Improving the expression of mini-proinsulin in Pichia pastoris

Increased expression of recombinant mini-proinsulin in Pichia pastoris in 2.5 l bioreactors was achieved by increasing the cultivation pH from 5.1 to 6.3, by decreasing the temperature from 28 to 22 degrees C, and by periodical addition of ammonium sulfate and EDTA to the culture broth. Using this procedure, mini-proinsulin reached nearly 0.3 g l(-1) in the culture supernatant after 160 h of growth.     

Methanol induction optimization for scFv antibody fragment production in Pichia pastoris

Fibronectin splice variant ED B (extracellular domain B) is a promising marker for angiogenesis in growing solid tumors. Currently, recombinant antibodies against ED B are being investigated concerning their potential use, for either therapeutic or diagnostic purposes. Single-chain antibody fragments directed against the ED B can be efficiently expressed in Pichia pastoris; thus, a recombinant strain of the methylotropic yeast P. pastoris was used for this work. Three different forms of scFv antibody fragment are found in the supernatant from this fermentation: covalent homodimer, associative homodimer, and monomer. Both homodimeric forms can be converted to the monomeric form (under reducing conditions) and be efficiently radiolabeled, whereas the monomeric form of scFv already present in the supernatant cannot. It was also found that the fraction of protein in the monomeric form is highly dependent on the mode of induction rather than scFv concentration. This suggests that the monomeric form of the scFv present in the supernatant might be a result of events occurring at the expression, secretion, or folding level. A high cell density fermentation protocol was developed by optimizing methanol induction, yielding the highest scFv antibody fragment production rate and product quality; cell concentration at the induction point and specific methanol uptake rate were found to be the most important control variables. A decrease in specific methanol uptake rate led to a higher specific production rate for the scFv antibody fragment (5.4 microg g(cell) h(-1)). Product quality, i.e., percentage of product in a homodimeric form, also increased with the decrease in methanol uptake rate. Furthermore, the volumetric productivity depended on cell concentration at the induction point, increasing with the increase of cell concentration up to 320 g L(-1) wet cell weight (WCW). The reduction of the methanol feeding rate for induction, and consequently of the oxygen uptake rate, have important consequences for optimizing product titers and quality and thus on the scale-up of this production process; hence one of the major limitations upon high cell density cultivation in bioreactors is keeping the high oxygen transfer rate required. From the results obtained, a scale-up strategy was developed based on the available oxygen transfer rates at larger scales, allowing the definition of the optimum biomass concentration for induction and methanol feeding strategy for maximization of product titer and quality.     

Modeling of mini-proinsulin production in Pichia pastoris using the AOX promoter

An unstructured model based on mass balance equations for a recombinant methylotrophic yeast Pichia pastoris Mut^S (Methanol Utilization Slow) strain expressing the mini-proinsulin (MPI), was successfully established in quasi-steady state fed-batch fermentations with varying total quantity of biomass in a 7 l fermenter. The model describes the relationships between the total biomass and induction time, both in the batch and fed-batch phases. In addition, good correlations were obtained when the total quantity of MPI was correlated with the total biomass, demonstrating that the product of interest is associated with growth in the methanol phase. The parameters of the fermentation model obtained are similar to those reported by other researchers.     

Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations.

In the last few years the Pichia pastoris expression system has been gaining more and more interest for the expression of recombinant proteins. Many groups have employed fermentation technology in their investigations because the system is fairly easy to scale up and suitable for the production in the milligram to gram range. A large number of heterologous proteins from different sources has been expressed, but the fermentation process technology has been investigated to a lesser extent. A large number of fermentations are carried out in standard bioreactors that may be insufficiently equipped to meet the demands of high-cell-density fermentations of methylotrophic yeasts. In particular, the lack of on-line methanol analysis leads to fermentation protocols that may impair the optimal expression of the desired products. We have used a commercially available methanol sensor to investigate in detail the effects of supplementary glycerol feeding while maintaining a constant methanol concentration during the induction of a Mut(+) strain of Pichia pastoris. Specific glycerol feed rates in the range of 38-4.2 mg. g(-1). h(-1) (mg glycerol per gram fresh weight per hour) were investigated. Expression of the recombinant scFv antibody fragment was only observed at specific feed rates below 6 mg. g(-1). h(-1). At low specific feed rates, growth was even lower than with methanol as the sole carbon source and the harvest expression level of the scFv was only half of that found in the control fermentation. These results show that glycerol inhibits expression driven by the AOX1 promoter even at extremely limited availability and demonstrate the benefits of on-line methanol control in Pichia fermentation research.     

Enhancement of alkaline phytase production in Pichia pastoris: influence of gene dosage, sequence optimization and expression temperature

Supplementation of animal feed with phytases has proven to be an effective strategy to alleviate phosphorous contamination of soil and water bodies. The inability of non-ruminant animals to digest phytates in corn and soybeans contributes to environmental contamination. Alkaline phytase from lily pollen (LlALP) exhibits unique catalytic and thermal stability properties that could be useful as a feed supplement. rLlALP2 was successfully expressed in Pichia pastoris; however, enzyme yields were modest (8-10 mg/L). In this paper, we describe our efforts to enhance rLlALP2 yield by investigating the influence of the following potential limiting factors: transgene copy number, codon bias, sequence optimization, and temperature during expression. Data presented indicate that increasing rLlAlp2 copy number was detrimental to heterologous expression, clones with one copy of wt-rLlAlp2 produced the highest activity, clones with two, four and seven or more copies produced 70%, 25% and 10% respectively, of enzyme activity implying that gene dosage is not limiting rLlALP2 yield. Use of a sequence-optimized rLlAlp2 increased the yield of the active enzyme by 25-50% in one/two copy clones, suggesting that translational efficiency is not a major bottleneck for rLlALP2 expression. Reducing the temperature during heterologous expression led to increases of 1.2-20-fold suggesting that protein folding and post-translational processes may be the dominant factors limiting rLlALP2 expression. Early knowledge of the transgene copy number allowed us to develop a more rational strategy for yield enhancement. Cumulatively, sequence optimization and temperature reduction led to the doubling of rLlALP2 enzyme activity in P. pastoris.     

毕赤酵母中表达透明颤菌血红蛋白提高重组脂肪酶的表达

解脂耶氏酵母胞外脂肪酶Lip2(YlLip2)是一种具有广泛应用前景的工业酶.为了改善高密度发酵生产Y1Lip2过程中的溶氧限制,提高Y1Lip2的表达量,将YlLip2基因lip2和透明颤菌血红蛋白(VHb)基因vgb分别置于AOXl启动子和PsADH2启动子的调控之下,进行YlLip2和VHb在毕赤酵母中的共表达.PsADH2启动子来源于树干毕赤酵母Pichia stipitis,在低氧条件下能被激活.SDS-PAGE和CO-差式光谱分析表明,Y1Lip2和VHb在重组菌中成功实现了共表达.在氧限制性条件下,VHb表达的细胞(VHb+,GS 115/9Klip2-pZPVT)与对照细胞(VHb-,GS 115/9Klip2)相比,摇瓶和10 L发酵罐中YlLip2表达量分别提高了25％和83％.此外,在低氧条件下,VHb+细胞在10 L发酵罐中的生物量也比VHb-细胞高.文中也获得了一株表达了VHb的并携带有多个lip2基因拷贝的克隆子GS 115/9Klip2-pZP VTlip2 49#,在低氧条件下,该克隆子在10L发酵罐中的最高脂肪酶水解活力达33 900 U/mL.因此,在毕赤酵母中用PsADH2启动子表达VHb,同时增加lip2基因的拷贝数是提高YlLip2表达量的一种有效策略.     

利用游离型表达质粒强化毕赤酵母表达木聚糖酶

巴斯德毕赤酵母是用途广泛的蛋白表达系统。目前用于毕赤酵母的质粒主要以整合型质粒为主,很少见到游离的质粒用于外源基因的表达。文中通过将来源于酵母自身的自主复制序列连接到酵母整合型表达载体pGAP中构成自主复制的游离型表达载体pGAPZαA-PARS,将该载体用于表达木聚糖酶基因。转化毕赤酵母后同传统的整合型表达菌株相比,以甘油为碳源时最高酶活达到343 U/mL,比整合型表达提高了45.9%。同时游离载体表达重组酶比活相对整合表达提高了81.2%。为了节约发酵成本,进一步研究了分别以甘油、葡萄糖、蔗糖、混合碳源(蔗糖︰甘油=1︰2)等不同碳源下游离型重组菌株的表达水平。发现甘油表达水平最高,蔗糖最低,但是以工业葡萄糖为碳源时产酶成本最低。由于pGAP载体不需要以甲醇为碳源,因而文中所构建的游离载体pGAPZαA-PARS极大促进了毕赤酵母在食品行业中的应用。同时,游离型载体可大幅度提高表达水平,为进一步研究提高GAP启动子的高效表达奠定了基础。     

Large-scale production and purification of recombinant Galanthus nivalis agglutinin (GNA) expressed in the methylotrophic yeast Pichia pastoris

The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris. Transformants of P. pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed. GNA was secreted at approximately 80 mg l^–1 at the 200 l scale and was purified to 95% homogeneity using hydrophobic interaction chromatography. The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity.     

Model-based optimization of biosurfactant production in fed-batch culture Azotobacter vinelandii

Fed-batch cultivation of Azotobacter vinelandii 21was optimized for biosurfactant production. Optimization of feed-rate time profile and concentration of nutrient medium components in feeding solution is based on a hybrid mathematical model consisting of mass-balance equations for biomass, biosurfactant, volume of cultural liquid and substrate components: glucose, ammonia nitrogen and phosphate phosphorus. The rate of cultural liquid emulsification activity growth as well as the rates of ammonia nitrogen and phosphate phosphorus consumption is modelled by means of artificial neural network, while the rates of the other biochemical transformations are modelled by adequate kinetic relationships.     

毕赤氏酵母醇氧化酶-2基因启动子突变体的分离和鉴定

巴斯毕赤我苯酵母表达系统已被广泛用于生产外源蛋白的寄主菌。利用该系统将外源基因整合交换到染色体上时,ＡＯＸ１基因被破力的甲醇利用缓慢,给发本报生产千古 定影响。在不改变现有表达系统前提下,从ＡＯＸＩ功能缺陷 株分离出Ｍｕｔ＾＋自发突变化突变体,通过突变体在甲醇培养基中生长曲线的测定,ＨＳＡ表达产物的聚丙烯酰胺凝胶电泳检测,证明突变体的甲醇利用能力和蛋白表达比原始菌株大大提高,突变体ＡＯＸ２基因上游     

Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris

The methylotrophic yeast Pichia pastoris is a powerful system for production of recombinant proteins, showing high ability to secrete properly folded proteins. A major plus is the strong AOX1 promoter highly induced by methanol. During growth on methanol, however, oxygen readily becomes limiting. In oxygen-limited cultivations of recombinant Pichia pastoris, the methanol concentration had a strong impact on the production of a single-chain antibody fragment (scFv). High methanol concentrations were required to compensate the lack of oxygen and fully induce recombinant protein production, at the same time reducing gratuitous biomass formation due to a lower biomass yield. Product concentrations of 60, 150, and 350 mg/L were obtained with methanol concentrations of 0.3, 1, and 3% (v/v). Moreover, accumulation of a putative product fragment that cannot be removed during affinity purification was prevented at high methanol concentrations. Cell vitality after 100 h was maintained above 98% and 96% of the culture with 0.3% and 3% methanol, respectively. In cultivations supplemented with oxygen, in contrast, methanol concentration between 0.3% and 3% did not influence the product yield of 300-400 mg/L. Thus, efficient recombinant protein production under oxygen-limitation seems to require high methanol concentrations, enabling product concentration as high as otherwise obtained only with expensive supply of pure oxygen.     

Single-chain antibody fragment production in Pichia pastoris: Benefits of prolonged pre-induction glycerol feeding

Secretory production of a single-chain antibody fragment (scFv) by recombinant Pichia pastoris using the methanol inducible AOX1 promoter is limited biochemically by retarded secretion, and economically by the high demand for pure oxygen. To address the problem, the adaptation phase with growth-limiting feeding of glycerol before the production phase was optimized. In a standard procedure with a short glycerol-feeding phase before induction, scFv accumulated in the supernatant only after 15 h. Conversely, scFv started to appear immediately in the medium upon methanol induction when the glycerol-feeding phase was extended to 18 h. Interestingly, despite a significantly lower cell density in the cultivation with extended glycerol feeding, the same amount of functional product of 300 mg/L was obtained about 30 h after the start of glycerol feeding with both methods. mRNA analysis revealed that the higher and faster production of the product was related to longer lasting induction of the scFv mRNA. Additional effects of a better adaptation of the secretion machinery may be suggested by higher expression of unfolded protein response-related genes KAR2 and PDI. A clear benefit of the longer glycerol-feeding phase was a 75% reduction of the consumption of both pure oxygen and methanol, and a significantly lower cell density, which would be beneficial for down-stream purification of the product.     

Cyclic fed-batch culture for production of human serum albumin in Pichia pastoris

Simple cyclic fed-batch culture (cfbc), consisting of a constant medium feed with periodic withdrawals of culture, resulted in a product yield (13.4 mg protein per gram biomass) similar to that obtained using the complex multiphase industrial production strategy (13.7 mg protein per gram biomass). In cfbc, productivity was ultimately limited by the rate at which the cells could assimilate methanol. Glycerol was inhibitory to growth at high concentrations. However, product yield continued to increase as the glycerol concentration was increased. In chemostat culture, dissolved oxygen concentration influenced product yield independently of any detectable influence on cell growth.     

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.     

洋葱伯克霍尔德菌脂肪酶的基因改造及其在毕赤酵母中组成型和诱导型的表达

【目的】克隆洋葱伯克霍尔德菌(Burkholderia cepacia)脂肪酶基因,实现其在毕赤酵母中快速、安全和稳定性的大量表达。【方法】首先设计引物扩增B.cepacia脂肪酶基因,然后应用生物信息学方法分析B.cepacia和毕赤酵母整体密码子使用情况、脂肪酶基因信号肽及密码子偏好性。在此基础上,运用overlap PCR对脂肪酶基因中低使用频率密码子进行改造并同时降低基因的G+C含量,获得优化的脂肪酶基因。再分别把原始和优化的脂肪酶基因导入载体pGAPZα和pPIC9K中,获得组成型表达载体pGAPlipW、pGAPlipO和诱导型表达载体pPIClipW、pPIClipO。分别将所得4种载体转入GS115中,得到一系列工程菌。经发酵和NTA树脂纯化后,对脂肪酶的酶学性质进行了初步研究。【结果】4种工程菌的脂肪酶活力分别为pPIClipW37.8U/mL,pPIClipO129.5U/mL,pGAPlipW40.2U/mL和pGAPlipO184.3U/mL。改造后脂肪酶活力比原始脂肪酶提高了4.6倍。酶学性质研究表明,脂肪酶在60℃时活力最高,在40℃-65℃范围内非常稳定;脂肪酶最适pH值为9.0,在pH6.0-pH10.0范围均表现很好的稳定性。【结论】通过overlap PCR改造后的脂肪酶显著提高了其在毕赤酵母中的表达效率,且GAP启动子比AOX1启动子更适合于B.cepacia脂肪酶的表达。大量表达的重组脂肪酶的性质与野生脂肪酶的性质相同,符合生产要求。     

甲醇/山梨醇共混流加控制溶氧改善毕赤酵母表达猪圆环病毒Cap蛋白的发酵性能

研究在重组毕赤酵母(GS115,Mut+)表达猪圆环病毒Cap蛋白的发酵过程中,甲醇毒害作用以及溶氧波动影响目的蛋白的正常表达。基于对甲醇和山梨醇代谢途径的分析,将C源流加的手动调节与反馈控制相结合,提出了1种新型的甲醇/山梨醇共混诱导策略,能够将溶氧稳定地控制于某一设定值,同时避免甲醇毒害作用。使用该策略将溶氧控制于20%的批次,C源(甲醇和山梨醇)添加过少,导致Cap蛋白表达量较低(54 mg/L);而将溶氧控制于10%的批次,C源流加速率适宜,Cap蛋白表达量达到198 mg/L,表达水平明显高于采用传统甲醇诱导策略(0 mg/L)和DO-stat诱导策略的批次(121 mg/L)。     

Transcriptional analysis for carbon metabolism and kinetic modeling for heterologous proteins productions by Pichia pastoris in induction process with methanol/sorbitol co-feeding

It is difficult to control concentrations of methanol/dissolved oxygen at high levels simultaneously in heterologous proteins productions by Pichia pastoris during induction phase. Two strains, a methanol utilization slow (Mut^S) type and a plus (Mut⁺) type were used with methanol/sorbitol co-feeding strategy to induce porcine interferon-α and human serum albumin-human granulocyte colony stimulating factor respectively, under the conditions of “methanol sufficient-oxygen limited (MS-OL)” and “methanol limited-oxygen sufficient (ML-OS)”. For the Mut^S/Mut⁺ strains, the target proteins titers under “MS-OL” were 6-fold/19.2% of those under “ML-OS”. The key genes in methanol metabolism of the Mut^S strain were up-regulated under “MS-OL”, but they were not differently expressed in the Mut⁺ strain. Methanol utilization rate (r_MeOH) of the Mut^S strain reduced when decreasing methanol concentration, but r_MeOH of the Mut⁺ strain unchanged unless methanol concentration decreased to a low-limit of 0.6 g/L. Finally, kinetic models were designed to describe the methanol/sorbitol co-feeding process.     

甲醇/山梨醇共混流加诱导改变毕赤酵母生产猪α干扰素过程的代谢产能途径强化发酵性能

旨在探讨毕赤酵母生产猪α干扰素过程的代谢产能规律及其对发酵性能的影响。在10 L罐下,开展了不同诱导条件下的毕赤酵母高效发酵生产猪α干扰素过程的代谢酶学和能量再生分析研究。结果表明:甲醇单独诱导条件下、将诱导温度从30℃降低到20℃,胞内醇氧化酶(AOX)、甲醛脱氢酶(FLD)和甲酸脱氢酶(FDH)的比活性增加显著,细胞的甲醇代谢和甲醛异化产能能力、猪α干扰素抗病毒活性大幅提高,最高抗病毒活性达到1.4×106IU/mL,约为30℃诱导条件下的10倍。30℃、甲醇/山梨醇共混流加下,主要供能途径由甲醇单独诱导时的甲醛异化代谢转向TCA循环,甲醛异化供能途径被弱化、毒副产物甲醛的生成积累得到抑制,走向目标蛋白合成途径的甲醇分配比例得到提高。此时,最高抗病毒活性达到1.8×107IU/mL,是30℃甲醇单独诱导下最高活性的100倍以上。更加重要的是,共混流加诱导可以在常温、使用空气供氧的条件下进行,发酵成本明显下降、整体发酵性能改善显著。     

Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris

Pichia pastoris, a methylotrophic yeast, is an efficient producer of recombinant proteins in which the heterologous gene is under the control of the methanol-induced AOX1 promoter. Hence, the accepted production procedure has two phases: In the first phase, the yeast utilizes glycerol and biomass is accumulated; in the second phase, the yeast utilizes methanol which is used both as an inducer for the expression of the recombinant protein and as a carbon source. Since the yeast is sensitive to methanol concentration, the methanol is supplied gradually to the growing culture. Three methanol addition strategies were evaluated for the purpose of optimizing recombinant endostatin production. Two strategies were based on the yeast metabolism; one responding to the methanol consumption using a methanol sensor, and the other responding to the oxygen consumption. In these two strategies, the methanol supply is unlimited. The third strategy was based on a predetermined exponential feeding rate, controling the growth rate at 0.02 h(-1), in this strategy the methanol supply is limited. Throughout the induction phase glycerol, in addition to methanol, was continuously added at a rate of 1 g L h(-1). Total endostatin production was similar in all three strategies, (400 mg was obtained from 3 L initial volume), but the amount of methanol added and the biomass produced were lower in the predetermined rate method. This caused the specific production of endostatin per biomass and per methanol to be 2 times higher in the predetermined rate than in the other two methods, making the growth control strategy not only more efficient but also more convenient for downstream processing.