A model-based feeding strategy for fed-batch fermentation of recombinant Pichia pastoris

A two stage, exponential feeding strategy with mixed glycerol/methanol substrate was used in a fed-batch recombinant Pichia pastorisfermentation. The feeding strategy was developed using a simple model based on mass balances, Monod-type growth kinetics, and constant specific heterologous protein production rate. The model accurately predicted cell growth, and demonstrated the usefulness of a rational, model-based approach for improving the productivity of recombinant P. pastoris fermentation.     

A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fedbatch fermentation

A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.     

Maximization of production of secreted recombinant proteins in Pichia pastoris fed-batch fermentation

Pontryagin's Maximum Principle has been applied for optimization of secreted proteins from Pichia pastoris fed-batch fermentation. The objective of this work is to maximize the total accumulated product per unit operation time under different given conditions and system constraints. To obtain optimal solutions, an automated curve-fitting software, Table Curve 2D, was employed to construct the necessary mathematical models and solve the complicated functions. In the solution processes, the end of the glycerol batch phase was defined as the initial state of the system, the end of the methanol fed-batch phase as the final state, the cell mass produced along with product accumulated as state variables, and the specific growth rate (mu) as the control variable. Initially, a relationship between the specific production rate (rho) and mu was established. Then, according to Pontryagin's Maximum Principle, the admissible range of mu and its trajectories for the optimal operations were determined. Four representative cases with different combinations of the operation time along with the initial and final states were evaluated. A close correlation was obtained between the predicted values of the model equation with the experimental results from the Pichia pastoris fed-batch fermentations producing secreted alpha-galactosidase. The approaches proposed here greatly simplify the computational processes and validate the optimization strategy as a generalized approach to maximize the yield from fed-batch fermentations.     

Production of recombinant protein in Pichia pastoris by fermentation

This protocol is applicable to recombinant protein expression by small-scale fermentation using the Pichia pastoris expression system. P. pastoris has the capacity to produce large quantities of protein with eukaryotic processing. Expression is controlled by a methanol-inducible promoter, which allows a biomass-generation phase before protein production is initiated. The target protein is secreted directly into a protein-free mineral salt medium, and is relatively easy to purify. The protocol is readily interfaced with expanded bed adsorption for immediate capture and purification of recombinant protein. The setting up of the bioreactor plus the fermentation itself takes 1 wk. Making the master and user seed lots takes approximately 2 wk for each individual clone.     

Two-stage glycerol feeding for enhancement of recombinant hG-CSF production in a fed-batch culture of Pichia pastoris

Recombinant hG-CSF was expressed in Pichia pastoris under the control of the AOX1 promoter. In this study, the glycerol feeding rate was adjusted to achieve the maximum attainable specific growth rate before induction. Using a two-stage glycerol feeding method, the specific growth rate was changed from a maximum value of 0.21 h⁻¹ (at the beginning of feeding) to 0.15 h⁻¹ prior to induction. With this approach, the final dry cell wt and rhG-CSF yield achieved was close to 120 g l⁻¹ and 320 mg l⁻¹, respectively. Our study found that the two-stage feeding method allowed the overall productivity of rhG-CSF to increase 2.9 times that of the conventional fed-batch method.     

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.     

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.     

利用Pichia pastoris生产S-腺苷甲硫氨酸的发酵工艺

在摇瓶中考察了重组Pichia pastoris发酵的诱导剂量,L-甲硫氨酸,以及pH对腺苷甲硫氨酸产量的影响.放大到3.7 L发酵罐和30 L发酵罐后,研究了重组细胞的发酵过程变化,对S-腺苷甲硫氨酸初步纯化.摇瓶中优化后的发酵条件是:每天添加1%甲醇诱导,L-甲硫氨酸为50mmol/L,培养基pH 5.0.培养144 h后SAM产量达到2.32 g/L.3.7 L发酵罐中发酵251 h后细胞浓度为120 g/L,SAM总量为15.18 g.放大到30 L发酵罐中,发酵225.5 h后细胞浓度约为120 g/L,SAM总量为145.05 g.纯化后SAM的纯度为93.5%,回收率为84.5%.     

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.     

Phytase production by fermentation of recombinant Pichia pastoris in monosodium glutamate wastewater

A novel method is proposed to produce both phytase and single-cell protein in recombinant Pichia pastoris fermentation using monosodium glutamate wastewater (MSGW) as the basal medium. Recombinant P. pastoris MR33 transformed with a phytase gene (AppA-m) from Escherichia coli was constructed and showed capability to utilize ammonium as the only nitrogen source. The fermentation medium was optimized in shake flasks by single-factor test and response surface methodology. A fed-batch system containing 30% MSGW, 50 g/l glucose, 1.58 g/l CaSO₄, 5.18 g/l MgSO₄ and 6.67 g/l KH₂PO₄ was developed in a 3.7-l bioreactor. The maximum phytase activity in the MSGW medium reached 3,380 U/ml, 84.2% of that in chemically defined medium, and the dry cell weight was 136 g/l. The single-cell protein (SCP; 46.66% dry cell weight) contains a variety of amino acids and is low in fat, which is ideal for utilization in animal feed. Thus, it is feasible to use MSGW medium for the production of enzymes that can be expressed in P. pastoris.     

Response surface optimization of fermentation conditions for xylanase production by recombinant Pichia pastoris

研究不同碳源、氮源和无机盐对毕赤酵母AX181菌株产木聚糖酶的影响.实验表明,分别采用葡萄糖和玉米浆干粉为碳源和氮源可以明显提高木聚糖酶的产量.无机盐单因子优化实验显示添加适量的(NH4)2SO4、KH2 PO4、MnSO4·H2O、FeSO4·7H2O也可以部分提高木聚糖酶产量.在此基础上利用响应面法优化毕赤酵母产木聚糖酶培养基,利用12次实验的Plackett - Burman设计实验筛选出影响产木聚糖酶的3个主要因素,即玉米浆干粉、MrSO4 ·H2O和FeSO4·7H2O.并进一步通过最陡爬坡路径逼近最大响应区域,采用中心组合实验设计确定最佳条件.优化后的产木聚糖酶培养基组分为(g/L):葡萄糖40.00,玉米浆干粉80.84,(NH4)2SO4 6.25,KH2PO4 1.25、MnSO4·H2O 0.35,FeSO4 ·7H2O 1.31.培养基优化后,实际产酶2 883.86 U/mL,是优化前YPD培养基产酶的2.51倍.     

响应面分析优化产木聚糖酶毕赤酵母基因工程菌培养条件

研究不同碳源、氮源和无机盐对毕赤酵母AX181菌株产木聚糖酶的影响。实验表明,分别采用葡萄糖和玉米浆干粉为碳源和氮源可以明显提高木聚糖酶的产量。无机盐单因子优化实验显示添加适量的（NH4）2SO4、KH2PO4、MnSO4·H2O、FeSO4·7H2O也可以部分提高木聚糖酶产量。在此基础上利用响应面法优化毕赤酵母产木聚糖酶培养基,利用12次实验的Plackett—Burman设计实验筛选出影响产木聚糖酶的3个主要因素,即玉米浆干粉、MnSO4·H2O和FeSO4·7H20。并进一步通过最陡爬坡路径逼近最大响应区域,采用中心组合实验设计确定最佳条件。优化后的产木聚糖酶培养基组分为（g/L）：葡萄糖40．00,玉米浆干粉80．84,（NH4）2SO46．25,KH2PO41．25、MnSO4·H2O0．35,FeS04-7H2O1．31。培养基优化后,实际产酶2883．86u／mL,是优化前YPD培养基产酶的2．51倍。     

A simple structured model for recombinant IDShr protein production in Pichia pastoris

A simple structured model is proposed for the methanol production phase of the iduronate 2-sulphate sulfatase recombinant enzyme (IDShr) in Pichia patoris Mut(+). The model is mainly focused in oxidative stress phenomenon due to methanol consumption and based on extracellular experimental information and the basic knowledge of methanol metabolism in Pichia pastoris yeast (P. pastoris). The model's prediction shows a reasonable accuracy as compared with the experimental data. Likewise, it was proved that this model is able to simulate the production of other recombinant protein in P. pastoris.     

Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation

An optimization method for repeated fed-batch fermentation was established with the aim of improving the recombinant human serum albumin (rHSA) production in Pichia pastoris. A simulation model for fed-batch fermentation was formulated and the optimal methanol-feeding policy calculated by dynamic programming method using five different methanol-feeding periods. The necessary state variables were collected from the calculated results and used for further optimization of repeated fed-batch fermentation. The optimal operation policy was investigated using the pre-collected state variables by estimating the overall profit per total methanol-feeding time. The calculated results indicated that the initial cell mass from the 2nd fed-batch fermentation on should be set at 35 or 40 g and methanol-feeding time at 264 h. In repeated fed-batch fermentation using the optimal operation policy, actual culture volume was in good agreement with the values simulated by model equations, but some discrepancy was observed in rHSA production. Minimum experiments were therefore carried out to re-evaluate rHSA production levels, which were then applied in re-calculations to determine the optimal operation policy. The optimal policy for repeated fed-batch fermentation established in the present study (i.e., 4-times-repeated fed-batch fermentation) achieved a 47% increase in annual rHSA production. Optimization of the culture period also brought about a 28% increase in annual rHSA production even in simple (not repeated) fed-batch fermentation.     

Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes

An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood β-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q _O/q _O,max), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90–95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.     

重组毕赤酵母产青霉素G酰化酶的分批发酵动力学研究

构建了重组毕赤酵母产青霉素G酰化酶的分批发酵动力学模型。实验考察了分批发酵过程中甘油消耗、甲醇浓度、菌体浓度、溶氧、补料时间对青霉素G酰化酶活力的影响。应用Matlab软件,对菌体生长、基质消耗和产物生成方程进行最优参数估算和非线性拟合,得到相应的动力学模型。模型的计算值与实验值能较好地拟合,表明所建模型能较好反映重组毕赤酵母产青霉素G酰化酶的分批发酵过程。