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.     

表达重组咖啡豆α-半乳糖苷酶的酵母工程菌的高密度发酵

用5 L发酵罐优化了重组咖啡豆α-半乳糖苷酶酵母工程菌pPIC9K-Gal/GS115(本室构建)的高密度发酵工艺.通过对发酵条件的优化,包括甘油补充量及补充时机、甲醇诱导量及诱导时机、溶氧控制、诱导时间等,重组咖啡豆α-半乳糖苷酶在毕赤酵母中得到了高效表达.利用所确定的最适条件进行发酵,菌体密度最终达到368 g/L以上,每批发酵液离心后可获得3.5 L的发酵上清,上清中的蛋白含量达到3 g/L以上,目的蛋白占上清总蛋白的50%以上,含量约为1.5 g/L,上清中α-半乳糖苷酶的活性维持在80 U/ml左右.确立工艺后又进行了3次发酵试验,证明了工艺的可行性和稳定性.为重组咖啡豆α-半乳糖苷酶在B→O血型改造和酶解大豆低聚糖方面的应用奠定了基础.     

人源性抗HBsAg Fab抗体的发酵生产研究

为了适应工业生产的需要,利用fed—batch方法,重组人源性抗HBsAg Fab抗体酵母工程菌在30L发酵罐中进行了高密度发酵,发酵最适温度30℃,pH值范围5．0～5．3,溶氧范围20％～30％。发酵液OD600值达到300时开始诱导,甲醇最佳诱导浓度为10mL／L。重组人源性抗HBsAg Fab抗体经离子交换层析纯化,纯化产品经SDS-PAGE、Western blot进行分析和ELISA方法进行活性测定。结果显示,重组Fab抗体在Fed-batch发酵系统中可高效表达,经过192h的发酵生产,重组人源性抗HBsAg Fab抗体的表达量可达412mg／L。发酵上清经过离子交换层析纯化,获得纯度为95％的重组Fab抗体,该Fab抗体经ELISA分析具有较高的HBsAg抗原亲和力和特异性。结果证实可以通过高密度发酵毕赤酵母工程菌来高效生产重组人源性抗HBsAg Fab抗体,为后续的工业化生产应用奠定了基础。     

重组人干扰素ω在对数生长期毕赤酵母中的高效表达特性研究

用不同比生长速率μ的毕赤酵母探讨其表达外源重组蛋白的差异性,通过起始pH值、甲醇诱导浓度和周期、菌体浓度、装液量等实验,优化具有较高μ的对数生长期毕赤酵母表达rhIFNω的摇瓶条件。结果表明,μ对毕赤酵母表达rhIFNω有显著影响。μ为0.1612h-1的毕赤酵母表达rhIFNω最高为558mg/L,较μ为0.1321、0.0505和0.0052h-1的毕赤酵母分别提高50%、68%和99%。对数生长期的毕赤酵母表达rhIFNω的最适摇瓶表达条件为:250mL摇瓶装入30mL BMMY,控制菌体浓度达到200~300g/L(WCW),起始pH值自然,每24h添加甲醇15g/L一次,诱导表达周期为4d。通过表达条件的优化,rhIFNω的表达量达到1070mg/L,较优化前提高149%。     

Multi-objective optimization in Aspergillus niger fermentation for selective product enhancement

A multi-objective optimization formulation that reflects the multi-substrate optimization in a multi-product fermentation is proposed in this work. This formulation includes the application of ε-constraint to generate the trade-off solution for the enhancement of one selective product in a multi-product fermentation, with simultaneous minimization of the other product within a threshold limit. The formulation has been applied to the fed-batch fermentation of Aspergillus niger that produces a number of enzymes during the course of fermentation, and of these, catalase and protease enzyme expression have been chosen as the enzymes of interest. Also, this proposed formulation has been applied in the environment of three control variables, i.e. the feed rates of sucrose, nitrogen source and oxygen and a set of trade-off solutions have been generated to develop the pareto-optimal curve. We have developed and experimentally evaluated the optimal control profiles for multiple substrate feed additions in the fed-batch fermentation of A. niger to maximize catalase expression along with protease expression within a threshold limit and vice versa. An increase of about 70% final catalase and 31% final protease compared to conventional fed-batch cultivation were obtained. Novel methods of oxygen supply through liquid-phase H₂O₂ addition have been used with a view to overcome limitations of aeration due to high gas–liquid transport resistance. The multi-objective optimization problem involved linearly appearing control variables and the decision space is constrained by state and end point constraints. The proposed multi-objective optimization is solved by differential evolution algorithm, a relatively superior population-based stochastic optimization strategy.     

Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry

This paper describes the establishment of flow cytometric methods for recombinant Pichia pastoris strains, and their application to a lab scale fed batch fermentation. Using a strain which secretes human trypsinogen, the viability and the product which remained associated to the cell were measured with propidium iodide and immunofluorescent staining, respectively. Viability decreases significantly below 70% during the methanol fed batch phase, indicating a stress situation triggered by the fermentation conditions. Cell associated product is accumulated earlier after methanol induction than secreted product. These data demonstrate that flow cytometry is a powerful tool for the analysis and optimization of recombinant protein production processes, and they indicate the need to further improve a widely used fermentation protocol for P. pastoris.     

High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system

A continuous fermentation process has been developed in Pichia pastoris (P. pastoris) with the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in order to produce large quantities of recombinant human chitinase (rh-chitinase) for preclinical studies as a potential high-dose antifungal drug. Expression levels of about 200 to 400 mg/L have been demonstrated in fed-batch fermentations using strains with either the traditional methanol-inducible or the constitutive GAP promoter. Proteolytic degradation of the enzyme was typically seen in fed-batch fermentations. Continuous production of the enzyme by P. pastoris with the GAP promoter was demonstrated in a 1.5-L working volume fermentor using either glucose or glycerol as the carbon source. The fermentation could be extended for >1 month with a steady-state protein concentration of approximately 300 mg/L. Cell densities were >400 g/L wet cell weight (WCW) (approximately 100 g/L dry cell weight [DCW]) at a dilution rate (D) of 0.83 day(-1) or 1.2 volume exchanges per day (VVD). No proteolytic degradation of the enzyme was seen in the continuous fermentation mode.     

Use of multi-staining flow cytometry to characterise the physiological state of Escherichia coli W3110 in high cell density fed-batch cultures.

High cell density fed-batch fermentations of Escherichia coli W3110 have been carried out at specific growth rates of less than 0.3 h-1, to investigate the effect of glucose limitation on the physiological state of individual cells. After an initial exponential batch phase, the feed rate was held constant and a final dry cell weight of approximately 50 g per litre was achieved. The fermentations were monitored by mass spectrometry whilst measurements of pH, DOC, CFU/mL, TCN, OD500nm and residual glucose concentrations were made. Satisfactory and reproducible results were obtained. Flow cytometric analysis of cells in broth samples, based on either of two multi-staining protocols, revealed a progressive change in cell physiological state throughout the course of the fermentations. From these measurements it was concluded that the loss in reproductive viability towards the end of the fed-batch process is due to cell death and not due to the formation of a "viable but nonculturable state" as had previously been reported. Since the presence of a high proportion of dead or dying cells at any time during a fermentation has a detrimental effect on the synthesis of any desired product it is proposed that an on-line flow cytometric analysis and control strategy could be used as a means of increasing overall process efficiency.     

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.     

Twenty-four-well plate miniature bioreactor high-throughput system: assessment for microbial cultivations

High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (micro)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the micro-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6 +/- 2.4, 46.5 +/- 4.6, 51.6 +/- 3.7, and 56.1 +/- 1.6 h(-1) at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively.     

Modelling the optimal timing in metabolic pathway activation—Use of Pontryagin's Maximum Principle and role of the Golden section

The time course of enzyme concentrations in metabolic pathways can be predicted on the basis of the optimality criterion of minimizing the time period in which an essential product is generated. This criterion is in line with the widely accepted view that high fitness requires high pathway flux. Here, based on Pontryagin's Maximum Principle, a method is developed to solve the corresponding constrained optimal control problem in an almost exclusively analytical way and, thus, to calculate optimal enzyme profiles, when linear, irreversible rate laws are assumed. Three different problem formulations are considered and the corresponding optimization results are derived. Besides the minimization of transition time, we consider an operation time in which 90% of the substrate has been converted into product. In that case, only the enzyme at the lower end of the pathway rather than all enzymes are active in the last phase. In all cases, biphasic or multiphasic time courses are obtained. The biological meaning of the results in terms of a consecutive just-in-time expression of metabolic genes is discussed. For the special case of two-enzyme systems, the role of the Golden section in the solution is outlined.     

Model-based optimization of viral capsid protein production in fed-batch culture of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

An optimized fed-batch cultivation process for the production of the polyoma virus capsid protein VP1 in recombinant Escherichia coli BL21 bacteria is presented. The optimization procedure maximizing the amount of desired protein is based on a mathematical model. The model distinguishes an initial cell growth phase from a protein production phase initiated by inducer injection. A new approach to model the target protein formation rate was elaborated, where product formation is primarily dependent on the specific biomass growth rate. Lower growth rates led to higher specific protein concentrations. The model was identified from a series of fed-batch experiments designed for parameter identification purposes and possesses good prediction quality. Then the model was used to determine optimal open-loop control profiles by manipulating the substrate feed rates in both phases as well as the induction time. Feed-rate optimization has been solved using Pontryagin's maximum principle. The solution was validated experimentally. A significant improvement of the process performance index was achieved.     

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.     

流式细胞术检测毕赤酵母发酵过程中胞内活性氧水平

以2′,7′-二氢二氯荧光黄双乙酸钠(DCFH-DA)和碘化丙锭(PI)为标记探针,通过DCFH-DA/PI双染色与PI单染色的对照,检测毕赤酵母胞内活性氧(reactive oxygen species,ROS)的水平及其影响。研究发现发酵过程细胞活性下降与胞内ROS积累相关。在甘油生长期,细胞几乎没有ROS积累,细胞活性接近100%。在甲醇诱导初期,部分细胞积累少量的ROS,细胞活性仍然很高,死亡细胞所占比例只有1.5%。在甲醇诱导后期,94.0%的细胞积累了大量的ROS,高含量的ROS造成细胞损伤,引起部分细胞丧失了活性,在总共29.1%的死亡细胞中,高ROS积累的死亡细胞占了25.4%。     

Production of recombinant humanized anti-HBsAg Fab fragment from Pichia pastoris by fermentation

In this report, we describe the high-yield secretory expression of the recombinant human anti-HBsAg Fab fragment from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of alcohol oxidase promoter) into the genome of the yeast cells. The fed-batch fermentations were carried out in a 5 L scale. Both chains of the Fab were successfully expressed upon methanol induction. The absorbance (OD600) of the broth can reach 350 approximately 500 at the end of fed-batch phase. After the induction, the expression level of the recombinant Fab (soluble) reached 420 approximately 458 mg/L. The recombinant Fab fragment was purified from the crude culture supernatant by ion exchange chromatography and the purity of the recombinant Fab fragment was over 95%. The affinity activities of the crude fermentation supernatant and the purified Fab were analyzed by indirect ELISA, which showed that the purified recombinant Fab fragment had high affinity activity with hepatitis B surface antigen.     

Fed-batch optimization of alpha-amylase and protease-producing Bacillus subtilis using Markov chain methods

A stoichiometry-based model for the fed-batch culture of the recombinant bacterium Bacillus subtilis ATCC 6051a, producing extracellular alpha-amylase as a desirable product and proteases as undesirable products, was developed and verified. The model was then used for optimizing the feeding schedule in fed-batch culture. To handle higher-order model equations (14 state variables), an optimization methodology for the dual-enzyme system is proposed by integrating Pontryagin's optimum principle with fermentation measurements. Markov chain Monte Carlo (MCMC) procedures were appropriate for model parameter and decision variable estimation by using a priori parameter distributions reflecting the experimental results. Using a simplified Metropolis-Hastings algorithm, the specific productivity of alpha-amylase was maximized and the optimum path was confirmed by experimentation. The optimization process predicted a further 14% improvement of alpha-amylase productivity that could not be realized because of the onset of sporulation. Among the decision variables, the switching time from batch to fed-batch operation (t(s)) was the most sensitive decision variable.     

A simple Pichia pastoris fermentation and downstream processing strategy for making recombinant pandemic Swine Origin Influenza A virus Hemagglutinin protein

The present Influenza vaccine manufacturing process has posed a clear impediment to initiation of rapid mass vaccination against spreading pandemic influenza. New vaccine strategies are therefore needed that can accelerate the vaccine production. Pichia offers several advantages for rapid and economical bulk production of recombinant proteins and, hence, can be attractive alternative for producing an effective influenza HA based subunit vaccine. The recombinant Pichia harboring the transgene was subjected to fed-batch fermentation at 10 L scale. A simple fermentation and downstream processing strategy is developed for high-yield secretory expression of the recombinant Hemagglutinin protein of pandemic Swine Origin Influenza A virus using Pichia pastoris via fed-batch fermentation. Expression and purification were optimized and the expressed recombinant Hemagglutinin protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot and MALDI-TOF analysis. In this paper, we describe a fed-batch fermentation protocol for the secreted production of Swine Influenza A Hemagglutinin protein in the P. pastoris GS115 strain. We have shown that there is a clear relationship between product yield and specific growth rate. The fed-batch fermentation and downstream processing methods optimized in the present study have immense practical application for high-level production of the recombinant H1N1 HA protein in a cost effective way using P. pastoris.     

重组人卵透明带ZP3蛋白在毕赤酵母中的表达

利用P.pastoris表达人卵透明带ZP3蛋白。设计特定引物从全长hZP3 cDNA上扩增含跨膜区序列的人卵透明带ZP3基因片段,并在N末端接上串联组氨酸编码序列的重组基因序列;扩增片段插入表达载体pPIC9K中;线性化后的重组质粒转入P.pastoris中,用高浓度G418筛选高拷贝菌株,然后甲醇诱导目的蛋白表达。用SDS-PAGE和Western blot分析表达产物。结果发现P.pastoris表达的人ZP3蛋白可以分泌到培养液中,并且可溶性好。纯化前后的重组人ZP3蛋白均能与兔抗猪ZP3蛋白抗体发生交叉反应,证实表达的目的蛋白具有反应原性。     

A Novel Approach for Secretion of Heterologous Proteins with Correct N-Terminal Processing by Using α-Factor Pre Sequence in Pichia pastoris

Numerous proteins have been secreted in P. pastoris by fusing the target gene with α-factor pre-pro sequence at Kex2 endopeptidase cleavage site. However, in some instances the product cannot be correctly processed due to aberrant cleavage by Kex2 endopeptidase such as aprotinin. In this study, an aprotinin gene was cloned into pPIC9K at the signal peptidase cleavage site through a single NheI restriction site designed at the 3'end of the α-factor signal sequence preregion, and transformed into GS115 host cell. By G418 resistance and ELISA assay, a high-yield recombinant was selected. After fed-batch cultivation in a 7-L bioreactor, the product was efficiently secreted into culture medium and accumulated up to ～ 4.7 mg L-1. MALDI-TOF/MS and N-terminal analyses confirmed its authenticity. Thus, a novel cloning strategy for secretion of aprotinin with correct N-terminal processing in P. pastoris has been developed which can be potentially applied to other proteins.