. Thus, quasi‐continuous feeding is achieved reducing the oscillation of the DOT signal to an amplitude of 20% and 40% for a dilution rate of 0.2 h^?1 and 0.5 h^?1, respectively. Fermentation conditions characterized by complete absence of oxygen limitation and without formation of overflow metabolites could be obtained for dilution rates from 0.1 h^?1 – 0.5 h^?1. Thus, sequential operation of three membranes minimizes oscillations in the DOT signal providing a nearly homogenous culture over time. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1348–1355, 2014     

Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G₄S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut⁺) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15°C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.