Fed-batch high-cell-density fermentation strategies for Pichia pastoris growth and production

Pichia pastoris is extensively used to produce various heterologous proteins. Amounts of biopharmaceutical drugs and industrial enzymes have been successfully produced by fed-batch high-cell-density fermentation (HCDF) of this cell factory. High levels of cell mass in defined media can be easily achieved and therefore large quantities of recombinant proteins with enhanced activities and lower costs can be obtained through HCDF technology. A robust HCDF process makes a successful transition to commercial production. Recently, efforts have been made to increase the heterologous protein production and activity by the HCDF of P. pastoris. However, challenges around selecting a suitable HCDF strategy exist. The high-level expression of a specific protein in P. pastoris is still, at least in part, limited by optimizing the methanol feeding strategy. Here, we review the progress in developments and applications of P. pastoris HCDF strategies for enhanced expression of recombinant proteins. We focus on the methanol induction strategies for efficient fed-batch HCDF in bioreactors, mainly focusing on various stat-induction strategies, co-feeding, and the limited induction strategy. These processes control strategies have opened the door for expressing foreign proteins in P. pastoris and are expected to enhance the production of recombinant proteins.     

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.     

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

The methanol utilization (Mut) phenotype in the yeast Pichia pastoris (syn. Komagataella spp.) is defined by the deletion of the genes AOX1 and AOX2. The Mut⁻ phenotype cannot grow on methanol as a single carbon source. We assessed the Mut⁻ phenotype for secreted recombinant protein production. The methanol inducible AOX1 promoter (P_AOX1) was active in the Mut⁻ phenotype and showed adequate eGFP fluorescence levels and protein yields (Y_P/X) in small-scale screenings. Different bioreactor cultivation scenarios with methanol excess concentrations were tested using P_AOX1HSA and P_AOX1vHH expression constructs. Scenario B comprising a glucose-methanol phase and a 72-hr-long methanol only phase was the best performing, producing 531 mg/L HSA and 1631 mg/L vHH. 61% of the HSA was produced in the methanol only phase where no biomass growth was observed, representing a special case of growth independent production. By using the Mut⁻ phenotype, the oxygen demand, heat output, and specific methanol uptake (q_methanol) in the methanol phase were reduced by more than 80% compared with the Mut^S phenotype. The highlighted improved process parameters coupled with growth independent protein production are overlooked benefits of the Mut⁻ strain for current and future applications in the field of recombinant protein production.     

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.     

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     

Shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris bioreactor cultures

Recently, we engineered a Pichia pastoris Mut⁺ strain to produce and secrete recombinant Litopenaeus vannamei trypsinogen. Despite the observed toxicity of the recombinant shrimp trypsinogen to the P. pastoris cell host, when high density cell cultures in shake flasks with alanine in the induction medium were used recombinant shrimp trypsinogen could be produced. To further improve the product yield, in this work, we evaluated L. vannamei trypsinogen production in P. pastoris using a bioreactor and two recombinant P. pastoris strains with different methanol utilization (Mut) phenotypes. The effect of pH and temperature during the induction step on the trypsinogen production was also evaluated. The results indicate that temperature, pH, and Mut phenotypes influence the production of the recombinant protein, with almost no observed effect on cell growth. All cultures with the Mut⁺ strain had significant operational difficulties, such as in lowering the induction temperature, maintaining dissolved oxygen (DO) above 20%, and maintaining the methanol concentration at a constant value, and showed a decrease in metabolic activity due to trypsinogen toxicity to the cell host. In the culture with the Mut^s strain, however, the temperature, methanol concentration, and DO could be more easily controlled, the temperature could be easily decreased, and the trypsinogen caused the lowest toxicity to the host cells. After 96 h of Mut^s strain induction (pH 6 and 25°C), about 250 mg/L recombinant trypsinogen was detected in the culture medium. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Production and purification of recombinant Blomia tropicalis group 5 allergen from Pichia pastoris culture

The gene of Blomia tropicalis group 5 allergen (Blo t 5) was cloned and expressed in Pichia pastoris KM71. Selected KM71 clones were cultivated in a fed-batch bioreactor feeding first glycerol then followed by methanol. Recombinant Blo t 5 constituted about 30% of the total broth protein after 60 h cultivation. The harvested broth was purified to >95% purity by a two-step anion exchange chromatography. The overall yield was 37 mg Blo t 5 per litre broth.     

A novel feeding strategy for enhanced protein production by fed-batch fermentation in recombinant Pichia pastoris

A novel feeding strategy for enhanced protein production of hepatitis B virus surface antigen (HBsAg) in fed-batch fermentation, recombinant Pichia pastoris, has been developed. A minimal salt medium was used to grow cells in the initial batch fermentation, followed by a glycerol+salts fed-batch phase. At the end of the fed-batch phase a dry cell weight of 130 g l⁻¹ was achieved. In the absence of basal salts, the same amount of glycerol feed resulted in only 90 g l⁻¹ cell dry weight. When a limited amount of casamino acids were also included every 24 h during methanol induction, there was a two-fold increase in expression levels of HBsAg. After 192 h of induction, the expression levels of HBsAg (soluble and insoluble) reached >1 g l⁻¹ using the Mut⁻ strain. Thus, the use of basal salts in the glycerol feed, along with the addition of limited amounts of casamino acids with the methanol feed, resulted in an increased expression of total HBsAg.     

Production of salmosin, a snake venom-derived disintegrin, in recombinant Pichia pastoris using high cell density fed-batch fermentation

Salmosin, a snake venom-derived disintegrin, was successfully expressed in the methylotrophic yeast Pichia pastoris and secreted into the culture supernatant, as a 6 kDa protein. High-cell density fermentation of recombinant P. pastoris was optimized for the mass production of salmosin. In a 5 L jar fermentor, recombinant P. pastoris was fermented in growth medium containing 5% (w/v) glycerol at the controlled pH of 5.0. After culturing for 21 h, glycerol feeding medium was fed at one time into the culture broth. After 7 h (a total of 28 h), induction medium that contained methanol was increasingly added until the culture time totaled 75 h. Finally, these optimized culture conditions produced a high cell density of recombinant P. pastoris (dry cell weight of 113.38 g/L) and led to the mass production of salmosin (a total protein concentration of 369.2 mg/L). The culture supernatant containing salmosin inhibited platelet aggregation, resulting in a platelet aggregation of 9% compared to that of 94% in the control experiment, without culture supernatant. These results demonstrate that recombinant salmosin in culture supernatant from high cell density fed-batch fermentation can serve as a platelet aggregation inhibitor.     

Effect of dilution rate and methanol‐glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastoris Mut+ phenotype in continuous culture

The induction using substrate mixtures is an operational strategy for improving the productivity of heterologous protein production with Pichia pastoris. Glycerol as a cosubstrate allows for growth at a higher specific growth rate, but also has been reported to be repressor of the expression from the AOX1 promoter. Thus, further insights about the effects of glycerol are required for designing the induction stage with mixed substrates. The production of Rhizopus oryzae lipase (ROL) was used as a model system to investigate the application of methanol‐glycerol feeding mixtures in fast metabolizing methanol phenotype. Cultures were performed in a simple chemostat system and the response surface methodology was used for the evaluation of both dilution rate and methanol‐glycerol feeding composition as experimental factors. Our results indicate that productivity and yield of ROL are strongly affected by dilution rate, with no interaction effect between the involved factors. Productivity showed the highest value around 0.04–0.06 h^?1, while ROL yield decreased along the whole dilution rate range evaluated (0.03–0.1 h^?1). Compared to production level achieved with methanol‐only feeding, the highest specific productivity was similar in mixed feeding (0.9 UA g‐biomass^?1 h^?1), but volumetric productivity was 70% higher. Kinetic analysis showed that these results are explained by the effects of dilution rate on specific methanol uptake rate, instead of a repressor effect caused by glycerol feeding. It is concluded that despite the effect of dilution rate on ROL yield, mixed feeding strategy is a proper process option to be applied to P. pastoris Mut⁺ phenotype for heterologous protein production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:707–714, 2015     

Secretion of pro- and mature Rhizopus arrhizus lipases by Pichia pastoris and properties of the proteins

The lipases of Rhizopus spp. share a high 1,3-regiospecificity toward triacylglycerols, which makes them important enzymes in lipid modification. In the present study, the extracellularly active production of recombinant Rhizopus arrhizus lipase was carried out with genes encoding the mature region (mRAL) and the mRAL having the prosequence (ProRAL) in Pichia pastoris. Two transformed P. pastoris clones containing the multicopy of mRAL and ProRAL genes were separately selected for the production of recombinant enzymes. In a fed-batch cultivation, where methanol feeding was controlled by an on-line methanol analyzer, the supernatant contained 91 mg/L recombinant pro-form lipase (rProRAL) and 80 mg/L recombinant mature lipase (rRAL) after 92 h of cultivation. rProRAL and rRAL were purified by ultrafiltration, SP-Sepharose Rast Flow chromatography, and Butyl-Sepharose Fast Flow chromatography. Molecular weights of rProRAL and rRAL are 32 kDa and 29 kDa, respectively. The amino-terminal analysis showed that the 32-kDa protein was mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL). The specific lipase activities of mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL) and rRAL were 1543 U/mg and 2437 U/mg. The rPro28RAL was more stable than rRAL at pH 4.0–7.0, whereas rRAL was more stable at pH 7.0–10.0. The rPro28RAL had the highest lipase activity toward tributyrin (C₄), whereas rRAL had the highest lipase activity toward tricaprylin (C₈).     

High expression of Trigonopsis variabilis -amino acid oxidase in Pichia pastoris

To explore a new approach of high expression of -amino acid oxidase (DAAO) in Pichia pastoris, a gene encoding DAAO from Trigonopsis variabilis (TvDAAO gene) deleted intron was prepared by PCR amplification and cloned into the intracellular expression vector pPIC3.5K. The expression plasmid pPIC3.5K-DAAO linearized by SalI was transformed into Pichia pastoris strain GS115 (his⁻mut⁺). By means of MM and MD plates and PCR, the recombinant P. pastoris strains (his⁺mut⁺) were obtained. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant strain PD27 with the highest expression of DAAO was screened through activity assay and its high-density fermentation was carried out in a 1-l fermentor. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant cells with high expression of DAAO were screened and the high-density fermentation was carried out in a 1-l fermentor. Interestingly, the DAAO expression level reached up to 473 U/g dry cell weight in fermentation yield. Finally, 1-hexanol was used to break recombinant cells and the specific activity of DAAO was 1.46 U/mg protein in crude extraction.     

Improved production of human type II procollagen in the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> in shake flasks by a wireless-controlled fed-batch system

Background  

Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.     

Production and Purification of the Heavy-Chain Fragment C of Botulinum Neurotoxin, Serotype B, Expressed in the Methylotrophic YeastPichia pastoris

A recombinant H_cfragment of botulinum neurotoxin, serotype B (rBoNTB(H_c)), has been successfully expressed in a Mut⁺strain of the methylotrophic yeastPichia pastorisfor use as an antigen in a proposed human vaccine. The fermentation process consisted of batch phase on glycerol, followed by glycerol and methanol fed-batch phases yielding a final cell mass of 60 g/L (dcw) and was easily scaled-up to 60 L. A multistep ion-exchange chromatographic purification process was employed to produce 99% pure H_cfragment. The final yield of the purified antigen was 390 mg per kilogram of wet cell mass. The purified H_cfragment of serotype B was stable, elicited an immune response in mice, and protected upon challenge with native botulin.     

Gas-phase degradation of VOCs using supported bacteria biofilms

Herein we report the use of Pseudomonas putida F1 biofilms grown on carbonized cellulosic fibers to achieve biodegradation of airborne volatile organic compounds (VOCs) in the absence of any bulk aqueous-phase media. It is believed that direct exposure of gaseous VOC substrates to biomass may eliminate aqueous-phase mass transfer resistance and facilitate VOC capture and degradation. When tested with toluene vapor as a model VOC, the supported biofilm could grow optimally at 300 p.p.m. toluene and 80% relative humidity, with a specific growth rate of 0.425 day⁻¹. During long-term VOC biodegradation tests in a tubular packed bed reactor, biofilms achieved a toluene degradation rate of 2.5 mg g_DCW⁻¹ h⁻¹ during the initial growth phase. Interestingly, the P. putida F1 film kept biodegrading activity even at the stationary nongrowth phase. The supported biofilms with a biomass loading of 20% (wt) could degrade toluene at a rate of 1.9 mg g_DCW⁻¹ h⁻¹ during the stationary phase, releasing CO₂ at a rate of 6.4 mg g_DCW⁻¹ h⁻¹ at the same time (indicating 100% conversion of substrate carbon to CO₂). All of these observations promised a new type of “dry” biofilm reactors for efficient degradation of toxic VOCs without involving a large amount of water.     

Effect of nitrate feeding strategies on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana

Controlled nitrate feeding strategies for fed-batch cultures of microalgae were applied for the enhancement of lipid production and microalgal growth rates. In particular, in this study, the effect of nitrate feeding rates on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana were investigated using three feeding modes (i.e., pulse, continuous, and staged) and under two light variations on both lipid productivity and fatty acid compositions. Higher nitrate levels negatively affected lipid production in the study. Increasing the light intensity increased the lipid contents of the microalgae in all three fed-batch feeding modes. A maximum of 58.3% lipid- to dry weight ratio was achieved when using pulse-fed cultures at an illumination of 200 μmol photons m⁻² s⁻¹ and 10 mg/day of nitrate feeding. This condition also resulted in the maximum lipid productivity of 44.6 mg L⁻¹ day⁻¹. The fatty acid compositions of the lipids consisted predominantly of long-chain fatty acids (C:16 and C:18) and accounted for 70% of the overall fatty acid methyl esters. Pulse feeding mode was found to significantly enhance the biomass and lipid production. The other two feeding modes (continuous and staged) were not ideal for lipid and biomass production. This study demonstrates the applicability of pulse feeding strategies in fed-batch cultures as an appropriate cultivation strategy that can increase both lipid accumulation and biomass production.     

<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     

Application of recurrent neural network for online prediction of cell density of recombinant Pichia pastoris producing HBsAg

Abstract

Artificial neural networking (ANN) seems to be a promising soft sensor for implementing current approaches of quality by design (QbD) and process analytical technologies (PAT) in the biopharmaceutical industry. In this study, we aimed to implement best-fitted ANN architecture for online prediction of the biomass amount of recombinant Pichia pastoris (P. pastoris) – expressing intracellular hepatitis B surface antigen (HBsAg) – during the fed-batch fermentation process using methanol as a sole carbon source. For this purpose, at the induction phase of methanol fed-batch fermentation, carbon evolution rate (CER), dissolved oxygen (DO), and methanol feed rate were selected as input vectors and total wet cell weight (WCW) was considered as output vector for the ANN. The obtained results indicated that after training recurrent ANN with data sets of four fed-batch runs, this toolbox could predict the WCW of the next fed-batch fermentation process at each specified time point with high accuracy. The R-squared and root-mean-square error between actual and predicted values were found to be 0.9985 and 13.73, respectively. This verified toolbox could have major importance in the biopharmaceutical industry since recombinant P. pastoris is widely used for the large-scale production of HBsAg.     

Temperature limited fed-batch technique for control of proteolysis in <Emphasis Type="Italic">Pichia pastoris</Emphasis> bioreactor cultures

Background  

A temperature limited fed-batch (TLFB) technique is described and used for Pichia pastoris Mut⁺ strain cultures and compared with the traditional methanol limited fed-batch (MLFB) technique. A recombinant fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced and secreted by this strain.