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     

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.     

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.     

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     

Dual-substrate inhibition kinetic studies for recombinant human interferon gamma producing Pichia pastoris

Pichia pastoris is considered as one of the prominent host extensively used as a platform for heterologous protein production. In the present study, the growth inhibition kinetics of recombinant P. pastoris expressing human interferon gamma was studied under different initial substrate concentrations of gluconate (10–100?g?L^?1) and methanol (2–50?g?L^?1) in modified FM22 medium. The highest specific growth rate of 0.0206 and 0.019?hr^?1 was observed at 60?g?L^?1 of gluconate and 10?g?L^?1 of methanol, respectively. Various three- and four-parametric Monod-variant models were chosen to analyze the inhibition kinetics. The model parameters as well as goodness of fit were estimated using nonlinear regression analysis. The three-parameter Haldane model was found to be best fit for both gluconate (R²?=?0.95) and methanol substrate (R²?=?0.96). The parameter sensitivity analysis revealed that µ_max, K_i, and K_s are the most sensitive parameters for both methanol and gluconate. Different substrate inhibition models were fitted to the growth kinetic data and the additive form of double Webb model was found to be the best to explain the growth kinetics of recombinant P. pastoris.     

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.     

The expression of the Cuphea palustris thioesterase CpFatB2 in Yarrowia lipolytica triggers oleic acid accumulation

The conversion of industrial by‐products into high‐value added compounds is a challenging issue. Crude glycerol, a by‐product of the biodiesel production chain, could represent an alternative carbon source for the cultivation of oleaginous yeasts. Here, we developed five minimal synthetic glycerol‐based media, with different C/N ratios, and we analyzed the production of biomass and fatty acids by Yarrowia lipolytica Po1g strain. We identified two media at the expense of which Y. lipolytica was able to accumulate ～5 g L^?1 of biomass and 0.8 g L^?1 of fatty acids (0.16 g of fatty acids per g of dry weight). These optimized media contained 0.5 g L^?1 of urea or ammonium sulfate and 20 g L^?1 of glycerol, and were devoid of yeast extract. Moreover, Y. lipolytica was engineered by inserting the FatB2 gene, coding for the CpFatB2 thioesterase from Cuphea palustris, in order to modify the fatty acid composition towards the accumulation of medium‐chain fatty acids. Contrary to the expected, the expression of the heterologous gene increased the production of oleic acid, and concomitantly decreased the level of saturated fatty acids. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:26–35, 2016     

Efficient secretion of inulin fructotransferase in Pichia pastoris using the formaldehyde dehydrogenase 1 promoter

Inulin fructotransferase (IFTase) has received considerable attention due to its ability to catalyse inulin hydrolysis to difructose anhydride (DFA III), a natural low-calorie functional sweetener. In the present study, for the first time, we describe the expression of IFTase in Pichia pastoris under the control of the formaldehyde dehydrogenase 1 promoter (PFLD1). Using this system, we achieved efficient secretion with four substrate fed-batch strategies in a 3-L fermenter. The co-feeding induction strategy with methylamine hydrochloride and methanol achieved the maximum extracellular IFTase activity of 62.72 U mL^?1, which was 3.2-fold higher than that obtained with the wild-type strain. In addition to methanol, carbon sources such as glucose and glycerol could also be utilised by PFLD1-controlled P. pastoris for IFTase production using methylamine hydrochloride induction. However, we found that glycerol and glucose should be strictly controlled at low concentrations of 0.5–1.5 % (v/v) and 1–1.5 % (w/v), respectively. The use of glycerol and glucose demonstrated that P. pastoris was also attractive for IFTase production via methanol-free cultivation strategies. This study may provide the basis for the industrial use of this recombinant IFTase for the production of DFA III.     

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     

Improvement of specific growth rate of <Emphasis Type="BoldItalic">Pichia pastoris</Emphasis> for effective porcine interferon-α production with an on-line model-based glycerol feeding strategy

Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor. The influence of the glycerol feeding strategy on the specific growth rate and protein production was investigated. The traditional DO-stat feeding strategy led to very low cell growth rate resulting in low dry cell weight (DCW) of about 90 g/L during the subsequent induction phase. The previously reported Artificial Neural Network Pattern Recognition (ANNPR) model-based glycerol feeding strategy improved the cell density to 120 g DCW/L, while the specific growth rate decreased from 0.15 to 0.18 to 0.03–0.08 h⁻¹ during the last 10 h of the glycerol feeding stage leading to a variation of the porcine interferon-α production, as the glycerol feeding scheme had a significant effect on the induction phase. This problem was resolved by an improved ANNPR model-based feeding strategy to maintain the specific growth rate above 0.11 h⁻¹. With this feeding strategy, the pIFN-α concentration reached a level of 1.43 g/L, more than 1.5-fold higher than that obtained with the previously adopted feeding strategy. Our results showed that increasing the specific growth rate favored the target protein production and the glycerol feeding methods directly influenced the induction stage. Consequently, higher cell density and specific growth rate as well as effective porcine interferon-α production have been achieved by our novel glycerol feeding strategy.     

Effect of initial bacteria concentration on hydrogen gas production from cheese whey powder solution by thermophilic dark fermentation

Dark fermentative hydrogen gas production from cheese whey powder solution was realized at 55°C. Experiments were performed at different initial biomass concentrations varying between 0.48 and 2.86 g L^?1 with a constant initial substrate concentration of 26 ± 2 g total sugar (TS) per liter. The highest cumulative hydrogen evolution (633 mL, 30°C), hydrogen yield (1.56 mol H₂ mol^?1 glucose), and H₂ formation rate (3.45 mL h^?1) were obtained with 1.92 g L^?1 biomass concentration. The specific H₂ production rate decreased with increasing biomasss concentration from the highest value (47.7 mL g^?1 h^?1) at 0.48 g L^?1 biomass concentration. Total volatile fatty acid concentration varied beetween 10 and 14 g L^?1 with the highest level of 14.2 g L^?1 at biomass concentration of 0.48 g L^?1 and initial TS content of 28.4 g L^?1. The experimental data were correlated with the Gompertz equation and the constants were determined. The most suitable initial biomass to substrate ratio yielding the highest H₂ yield and formation rate was 0.082 g biomass per gram of TS. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 931–936, 2012     

Biomass soft sensor for a Pichia pastoris fed-batch process based on phase detection and hybrid modeling

A common control strategy for the production of recombinant proteins in Pichia pastoris using the alcohol oxidase 1 (AOX1) promotor is to separate the bioprocess into two main phases: biomass generation on glycerol and protein production via methanol induction. This study reports the establishment of a soft sensor for the prediction of biomass concentration that adapts automatically to these distinct phases. A hybrid approach combining mechanistic (carbon balance) and data-driven modeling (multiple linear regression) is used for this purpose. The model parameters are dynamically adapted according to the current process phase using a multilevel phase detection algorithm. This algorithm is based on the online data of CO₂ in the off-gas (absolute value and first derivative) and cumulative base feed. The evaluation of the model resulted in a mean relative prediction error of 5.52% and R² of .96 for the entire process. The resulting model was implemented as a soft sensor for the online monitoring of the P. pastoris bioprocess. The soft sensor can be used for quality control and as input to process control systems, for example, for methanol control.     

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.     

Control of recombinant human endostatin production in fed-batch cultures of Pichia pastoris using the methanol feeding rate

Endostatin is a 20 kDa carboxyl-terminal fragment of collagen XVIII that strongly inhibits angiogenesis and tumor growth. The methylotrophic yeast, Pichia pastoris, is a robust expression system that can be used to study methods to improve the yields of rhEndostatin. We expressed rhEndostatin in P. pastoris under the control of the alcohol oxidase 1 (aox 1) promoter (Mut⁺ phenotype) as a model, and used a cell biomass of about 50 g l^–1 dry cell wt as a starting point for the induction phase and varied the methanol feed rate at 8 ml l^–1 h^–1, 11 ml l^–1 h^–1 and 15 ml l^–1 h^–1. While the cell growth rate was proportional to the rate of methanol delivery, protein production rate was not. These findings could be used to guide parameters for large-scale production of recombinant proteins in the P. pastoris system.     

Effects of oxygen volumetric mass transfer coefficient on transglutaminase production by Bacillus circulans BL32

The effects of oxygenation in cultures of Bacillus circulans BL32 on transglutaminase (TGase) production and cell sporulation were studied by varying the agitation speed and the volume of aeration. Kinetics of cultivations has been studied in batch systems using a 2 L bioreactor, and the efficiency of agitation and aeration was evaluated through the oxygen volumetric mass transfer coefficient (k_La). It was adopted a two-stage aeration rate control strategy: first stage to induce biomass formation, followed by a second stage, in which cell sporulation was stimulated. A correlation of TGase production, spores formation, and oxygen concentration was established. Under the best conditions (500 rpm; 2 vvm air flow, followed by no air supply during stationary phase; k_La of 33.7 h⁻¹), TGase production reached a volumetric production of 589 U/L after 50 h of cultivation and the enzyme yield was 906 U/g cells. These values are 61% higher than that obtained in shaker cultures and TGase productivity increased 82%, when k_La varied from 4.4 to 33.7 h⁻¹. The maximal cell concentration increased four times in relation to shaker cultures and the cultivation time for the highest TGase activity was reduced from 192 h to just 50 h. These results show the importance of bioprocess design for the production of microbial TGase, especially concerning the oxygen supply of cultures and the induction of cell sporulation.     

Novel Strategy of Using Methyl Esters as Slow Release Methanol Source during Lipase Expression by mut+ Pichia pastoris X33

One of the major issues with heterologous production of proteins in Pichia pastoris X33 under AOX1 promoter is repeated methanol induction. To obviate repeated methanol induction, methyl esters were used as a slow release source of methanol in lipase expressing mut⁺ recombinant. Experimental design was based on the strategy that in presence of lipase, methyl esters can be hydrolysed to release their products as methanol and fatty acid. Hence, upon break down of methyl esters by lipase, first methanol will be used as a carbon source and inducer. Then P. pastoris can switch over to fatty acid as a carbon source for multiplication and biomass maintenance till further induction by methyl esters. We validated this strategy using recombinant P. pastoris expressing Lip A, Lip C from Trichosporon asahii and Lip11 from Yarrowia lipolytica. We found that the optimum lipase yield under repeated methanol induction after 120 h was 32866 U/L, 28271 U/L and 21978 U/L for Lip C, Lip A and Lip 11 respectively. In addition, we found that a single dose of methyl ester supported higher production than repeated methanol induction. Among various methyl esters tested, methyl oleate (0.5%) caused 1.2 fold higher yield for LipA and LipC and 1.4 fold for Lip11 after 120 h of induction. Sequential utilization of methanol and oleic acid by P. pastoris was observed and was supported by differential peroxisome proliferation studies by transmission electron microscopy. Our study identifies a novel strategy of using methyl esters as slow release methanol source during lipase expression.     

Coexpression of a carbonyl reductase and glucose 6-phosphate dehydrogenase in Pichia pastoris improves the production of (S)-1-phenyl-1,2-ethanediol

Abstract

To develop an efficient biocatalyst to produce optically active (S)-phenyl ethanediol (PED), a carbonyl reductase SCRII and glucose 6-phosphate dehydrogenase were coexpressed intracellularly in Pichia pastoris. The recombinant enzyme PpSCRII was purified with a specific activity of 8.32 U mg^?1, over 36% higher than that of Escherichia coli SCRII. The recombinant cells P. pastoris/SCRIIG catalyzed the reduction of 2-hydroxyacetophenone to give (S)-PED with optical purity of >99% in a yield of 96.3%. The yield was improved by 19.9% and 25.7% over E. coli BL21/SCRII and Candida parapsilosis, respectively, when the reaction duration was shorted from 48 h to 24 h. When using glucose 50 g L^?1 as co-substrate, these P. pastoris/SCRIIG cells could be reused ten times and the optical purity and yield of (S)-PED kept at >99% enantiomeric excess and >85%, respectively.     

High-level expression of a truncated 1,3-1,4-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucanase from <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by optimization of codons and fermentation

1,3-1,4-β-d-glucanase is an important endoglycosidase in the brewing and animal feed industries. To achieve high-level expression of recombinant glucanase in Pichia pastoris, we designed sequences encoding the α-factor signal peptide from Saccharomyces cerevisiae and the truncated 1,3-1,4-β-d-glucanase from Fibrobacter succinogenes as a whole. The codons encoding the 52 amino acids of the signal peptide and 106 residues of the glucanase protein were optimized for expression in P. pastoris; 189 nucleotides were changed. The G + C content was adjusted to 48–49%, and AT-rich stretches were eliminated to avoid premature termination. The messenger ribonucleic acid secondary structure near the AUG start codon was also adjusted to ensure efficient translation; the resulting glucanase production was twofold higher compared with that achieved with gene structure optimization alone. We also propose a new fermentation strategy for the induction phase, in which 5/95% glycerol/methanol mixed feed was used in days 1–3 and 100% methanol was used on days 4–6. By comparison with methanol feed and glycerol/methanol-mixed feed alone, the yield of recombinant glucanase increased by 38.5 and 16.5%, respectively. The expressed optimized recombinant 1,3-1,4-β-d-glucanase constituted ~90% of the total secreted protein, reaching up to 3 g l⁻¹ in the medium.