Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica

The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter. Methanol concentrations during the induction phase directly affect cellular growth and protein yield. Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P. methanolica expressing the human transferrin N-lobe protein. The PMAD18 P. methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype. Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration. Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time.     

Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris

To improve the specific production rate of Rhizopus oryzae lipase (ROL) in Pichia pastoris, a protein that triggers the unfolded protein response in P. pastoris, the effect of sorbitol/methanol mixed substrates was tested in batch and fed-batch cultures. Remarkably, a different substrate consumption behaviour was observed depending on the host's phenotype (Mut(+) or Mut(s)) in batch cultures: when the methanol assimilation capacity is genetically reduced (Mut(s) phenotype), both substrates were consumed simultaneously, allowing not only a higher specific growth rate but also higher lipase levels (8.7-fold) compared to those obtained by cells growing on methanol as a sole carbon source in batch culture. This effect was not observed in Mut(+) phenotype, where the two substrates were consumed sequentially and the levels of heterologous product were only slightly higher (1.7-fold). A mixed substrate strategy was also applied to a Mut(s) fed-batch culture at a low methanol concentration set-point (0.5 gl(-1)). This resulted in a 2.2-fold increase in the heterologous protein level achieved, compared with the methanol-only feeding strategy. In addition, sorbitol co-feeding permitted the achievement of higher specific growth rates, and avoided the drastic decrease of the specific production rate observed after the start of the induction phase when methanol was used as sole carbon source This resulted in a significant increase in the overall bioprocess volumetric productivity (2.2-fold) and specific productivity (1.7-fold). Moreover, whereas increased ROL gene dosage in Mut(s) strains have been previously reported to be deleterious for P. pastoris cells growing on methanol, sorbitol co-feeding allowed for sustained cell growth and lipase production.     

Design of methanol Feed control in Pichia pastoris fermentations based upon a growth model

The methylotrophic yeast Pichia pastoris is an effective system for recombinant protein productions that utilizes methanol as an inducer, and also as carbon and energy source for a Mut(+) (methanol utilization plus) strain. Pichia fermentation is conducted in a fed-batch mode to obtain a high cell density for a high productivity. An accurate methanol control is required in the methanol fed-batch phase (induction phase) in the fermentation. A simple "on-off" control strategy is inadequate for precise control of methanol concentrations in the fermentor. In this paper we employed a PID (proportional, integral and derivative) control system for the methanol concentration control and designed the PID controller settings on the basis of a Pichia growth model. The closed-loop system was built with four components: PID controller, methanol feed pump, fermentation process, and methanol sensor. First, modeling and transfer functions for all components were derived, followed by frequency response analysis, a powerful method for calculating the optimal PID parameters K(c) (controller gain), tau(I) (controller integral time constant), and tau(D) (controller derivative time constant). Bode stability criteria were used to develop the stability diagram for evaluating the designed settings during the entire methanol fed-batch phase. Fermentations were conducted using four Pichia strains, each expressing a different protein, to verify the control performance with optimal PID settings. The results showed that the methanol concentration matched the set point very well with only small overshoot when the set point was switched, which indicated that a very good control performance was achieved. The method developed in this paper is robust and can serve as a framework for the design of other PID feedback control systems in biological processes.     

Dissolved-oxygen-stat controlling two variables for methanol induction of rGuamerin in Pichia pastoris and its application to repeated fed-batch

A DO-stat control strategy for two variables was introduced to the rGuamerin production process in Pichia pastoris and applied to repeated fed-batch culture. Two interrelated variables, namely the ratio of partial pressure of pure O2 in the inlet air-stream and the methanol feed rate, were controlled simultaneously. By using this control strategy, methanol feeding for induction could be controlled automatically while efficiently controlling the dissolved oxygen level. As a result, the cell concentration reached more than 140 g l(-1) and rGuamerin expression level 450 iu l(-1). rGuamerin was secreted into the culture medium and reached a level that was 40% higher than achieved in a fed-batch process using manual control of the methanol feeding rate. Repeated rGuamerin induction was achieved by repeating the methanol feeding and withdrawing the culture broth during extended production. During more than 250 h of culture, expression of rGuamerin was maintained at an average of about 430 iu l(-1 )(473 mg l(-1)), without causing the cell density to decrease. In addition to the rGuamerin production process, the proposed control system might be applied to cultivation of other methylotrophic yeasts in the production of therapeutic proteins.     

An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris

The expression of a humanized single-chain variable domain fragment antibody (A33scFv) was optimized for Pichia pastoris with yields exceeding 4 g L(-1). A33scFv recognizes a cell surface glycoprotein (designated A33) expressed in colon cancer that serves as a target antigen for immunotherapy of colon cancer. P. pastoris with a MutS phenotype was selected to express A33scFv, which was cloned under regulation of the methanol-inducible AOX1 promoter. We report the optimization of A33scFv production by examining methanol concentrations using fermentation technology with an on-line methanol control in fed-batch fermentation of P. pastoris. In addition, we examined the effect of pH on A33scFv production and biomass accumulation during the methanol induction phase. A33scFv production was found to increase with higher methanol concentrations, reaching 4.3 g L(-1) after 72 h induction with 0.5% (v/v) methanol. Protein production was also greatly affected by pH, resulting in higher yields (e.g., 4.88 g L(-1)) at lower pH values. Biomass accumulation did not seem to vary when cells were induced at different pH values, but was greatly affected by lower concentration of methanol. Purification of A33scFv from clarified medium was done using a two-step chromatographic procedure using anion-exchange and hydrophobic interaction chromatography, resulting in 25% recovery and >90% purity. Pure A33scFv was tested for functionality using surface plasmon resonance and showed activity against immobilized A33 antigen. Our results demonstrate that functional A33scFv can be produced in sufficient quantities using P. pastoris for use in further functionality studies and diagnostic applications.     

Improving the monitoring of methanol concentration during high cell density fermentation of Pichia pastoris

The Pichia pastoris expression system is widely used for the production of recombinant proteins. A simple and efficient experimental set-up allowing on-line monitoring of the methanol concentration during the fermentation of P. pastoris based on the detection of the methanol vapor concentration in the exhaust air from fermenter by a tin dioxide (SnO2) semiconductor sensor is described. An experimental procedure to allow precise calibration of the system and to reduce methanol sensor's interferences (>95% reduction) are also presented and discussed. Accuracy and measurement error were estimated about 0.05 g x l(-1) and 6%, respectively. The efficient monitoring of methanol will help to advanced control of recombinant protein production and process optimization.     

Monitoring and control of methanol concentration during polysaccharide fermentation using an on-line methanol sensor

Combining principles of membrane separation and semiconductor gas sensor technology, we constructed a methanol sensor to follow methanol concentrations on-line. A length of silicone tubing allowed for mass transfer of methanol from the fermentation medium to a carrier gas which then flowed over a semiconductor gas sensor for detection. The sterilizable sensor demonstrated excellent ability in following methanol concentrations during the batch production of a polysaccharide by the organism Methylomonas mucosa, even as the fermentation broth became increasingly viscous. During fed-batch control by feeding methanol to the fermentation to maintain setpoint methanol levels, a drift in the sensor signal was noted and quantified. A drift factor was determined which, after it was incorporated into the calibration calculations, improved methanol concentration control greatly. Methanol concentration was held constant over a range of set point concentrations during fedbatch fermentations.     

Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris

A single-chain antibody fragment directed against fimbriae of enterotoxigenic Escherichia coli was produced by recombinant Pichia pastoris under control of the methanol-inducible AOX1 promoter. In high-cell-density cultivation on defined medium, methanol-limited and methanol-saturated conditions were compared. After batch and fed-batch phase on glycerol, the methanol concentration was controlled to 1% (v/v) or methanol was fed with an exponentially increasing rate. Whereas methanol limitation impaired cell integrity and product quality, finally yielding no active product as a result of degradation, oxygen limitation was acceptable. To postpone the onset of limitation, the inlet air was enriched by pure oxygen. Because of faster methanol consumption, however, the process became sensitive to fluctuations in the feeding rate, and complete arrest of metabolism encountered upon small perturbations shortened the active production period. Without additional oxygen supply, the process was robust. Loss of culture integrity was monitored by flow cytometry and was found to precede changes in metabolic rates; it can thus serve as a sensitive indicator of forthcoming problems. Single-step downstream processing from the culture supernatant by His-affinity chromatography was efficient when antifoam agent that coagulates upon pH titration was omitted and yielded 1 g of purified lyophilized product from 6 L initial culture volume.     

甲醇抑制时重组毕赤酵母发酵的特性研究

本文对毕赤酵母进行了恒化培养研究。以甲醇为唯一碳源时,在稀释率较低时(D<0.048 h^-1),连续培养系统操作很稳定。但在稀释率高时(D>0.048h^-1),连续培养系统的定态点不止一个,实验不能维持,故采用比生长速率恒定的分批流加培养进行研究。结果表明,毕赤酵母的生长符合Andrew普遍化底物抑制模型。综合考虑水蛭素的生成、底物的消耗,在生产中维持甲醇浓度为限制性浓度(0.5 g/L),且维持比生长速率为0.02 h^-1时,水蛭素Hir65的比生成速率达到最大值0.2 mg/(g·h)且甲醇的比消耗速率为0.04 g/(g·h)。     

不同甲醇流加策略对重组毕赤醇母高密度发酵生产水蛭素的影响

考察了不同甲醇流加策略对毕赤酵母高密度发酵生产水蛭素的影响。溶氧控制法不能有效地防止甲醇的过量流加。气相色谱离线检测法虽然防止了甲醇流加过量 ,但甲醇浓度的波动较大。利用甲醇传感器在线检测控制甲醇的流加可维持较恒定的甲醇浓度。在流加甲醇的同时 ,以限制性速率流加甘油可以增加表达期间的能量供应 ,提高产物的表达量。经优化后 ,采用甲醇甘油混合流加时细胞干重达到 16 2g L ,水蛭素活性达到 2 4×10 4ATU mL ,即 1 7g L。     

Evaluation of metals in a defined medium for Pichia pastoris expressing recombinant beta-galactosidase

Culture growth and recombinant protein yield of the Pichia pastoris GS115 methanol utilization positive system were studied in response to the types and levels of metals present in the growth medium and the supplemental salts typically used for these fermentations. Magnesium and zinc were both required to support cell growth but at significantly reduced levels compared to the control. However, supplementation with calcium, cobalt, iron, manganese, iodine, boron, and molybdenum were not required to sustain cell mass. When the medium was reformulated with only zinc and magnesium, the cells grew to 12-15 generations, which are expected for high cell density fed-batch fermentations. Product yields of the recombinant protein beta-galactosidase were significantly influenced by the trace metal concentrations. By using response surface and full factorial designs, maximum protein yield occurred when the concentration of zinc salt was limited to the level necessary only to support cell mass while protein yield positively correlated to increasing levels of the remaining trace metal salts. These studies are the first to show that excess trace metals must be optimized when developing P. pastoris based fed-batch fermentations.     

甲醇浓度对毕赤酵母表达重组人复合α干扰素分离纯化得率的影响

研究了毕赤酵母Pichia pastoris表达的重组人复合α干扰素(cIFN)时不同诱导甲醇浓度对cIFN分离纯化得率的影响,并分析了原因.在5L罐中采用0.25、0.50和0.75％(W/V)三个甲醇浓度诱导时,在0.75％高甲醇浓度诱导下cIFN表达水平最高,达到2.06 g/L,是0.25％低甲醇浓度诱导的1.24倍,但是低甲醇浓度诱导下cIFN分离纯化得率却高于高甲醇诱导浓度下3.75倍.另外,低甲醇浓度下发酵上清液cIFN抗病毒活性为2.85×108IU/mL,较高甲醇浓度提高了4.48倍.进一步采用SDS-PAGE和Native-PAGE免疫印迹分析不同条件下发酵液中cIFN存在状态,发现在高甲醇浓度下cIFN容易形成大量的聚合体,分别为共价聚合和非共价聚合,而cIFN单体含量较少,但是低甲醇浓度诱导下情况完全相反.最终在0.25％甲醇诱导下分离纯化1L发酵上清液可得0.73 g单体cIFN,是0.75％甲醇诱导下的3.84倍.     

A simple method to monitor and control methanol feeding of Pichia pastoris fermentations using mid-IR spectroscopy

Mid-infrared FTIR spectroscopy is an efficient tool for the monitoring of bioprocesses, since it is fast and able to detect many compounds simultaneously. However, complex and time-consuming calibration procedures are still required, and have inhibited the spreading of these instruments. A simple and quick method to calibrate a FTIR instrument was developed for the control of fed-batch fermentations of the methylotrophic yeast Pichia pastoris. Based on the assumptions that (1) only substrate concentration may change significantly during a fed-batch process and (2) absorbance can be considered as proportional to concentration, a linear two-point calibration was implemented. Long-term instability of the instrument had to be addressed in order to get accurate results: two fixed points, on both sides of substrate absorbance peak, were used to perform on-line a linear correction of the signal drift. Fed-batch experiments at constant methanol (substrate) concentration ranging from 0.8 to 15gl(-1) were carried out. Off-line HPLC control analysis showed a good agreement with on-line FTIR data, with standard error of prediction values < 0.12gl(-1). Even though methanol acts both as carbon source and inducer of protein expression, no significant effect was observed on the level of protein expression in the recombinant strain used.     

恒细胞密度发酵策略提高重组毕赤酵母生产碱性果胶酶的表达效率

为了提高重组毕赤酵母生产碱性果胶酶(Alkaline polygalacturonate lyase,PGL)的比速率,开发了一种新的恒细胞密度发酵策略。通过不同的甲醇流加方式,实现发酵过程细胞密度的合理控制。实验结果表明:控制细胞密度为75 g/L的策略为最优,最终单位发酵液体积生产强度和单位菌体生产强度为6.11 U/(mL.h)和81.5 U/(g.h),分别比传统高密度发酵提高了42.1%和191.2%,最终PGL酶活为441.9 U/mL。此外,该策略还具有提高细胞活性和降低蛋白酶降解作用等优势。     

Production and separation of manganese peroxidase from heme amended yeast cultures

A method for the production and concentration of the lignin-degrading enzyme, manganese peroxidase (rMnP), was developed using the yeast Pichia pastoris in high cell density, fed-batch cultivations. A gene encoding manganese peroxidase (mnp1) from the white-rot fungus Phanerochaete chrysosporium was cloned into a protease deficient (pep4-) strain of the methylotrophic yeast P. pastoris. Heme is an important cofactor for active rMnP production, and amendment of yeast cultures with heme increased active rMnP concentrations. In both shake-flasks and fed-batch bioreactors, the relationship between heme concentration and rMnP activity was logarithmic, with increasing heme concentrations resulting in progressively lesser increases in enzyme activity. Scale-up from shake-flasks to 2 L fed-batch cultivations increased rMnP activities from 200 U/L to 2,500 U/L, with addition of 0.1 g/L heme (added heme per liquid volume) at the beginning of the fed-batch phase resulting in higher enzyme activities than addition at the beginning of the batch phase. A combination of centrifugation, acetone precipitation, dialysis, and freeze drying was found to be effective for concentrating the rMnP from 2,500 U/L in the P. pastoris bioreactor culture to 30,000 U/L in 0.1 M potassium phosphate buffer pH 6. The rMnP recovery yield was 60% and the purity was 1-4%. By using 0.1 g/L heme during the fed-batch cultivation, the heme content of the final enzyme preparation could be reduced by 97%, and had sufficiently high rMnP activity and low enough color to be suitable for pulp bleaching experiments.     

L-蛋氨酸及甲醇浓度对毕赤酵母摇瓶发酵生产S-腺苷蛋氨酸的影响

利用甲醇传感器及高效液相色谱检测毕赤酵母摇瓶发酵过程的甲醇浓度及S-腺苷蛋氨酸(SAM)浓度,发现L-蛋氨酸浓度及甲醇浓度对毕赤酵母细胞生长及合成S-腺苷蛋氨酸具有影响,据此对摇瓶发酵过程的L-蛋氨酸浓度及甲醇浓度进行优化。优化结果表明:当L-蛋氨酸浓度为7.5 g/L时,最适于SAM积累,产量达到0.83 g/L;进而利用甲醇传感器对发酵过程的甲醇浓度进行检测及控制,考察不同甲醇浓度对SAM产量的影响,毕赤酵母产SAM的最佳甲醇浓度为15 g/L,在此浓度下SAM的产量达到1.41 g/L,比对照实验增加了21%。     

Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter

A Rhizopus oryzae lipase gene has been expressed in Pichia pastoris as a reporter using the formaldehyde dehydrogenase 1 promoter (PFLD1) of this organism, which has been reported to be strongly and independently induced by either methanol as sole carbon source or methylamine as sole nitrogen source. Levels of lipase expressed and secreted under the control of the PFLD1 at different induction conditions have been compared to those obtained with the commonly used alcohol oxidase 1 promoter (PAOX1) in small (shake flask) and 1l bioreactor batch cultures. PFLD1-controlled heterologous gene expression was strongly repressed by excess of either glycerol or glucose-but not sorbitol-during growth using methylamine both as sole nitrogen source and inducing substrate. Co-induction of PFLD1 with methanol and methylamine resulted in a synergistic effect on extracellular lipase expression levels. In all tested conditions, the substitution of ammonium for methylamine as carbon source provoked a clear decrease in the specific growth rate and yield of biomass per gram of carbon source. Overall, this study demonstrates that the PFLD1 promoter is at least as efficient as the PAOX1 for extracellular expression of heterologous proteins in P. pastoris bioreactor cultures and provides a first basis for the further design of methanol-free high cell density fed-batch cultivation strategies for controlled overproduction of foreign proteins in P. pastoris.     

Production of a Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies

Different cultivation strategies have been compared for the production of Rhizopus oryzae lipase (ROL) from Pichia pastoris. Several drawbacks have been found using a methanol non-limited fed-batch. On the one hand, oxygen limitation appeared at early cell dry weights and, on the other hand, high cell death was observed. A temperature limited fed-batch has been proposed to solve both problems. However, in our case study a methanol non-limited fed-batch results in better productivities. Finally, a lower salt medium were used to overcome cell death problems and a temperature limited fed-batch was applied thereafter to solve oxygen transfer limitations. This combined strategy has resulted in lower productivities when compared to a methanol non-limited fed-batch. However the culture could be longer prolonged and a 1.3-fold purer final product was obtained mainly due to cell death reduction.     

Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.