Sorbitol as a non-repressing carbon source for fed-batch fermentation of recombinant Pichia pastoris

Glycerol/methanol and sorbitol/methanol mixed-feed fermentation strategies for the production of recombinant proteins by Pichia pastoris were compared in order to examine sorbitol's potential as a carbon source. Although P. pastoris does have a lower cell yield on sorbitol than on glycerol, the specific rate of product formation is higher (60 g protein g^–1 dry wth for sorbitol/methanol, vs 45 g protein g^–1 dry wth for glycerol/methanol), resulting in comparable final recombinant expression levels. Importantly, the presence of residual sorbitol in the growth medium appears to be less repressive to the alcohol oxidase promoter in this organism, providing a more forgiving means of operating mixed-feed fed-batch recombinant P. pastoris fermentations.     

Fluorescence-based real time monitoring and diagnostics of recombinant Pichia pastoris cultivations in a bioreactor

This study describes the application of the multivariate curve resolution (MCR) analysis technique for real-time analysis of culture fluorescence during recombinant Pichia pastoris cultivation in a bioreactor. Fluorescence spectra were acquired with an on-line dual excitation wavelength fluorometer and then used to develop a real time MCR-based bioprocess monitoring and diagnostics tool. Initial bioreactor experiments using two similar recombinant antibody secreting P. pastoris cell lines showed significant differences in protein production. To distinguish between the contributions of operating conditions and the specific cell line's genetic composition to the observed differences in protein production, the bioreactor experiments were repeated and accompanied by real time MCR analysis. The tests demonstrated high sensitivity of MCR-derived “pure concentration” profiles to growth as well as to initial conditions, thus enabling real-time cultivation process trend diagnostics and fault detection. © 2018 Her Majesty the Queen in Right of Canada © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2761, 2019.     

A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures

The secreted proteome of Pichia pastoris X-33 was investigated in methanol-induced cultures with a goal to enhance the secretion and purification of recombinant proteins. In a fed-batch fermentation at 30 °C, more host proteins were found in greater concentrations compared to cultures grown at 25 °C. Protein samples collected directly from the culture media at 25 °C, as well as separated by two-dimensional (2D) gel, were subjected to ESI-MS/MS analysis. A total of 75 proteins were identified in the media from different conditions including pre- and post-methanol induction and in a strain overexpressing a recombinant schistosomiasis vaccine, Sm14-C62V. The identified proteins include native secreted proteins and some intracellular proteins, most of which have low isoelectric points (pI < 6). 2D gel analyses further revealed important characteristics, such as abundance, degradation, and glycosylation of these identified proteins in this proteome. Cell wall-associated proteins involved in cell wall biogenesis, structure, and modification comprised the majority of the secreted proteins which have been identified. Intracellular proteins such as alcohol oxidase and superoxide dismutase were also found in the proteome, suggesting some degree of cell lysis. However, both protocols show that their concentrations are significantly lower than the native secreted proteins. This study identifies proteins secreted or released into the culture media in the methanol-induced fermentation cultures of P. pastoris X-33 and suggests potential biotechnology applications based on the discovery of this proteome.     

Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources

A recombinant strain of Pichia pastoris with a phenotype of Mut^S was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O₂ concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l^–1 after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.     

Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production

To improve the growth of recombinant Pichia pastoris with a phenotype of Mut^S and expression of angiostatin, the effects of glycerol, sorbitol, acetate and lactic acid which were, respectively, added together with methanol in the expression phase, were studied in a 5-l fermentor. Methanol concentration was automatically controlled at 5 g/l by a methanol monitor and control system, while the feeding of the other carbon source was manually adjusted. The angiostatin production level was 108 mg/l when glycerol was added at an initial rate of 2.3 g/h and gradually increased to 9.9 g/h within an induction period of 96 h. The angiostatin concentration was 141 mg/l as sorbitol was used, while only 52 mg/l were obtained on acetate. The highest angiostatin production of 191 mg/l was achieved as lactic acid was used; whose feeding rate was gradually increased from 2.6 to 11.3 g/h. Lactic acid accumulated during the induction phase and reached 6.3 g/l at the end of fermentation. However, the accumulation of lactic acid did not interfere with angiostatin production, indicating that lactic acid to be a non-repressive carbon source. The average productivity and specific productivity of angiostatin obtained on lactic acid and methanol were, respectively, 2.96 and 0.044 mg/(g h), 1.7- and 2.5-fold of those obtained in the fermentation fed with glycerol and methanol.     

混合碳源流加对重组毕赤酵母生产碱性果胶酶的影响

为提高重组毕赤酵母生产碱性果胶酶(PGL)的产量和生产强度,在诱导期采用多种碳源与甲醇混合添加的模式。实验结果发现:甘油、山梨醇、乳酸与甲醇的混合添加均可以提高PGL的产量,其中山梨醇与甲醇的混合流加效果最为显著。研究表明,通过双碳源混合流加可以提高细胞活力,增强醇氧化酶活力,提高毕赤酵母表达外源蛋白效率。当山梨醇的流速为3.6g/(h·L)时,PGL酶活可达1593U/mL,生产强度为16.7U/(mL·h),比对照分别提高了84.6%和45.2%,实现了碱性果胶酶的高效生产。     

A robust fed-batch feeding strategy independent of the carbon source for optimal polyhydroxybutyrate production

A three-stage control strategy independent of the organic substrate was developed for automated substrate feeding in a two-phase fed-batch culture of Cupriavidus necator DSM 545 for the production of the biopolymer polyhydroxybutyrate (PHB). The optimal feeding strategy was determined using glucose as the substrate. A combined substrate feeding strategy consisting of exponential feeding and a novel method based on alkali-addition monitoring resulted in a maximal cell concentration in the biomass growth phase. In the PHB accumulation phase, a constant substrate feeding strategy based on the estimated amount of biomass produced in the first phase and a specific PHB accumulation rate was implemented to induce PHB under limiting nitrogen at different biomass concentrations. Maximal cell and PHB concentrations of 164 and 125 g/L were obtained when nitrogen feeding was stopped at 56 g/L of residual biomass; the glucose concentration was maintained within its optimal range. The developed feeding strategy was validated using waste glycerol as the sole carbon source for PHB production, and the three-stage control strategy resulted in a PHB concentration of 65.6 g/L and PHB content of 62.7% while keeping the glycerol concentration constant. It can thus be concluded that the developed feeding strategy is sensitive, robust, inexpensive, and applicable to fed-batch culture for PHB production independent of the carbon source.     

Enhancing the production of Fc fusion protein in fed-batch fermentation of Pichia pastoris by design of experiments

This study focuses on the feasibility of producing a therapeutic Fc fusion protein in Pichia pastoris (P. pastoris) and presents an optimization design of experiment (DOE) strategy in a well-defined experimental space. The parameters examined in this study include pH, temperature, salt supplementation, and batch glycerol concentration. The effects of these process conditions were captured by statistical analysis focusing on growth rate and titer responses. Batch medium and fermentation conditions were also investigated prior to the DOE study in order to provide a favorable condition to enable the production of this Fc fusion protein. The results showed that approximately 373 mg/L of the Fc fusion protein could be produced. The pH was found to be particularly critical for the production of this Fc fusion protein. It was significantly higher than the conventional, recommended pH for P. pastoris fermentation. The development of this process shows that protein production in P. pastoris is protein specific, and there is not a set of pre-defined conditions that can work well for all types of proteins. Thorough process development would need to be performed for every type of protein in order for large-scale production in P. pastoris to be feasible.     

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.     

EMG gait data from indwelling electrodes is attenuated over time and changes independent of any experimental effect

The effect of time on the validity of electromyography (EMG) signals from indwelling fine-wire electrodes has not been explored. This is important because experiments using intramuscular electrodes are often long and biochemical and mechanical factors, may impair measurement accuracy over time. Measures over extended periods might therefore be erroneous. Twelve healthy participants (age = 33 ± 8 years) walked for 50 min at a controlled speed. Fine-wire electrodes were inserted into tibialis anterior and a surface EMG sensor attached near the fine-wire insertion site. EMG signals progressively and significantly decreased with time with the fine-wire electrode, but not the surface electrode. For the fine-wire electrode, after 25 min mean amplitude had reduced by 11% (p < 0.001) and after 50 min by 16% (p < 0.001), and peak amplitude reduced 22% at 20 min (p = 0.006) and 37% at 50 min (p < 0.001). Reduced amplitude with indwelling EMG without concurrent changes in surface EMG signal suggests an important inconsistency in data from fine-wire EMG electrodes. Changes in EMG signal will occur over time independent of the experimental condition and this questions their use in experiments of more than 30 min. These results should impact on experimental study design. They also invite reinterpretation of prior literature and sensor innovation to improve measurement performance.     

X-ray crystallography and mass spectroscopy reveal that the N-lobe of human transferrin expressed in Pichia pastoris is folded correctly but is glycosylated on serine-32

The ferric form of the N-lobe of human serum transferrin (Fe(III)-hTF/2N) has been expressed at high levels in Pichia pastoris. The Fe(III)-hTF/2N was crystallized in the space group P41212, and X-ray crystallography was used to solve the structure of the recombinant protein at 2.5 A resolution. This represents only the second P. pastoris-derived protein structure determined to date, and allows the comparison of the structures of recombinant Fe(III)-hTF/2N expressed in P. pastoris and mammalian cells with serum-derived transferrin. The polypeptide folding pattern is essentially identical in all of the three proteins. Mass spectroscopic analyses of P. pastoris- hTF/2N and proteolytically derived fragments revealed glycosylation of Ser-32 with a single hexose. This represents the first localization of an O-linked glycan in a P. pastoris-derived protein. Because of its distance from the iron-binding site, glycosylation of Ser-32 should not affect the iron-binding properties of hTF/2N expressed in P. pastoris, making this an excellent expression system for the production of hTF/2N.     

Impact of NH4+ nitrogen source on the production of Rhizopus oryzae lipase in Pichia pastoris

BackgroundPichia pastoris is a highly successful system for heterologous expression. During the induction stage, the ammonium ion released into the fermentation broth has a deep impact on cell growth and protein expression. The impact of NH₄⁺ concentration on the expression of the Rhizopus oryzae lipase proAROL in P. pastoris was investigated.ResultsThe lipase activity under the optimum NH₄⁺ concentration of 440 mmol/L reached 12,019 U/mL. Increased concentrations of NH₄⁺ in the broth prevented the protease production, resulting in higher specific lipase activity in the supernatant. Furthermore, analysis of carbon metabolism and energy regeneration pattern revealed that under the definite NH₄⁺ concentrations more carbon source (methanol) was consumed with surged AOX activity and then the higher energy and amino acid precursors demand for recombinant protein synthesis is compensated for by the TCA cycle.ConclusionsIn this study, the R. oryzae lipase activity reaches the highest level ever reported under optimized NH₄⁺ concentration and the analysis of the carbon metabolism provides useful information for future optimization of protein production by P. pastoris in a molecular level.     

Combined effect of the methanol utilization (Mut) phenotype and gene dosage on recombinant protein production in Pichia pastoris fed-batch cultures

An important number of heterologous proteins have been produced in the methylotrophic yeast Pichia pastoris using the alcohol oxidase promoter. Two factors that drastically influence protein production and cultivation process development in this system are gene dosage and methanol assimilation capacity of the host strain (Mut phenotype). Using a battery of four strains which secrete a Rhizopus oryzae lipase (ROL), the combined effects of gene dosage and Mut phenotype on recombinant protein production in Pichia pastoris was studied in fed-batch cultures. Regarding the effect of phenotype, the specific productivity and the Y(P/X) were 1.29- and 2.34-fold higher for Mut(s)ROL single copy strain than for Mut+ROL single copy strain. On the contrary, the productivity of Mut+ROL single copy strain was 1.34-fold higher than Mut(s)ROL single copy strain. An increase in ROL gene dosage seems to negatively affect cell's performance in bioreactor cultures, particularly in Mut(s) strains. Overall, the Mut(s) strain may be still advantageous to use because it allows for easier process control strategies.     

The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation

DNA replication in Saccharomyces cerevisiae proceeds according to a temporal program. We have investigated the role of the telomere-binding Ku complex in specifying late replication of telomere-proximal sequences. Genome-wide analysis shows that regions extending up to 80 kb from telomeres replicate abnormally early in a yku70 mutant. We find that Ku does not appear to regulate replication time by binding replication origins directly, nor is its effect on telomere replication timing mediated by histone tail acetylation. We show that Ku instead regulates replication timing through its effect on telomere length, because deletion of the telomerase regulator Pif1 largely reverses the short telomere defect of a yku70 mutant and simultaneously rescues its replication timing defect. Consistent with this conclusion, deleting the genome integrity component Elg1 partially rescued both length and replication timing of yku70 telomeres. Telomere length-mediated control of replication timing requires the TG(1-3) repeat-counting component Rif1, because a rif1 mutant replicates telomeric regions early, despite having extended TG(1-3) tracts. Overall, our results suggest that the effect of Ku on telomere replication timing results from its impact on TG(1-3) repeat length and support a model in which Rif1 measures telomere repeat length to ensure that telomere replication timing is correctly programmed.     

毕赤酵母表达系统在外源蛋白表达中的研究及应用

巴斯得毕赤酵母（Pichia pastoris）表达系统作为一个日臻完善的外源蛋白真核表达系统由于它所具有的一些其它表达系统不可比拟的优势而得到越来越广泛的应用。分别从该表达系统的优点、外源基因整合及调控机理、表达蛋白糖基化及翻译后修饰等方面综述了其在外源蛋白表达中的研究进展及应用。     

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

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

A non-ionic surfactant reduces the induction time and enhances expression levels of bubaline somatotropin in Pichia pastoris

This study describes a simple approach for enhanced secretory expression of bubaline somatotropin (BbST) in the methylotropic yeast Pichia pastoris. A Mut^s Pichia transformant carrying multi-copy, non-codon optimized BbST cDNA sequence, expressed and secreted the recombinant protein into the culture medium to a level of 25 % of the total proteins in the culture supernatant, after 120 h of induction. Inclusion of polysorbate-80 in the inducing medium resulted in a significant improvement in the BbST expression (up to 45 % of the total culture supernatant proteins) with concomitant reduction in the induction time to 48 h. The amount of BbST obtained was 148 mg/L, which was around fivefold higher than that obtained without the surfactant. BbST was purified to near homogeneity by FPLC on Q-sepharose FF anion-exchange column. Protein authenticity was judged by SDS-PAGE and western blot analyses. A bioassay based on proliferation of Nb2 rat lymphoma cell lines confirmed that the purified, recombinant BbST is biologically active. Use of polysorbate-80 in combination with methanol, during the induction phase, is likely to have general applicability in lowering the induction time and enhancing the secretory expression of other commercially important proteins in Mut^s strains of P. pastoris.