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。     

甲醇周期诱导控制强化毕赤酵母生产猪α干扰素

利用甲醇营养型毕赤酵母生产猪α干扰素(pIFN-α),诱导过程一般在高细胞密度、定值控制甲醇浓度于5～10g/L下进行,此时、溶解氧浓度(DO)自然下降到接近于0的水平。如果高好氧的毕赤酵母长期处在高甲醇/低DO的诱导浓度环境会导致其代谢活性恶化,胞内甲醇积累严重,pIFN-α表达生产效率低。为此,提出了一种甲醇周期诱导控制策略来强化pIFN-α生产。先将甲醇控制于高浓度达7h,再降低甲醇流加速率,将DO控制在20%左右约4h,一共重复6个循环。采用上述周期控制策略,毕赤酵母代谢活性可以长期维持在较高水平;胞内甲醇处于极低水平(≤0. 003g/g DCW),解除了甲醇毒性效应; pIFN-α活性达到3. 90×10~7IU/ml的最高水平,是定值控制甲醇浓度时的1. 86倍。     

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)。     

猪干扰素-γ基因在毕赤酵母中的分泌表达

将去除信号肽的猪干扰素-γ（PoIFN-γ）基因置于酿酒酵母α因子分泌信号的DNA序列后, 构建成pPIC9K-α-PoIFN-γ分泌型重组表达载体, 电转化导入毕赤酵母GS115中,经G418筛选后获得2株多拷贝插入的重组子。SDS-PAGE和Western blot分析结果表明,所获得的重组子能够分泌表达出17kD和23kD左右的PoIFN-γ特异蛋白,其表达量为108mg/L,占培养液总蛋白的60%。实验首次在毕赤酵母表达系统中实现了PoIFN-γ基因的分泌表达。     

毕赤酵母表达猪干扰素-γ基因及其抑制蓝耳病病毒效果

为了研究和应用猪重组干扰素-γ（rPoIFN-γ）预防和治疗病毒性疫病,将大白猪PoIFN-γ基因插入到酵母整合载体pHIL-S1、构建了重组GS115工程菌（pHIL-S1/rPoIFN-γ）。经过SDSPAGE、Western blot分析和抗滤泡性口炎病毒（VSV）活性测定,证实rPoIFN-γ分子量为18 kD,在GS115中的表达量为18%;其抗VSV活性为450～540 u/mL。用rPoIFNγ处理猪肺巨噬细胞系Marc145后,经细胞病变抑制法（CPE50）测定,rPoIFNγ可以抵抗蓝耳病病毒（PRRSV）感染。结果显示酵母表达的rPoIFN-γ是有应用价值的抗病毒生物工程制剂。     

呼吸商监控毕赤酵母表达猪α干扰素活性水平的研究

在5L发酵罐下开展毕赤酵母流加培养表达pIFN-α研究。通过多批次研究,结果表明：诱导阶段,RQ稳定情况可以作为反映底物甲醇浓度水平是否合适以及pIFN-α活性高低的监控指标。当甲醇浓度在8～12g/L水平时,RQ在整个诱导阶段能稳定在0.4～0.5之间某个特定值上,同时pIFN-α最高活性都能维持在106IU/mL水平上;而当甲醇浓度在0～5g/L或15～20g/L范围内时,RQ很不稳定,分别在0.15～0.4和0.4～0.8区间上下振动,相应的pIFN-α最高活性也只在10^3IU/mL和10^4IU/mL水平上。     

发酵条件对毕赤酵母表达重组人干扰素ω糖基化的影响

发酵条件是影响毕赤酵母 (P .pastoris)表达外源重组糖蛋白时糖基化的重要因素。通过菌体浓度、起始pH值、甲醇诱导浓度和周期、装液量等摇瓶发酵实验 ,研究不同发酵条件对毕赤酵母表达分泌型重组人干扰素ω(rhIFNω)过程中糖基化的影响 ;同时 ,在连续培养过程中考察pH值变化对rhIFNω糖基化的影响和分批发酵过程中rhIFNω糖基化的变化。结果表明 ,控制菌体密度 250g L(WCW)、起始pH值 6 0、装液量小于 30mL、甲醇诱导浓度 15g L、甲醇诱导 3次 (每 24h诱导一次 )等发酵条件 ,有利于摇瓶发酵过程中rhIFNω的糖基化 ;控制pH值 70～75可促进rhIFNω的糖基化 ;分批发酵过程中 ,糖基化与非糖基化rhIFNω的含量有同比变化趋势 ,但糖基化rhIFNω所占比例明显低于摇瓶发酵实验的结果 ,其原因有待进一步研究。     

降低诱导温度提高毕赤酵母甲醇代谢关键酶基因转录水平和猪-α干扰素生产性能

以一株表达猪-α干扰素(pIFN-α)重组毕赤酵母为研究对象,考察了诱导温度对pIFN-α表达、细胞代谢活性及甲醇代谢关键酶的影响.在5 L发酵罐开展毕赤酵母高密度流加培养生产表达pIFN-α研究,利用实时PCR技术对不同诱导温度、诱导稳定期的胞内甲醇代谢关键酶(包括解毒酶)基因转录水平进行定量分析.低温(20℃)可以...     

毕赤酵母表达猪干扰素—γ基因及其抑制蓝耳病毒效果

为了研究和应用猪重组干扰素-γ（rPoIFN-γ)预防和治疗病毒性疫苗,将大白猪PoIFN-γ基因插入到酵母整合载体pHIL-S1,构建了重组GS115工程菌（pHIL-S1/rPoIFN-γ)。经过SDS－PAGE,Western blot分析和抗滤泡性口炎病毒（VSV）活性测定,证实了rPoIFN-γ分子量为18kD,在GS115中的表达量为18％。其抗VSV活性为450－540u/mL。用rPoIFN-γ处理猪肺巨噬细胞系Marc-145后,经细胞病变抑制法（CPE50）测定,rPoIFN-γ可以抑抗蓝耳病病毒（PRRSV）感染。结果显示酵母表达的rPoIFN-γ是有应用价值的抗病毒生物工程制剂。     

重组毕赤酵母产猪IFNα的诱导条件

采用Plackett-Burman实验设计,选取甲醇、酵母粉、蛋白胨、K2HPO4.12H2O、MgSO4、(NH4)2SO4、pH和PTM1这8个对重组猪IFNα表达量有影响的相关因子进行筛选,用SAS软件进行统计分析,分析表明:甲醇、蛋白胨以及K2HPO4.12H2O对重组猪IFNα表达量具有显著性影响;之后采用Box-Behnken设计及响应面分析法确定最佳的诱导条件。在最优条件下,重组猪IFNα表达量比优化前提高了2.46倍。     

Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rate

The intracellular metabolic fluxes through the central carbon pathways in the bioprocess for recombinant human erythropoietin (rHuEPO) production by Pichia pastoris (Mut⁺) were calculated to investigate the metabolic effects of dual carbon sources (methanol/sorbitol) and the methanol feed rate, and to obtain a deeper understanding of the regulatory circuitry of P. pastoris, using the established stoichiometry‐based model containing 102 metabolites and 141 reaction fluxes. Four fed‐batch operations with (MS‐) and without (M‐) sorbitol were performed at three different constant specific growth rates (h^?1), and denoted as M‐0.03, MS‐0.02, MS‐0.03, and MS‐0.04. Considering the methanol consumption pathway, the M‐0.03 and MS‐0.02 conditions produced similar effects and had >85% of formaldehyde flux towards the assimilatory pathway. In contrast, the use of the dual carbon source condition generated a shift in metabolism towards the dissimilatory pathway that corresponded to the shift in dilution rate from MS‐0.03 to MS‐0.04, indicating that the methanol feed exceeded the metabolic requirements at the higher µ₀. Comparing M‐0.03 and MS‐0.03 conditions, which had the same methanol feeding rates, sorbitol addition increased the rHuEPO synthetic flux 4.4‐fold. The glycolysis, gluconeogenesis, and PPP pathways worked uninterruptedly only at MS‐0.02 condition. PPP and TCA cycles worked with the highest disturbances at MS‐0.04 condition, which shows the stress of increased feeding rates of methanol on cell metabolism. Biotechnol. Bioeng. 2010; 105: 317–329. © 2009 Wiley Periodicals, Inc.     

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.     

采用混合策略提高葡萄糖氧化酶在毕赤酵母中的表达水平

为了提高葡萄糖氧化酶 (GOD) 在毕赤酵母中的表达水平,提出了甲醇/山梨醇混合碳源诱导和共表达分子伴侣二硫键异构酶 (PDI) 和透明颤菌血红蛋白 (VHb) 两种策略。利用对照菌株X33/pPIC9k–GOD 在5 L发酵罐放大培养时,采用甲醇/山梨醇混合碳源诱导,GOD最终酶活为456 U/mL,比只采用甲醇作为单一碳源诱导时GOD最终酶活提高了20%。利用整合伴侣蛋白菌株X33/pPIC9k-GOD/pPICZ-PDI-VHb在5 L发酵罐进行高密度发酵,采用甲醇/山梨醇混合碳源诱导,GOD最终酶活达到716 U/mL,蛋白浓度为7.4 g/L。研究结果对提高外源蛋白在毕赤酵母中的表达有重要参考价值。     

不同猪胰岛素前体基因拷贝数Mut~+型毕赤酵母的摇瓶发酵研究

毕赤酵母是优秀的外源蛋白的表达系统之一。本文对Mut~+型的不同PIP基因拷贝数的毕赤酵母进行了摇瓶试验。研究了生长特性以及对外源蛋白表达量的影响等。发现了低拷贝和高拷贝的蛋白表达量、生长情况有差异。G12重组菌的PIP表达量最高为181.6mg/L,是单拷贝重组菌表达量的12.6倍。对于基因拷贝数低于12的菌株,PIP表达水平与PIP基因拷贝数成线性关系(r=0.996)。     

发酵重组Pichia pastoris生产腺苷甲硫氨酸的研究

在5L发酵罐中对高产S腺苷甲硫氨酸的重组Pichia pastoris发酵进行了研究。重组菌在pH5．0生长,然后调为pH6．0积累腺苷甲硫氨酸,在30℃、溶氧5％及流加甲硫氨酸和尿素的条件下培养82h后,产量达4．3g／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.     

Metabolic engineering of Pichia pastoris for malic acid production from methanol

The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP‐CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45‐fold increase compared to the parent strain. To improve the efficiency of site‐directed gene knockout, NHEJ‐related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to by‐product generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose‐6‐phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value‐added chemicals from methanol.