碱性果胶酶在重组毕赤酵母中高效表达的关键因素研究

为提高重组毕赤酵母生产碱性果胶酶的产量和生产强度, 在摇瓶条件下优化了重组毕赤酵母生产碱性果胶酶的关键因素。结果表明, 以下条件：初始甘油浓度40 g/L、初始甲醇浓度3.1 g甲醇/g DCW、每24 h添加0.51 g甲醇/g DCW、诱导表达周期72 h、250 mL三角瓶诱导培养基装液量30 mL、初始pH 6.0, 最适于菌体生长与产物表达。在此基础上, 7 L罐上通过恒速流加甘油进一步提高细胞密度, 诱导阶段甲醇采取前期恒速流加和后期DO-stat, 发酵结束菌体干重达80 g/L, 酶活为217 U/mL, 比摇瓶结果提高了66.2%。     

碱性果胶酶在重组毕赤酵母中高效表达的关键因素研究

为提高重组毕赤酵母生产碱性果胶酶的产量和生产强度,在摇瓶条件下优化了重组毕赤酵母生产碱性果胶酶的关键因素。结果表明,以下条件：初始甘油浓度40g／L、初始甲醇浓度3.1g甲醇／gDCW、每24h添加0.51g甲醇／gDCW、诱导表达周期72h、250mL三角瓶诱导培养基装液量30mL、初始pH6,0,最适于菌体生长与产物表达。在此基础上,7L罐上通过恒速流加甘油进一步提高细胞密度,诱导阶段甲醇采取前期恒速流加和后期DO-stat,发酵结束菌体干重达80g／L,酶活为217U／mL,比摇瓶结果提高了66.2％。     

重组毕赤酵母高密度发酵生产碱性果胶酶的策略

重组Pichia pastoris GS115表达碱性果胶酶的诱导阶段, 最佳初始菌体浓度和甲醇诱导浓度分别为122 g/L和20 g/L, 两者之间最佳比值范围是0.16~0.20 g/g (甲醇/菌体浓度). 在此基础上通过生长阶段甘油的指数流加, 以及诱导阶段基于甲醇比消耗速率和溶氧等参数进行甲醇流加的方式, 将甲醇与菌体浓度比例控制在0.171~0.195 g/g之间. 此时, 酶活达到430 u/mL, 生产强度为4.34 u/mL/h, 实现了碱性果胶酶高效生产。     

重组毕赤酵母高密度发酵生产碱性果胶酶的策略

重组Pichia pastoris GS115表达碱性果胶酶的诱导阶段, 最佳初始菌体浓度和甲醇诱导浓度分别为122 g/L和20 g/L, 两者之间最佳比值范围是0.16~0.20 g/g (甲醇/菌体浓度). 在此基础上通过生长阶段甘油的指数流加, 以及诱导阶段基于甲醇比消耗速率和溶氧等参数进行甲醇流加的方式, 将甲醇与菌体浓度比例控制在0.171~0.195 g/g之间. 此时, 酶活达到430 u/mL, 生产强度为4.34 u/mL/h, 实现了碱性果胶酶高效生产。     

恒细胞密度发酵策略提高重组毕赤酵母生产碱性果胶酶的表达效率

为了提高重组毕赤酵母生产碱性果胶酶(Alkaline polygalacturonate lyase,PGL)的比速率,开发了一种新的恒细胞密度发酵策略。通过不同的甲醇流加方式,实现发酵过程细胞密度的合理控制。实验结果表明:控制细胞密度为75 g/L的策略为最优,最终单位发酵液体积生产强度和单位菌体生产强度为6.11 U/(mL.h)和81.5 U/(g.h),分别比传统高密度发酵提高了42.1%和191.2%,最终PGL酶活为441.9 U/mL。此外,该策略还具有提高细胞活性和降低蛋白酶降解作用等优势。     

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。     

双碳源流加对重组毕赤酵母高效表达葡萄糖氧化酶的影响

葡萄糖氧化酶(GOD)是一种具有广泛应用前景的工业酶.为了实现葡萄糖氧化酶的高效生产,提高重组毕赤酵母生产GOD的产量和增强生产强度,对重组毕赤酵母诱导阶段的初始菌体浓度和甲醇浓度进行了优化.在此基础上,诱导期采用了双碳源(甘油、山梨醇和甘露醇)与甲醇混合流加的模式.研究发现,最佳诱导前初始菌体浓度和甲醇浓度分别为100 g/L和18 g/L,此时GOD产量为427.6 U/mL.在诱导阶段采用甘油、山梨醇和甘露醇与甲醇的混合添加均可以提高GOD产量,其中甘露醇与甲醇的混合流加效果最为显著.当甲醇与甘露醇混合流加的比例为20∶1(W/W)时,诱导156h GOD产量和生产强度分别可达711.3 U/mL和4.60 U/(mL·h),比甲醇单一流加策略结果分别提高了66.3％和67.9％.此外采用合适的甘露醇混合流加策略不但不会抑制AOX1启动子的表达,甚至有一定促进作用,AOX酶活性为8.8 U/g(对照为5.2 U/g).双碳源流加方式还能推广到毕赤酵母其他表型中,为该系统高效表达外源蛋白提供一种新策略.     

Overproduction of alkaline polygalacturonate lyase in recombinant Escherichia coli by a two-stage glycerol feeding approach

This work aims to achieve the overproduction of alkaline polygalacturonate lyase (PGL) with recombinant Escherichia coli by a two-stage glycerol feeding approach. First, the PGL coding gene from Bacillus subtilis WSHB04-02 was expressed in E. coli BL21 (DE3) under the strong inducible T7 promoter of the pET20b (+) vector. And then the influence of media composition, induction temperature, and inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration on cell growth and PGL production was investigated. Finally, a two-stage glycerol feeding strategy was proposed and applied in a 3-L fermenter, where cultivation was conducted at a controlled specific growth rate (μset=0.2) during pre-induction phase, followed by a constant glycerol feeding rate of 12 ml h(-1) at post-induction phase. The total PGL yield reached 371.86 U mL(-1), which is the highest PGL production by recombinant E. coli expression system.     

重组毕赤酵母生产β-葡萄糖苷酶发酵条件初步研究

为提高重组毕赤酵母(P.pastoris KM71/pPIC9K-bgl)生产β-葡萄糖苷酶的产量,在摇瓶条件下对重组P.pastoris产β-葡萄糖苷酶的发酵过程进行了优化,得到最佳的条件:生长阶段甘油浓度为30 g/L,接种量为10%,诱导阶段甲醇的初浓度为4%,过程补加甲醇0.5%,诱导温度30℃,pH7.5,诱导周期120 h,酶活可达到245 U/mL。在此基础上,在3 L发酵罐上进行初步放大,流加甘油提高细胞密度至OD_(600)为170,开始流加甲醇诱导,最终BGL酶活达到1 175 U/mL。比摇瓶提高了4.8倍,为β-葡萄糖苷酶工业化生产打下了坚实的基础。     

甘油及甲醇补料策略对毕赤酵母表达猪α-干扰素的影响

本文研究了甘油及甲醇补料策略对重组毕赤酵母细胞生长及猪α-干扰素(p IFN-α)表达的影响。结果表明,甘油采取不同的补料策略,以及甲醇浓度控制于不同水平时,细胞生长速度不同,p IFN-α抗病毒活性水平也明显不同。高密度发酵阶段,甘油采用指数方式流加控制时,相较于40 g/L/h、10 g/L/h,恒速流加组的细胞浓度最先达最高水平132 g/L,后一直保持稳定,且发酵液中几乎无残留甘油;与之相应的p IFN-α抗病毒活性水平最高。诱导表达阶段,甲醇浓度须控制并恒定于12 g/L时,p IFN-α抗病毒活性最高水平能达到5.95×106IU/m L,而当甲醇浓度稳定于3.5 g/L、16.0 g/L、或者浓度波动较大(10.2~13.8 g/L)时,p IFN-α抗病毒活性均远低于最高水平。     

不同甲醇流加策略对重组毕赤醇母高密度发酵生产水蛭素的影响

考察了不同甲醇流加策略对毕赤酵母高密度发酵生产水蛭素的影响。溶氧控制法不能有效地防止甲醇的过量流加。气相色谱离线检测法虽然防止了甲醇流加过量 ,但甲醇浓度的波动较大。利用甲醇传感器在线检测控制甲醇的流加可维持较恒定的甲醇浓度。在流加甲醇的同时 ,以限制性速率流加甘油可以增加表达期间的能量供应 ,提高产物的表达量。经优化后 ,采用甲醇甘油混合流加时细胞干重达到 16 2g L ,水蛭素活性达到 2 4×10 4ATU mL ,即 1 7g L。     

表达血管生长抑制素的重组毕赤酵母在诱导阶段混合碳源的流加

为进行高密度发酵并实现外源基因的高表达,在表型为Mut^S的重组毕赤酵母（Pichia pastoris）表达人血管生长抑制素的诱导阶段,采用了甘油甲醇混合补料的培养方式。以溶氧水平作为甘油代谢指针来控制甘油限制性流加既可维持一定菌体生长,又不会发生发酵液中残余甘油及有害代谢产物（乙醇）阻遏蛋白表达。当表达阶段的菌体平均比生长速率控制于0.012h^-1,菌体浓度达150 g/L,血管生长抑制素浓度最高达到108 mg/L,血管生长抑制素的平均比生产速率为0.02 mg/(g·h),菌体关于甘油的表观得率为0.69 g/g,菌体关于甲醇的表观得率为0.93g/g,较没有采用甘油限制性流加时都有所提高。     

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。     

毕赤酵母发酵产S-腺苷蛋氨酸的甘油-甲醇混合流加策略

在毕赤酵母发酵生产S-腺苷蛋氨酸(SAM)的诱导阶段,以不同甘油-甲醇比例的甘油-甲醇混合培养基进行诱导培养,结果表明以10%(w/v)甘油含量的甘油-甲醇混合培养基进行诱导培养时最有利于SAM的表达,SAM产量达6.09 g/L,比0%甘油含量条件下的SAM产量提高了20.4%。对诱导方式进行优化,先以100%甲醇诱导24 h,然后再连续流加10%(w/v)甘油含量的甘油-甲醇混合培养基,SAM产量可达7.94 g/L,在此基础上,进一步改进诱导方式,SAM产量得到进一步的提高,达到9.80 g/L。     

Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris

The lipases of the Rhizopus species family are important and versatile enzymes that are mainly used in fat and oil modification due to their strong 1,3-regiospecificity. Inexpensive synthetic medium was used for the production of Rhizopus oryzae lipase in the methylotrophic yeast Pichia pastoris. Methanol accumulation inside the bioreactor has previously been shown to negatively influence the production level. Three different methanol fed-batch strategies for maintaining the methanol concentration within optimal limits have been assayed in high-density cultures. One methanol feeding strategy, which is based on the monitoring of the methanol concentration by gas chromatography, resulted in a 2.5-fold higher productivity compared to an initial cultivation, where the feeding rate was adjusted according to the dissolved oxygen concentration (DO) in the supernatant. Finally, productivity could be further increased by introducing a transition phase that involved the simultaneous feeding of glycerol and methanol followed by a single methanol feed. This optimized strategy resulted in the highest productivity (12888 U l(-1) h(-1)), which is 13.6-fold higher than the DO-based strategy.     

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.     

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.     

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.     

费希尔曲霉脂肪酶在毕赤酵母中的优化表达及高密度发酵

【背景】脂肪酶广泛应用于纺织、食品、药品、皮革等工业领域,其在微生物中的异源表达研究进一步促进了脂肪酶产品的生产和应用。【目的】实现来源于费希尔曲霉的脂肪酶在毕赤酵母中的高效异源表达,探究其合适的表达及发酵条件,提高产量,降低成本。【方法】对费希尔曲霉的脂肪酶编码基因进行密码子优化后,应用pPIC9k质粒整合到毕赤酵母GS115基因组上,构建高产脂肪酶Lip605的毕赤酵母工程菌;并通过响应面发酵条件优化、筛选最适伴侣蛋白和高密度发酵相结合的方法,综合提高脂肪酶表达量。【结果】确定高产脂肪酶毕赤酵母工程菌的最优摇瓶发酵产酶条件为:甲醇3.103%(体积比),生物素0.4 mg/L,酵母粉11.5 g/L,酵母基础氮源培养基(yeast nitrogen base,YNB) 13.4 g/L,初始pH 6.4,装液量50 mL/250 mL,转速220 r/min,温度24°C,培养时间40 h。优化后的胞外脂肪酶酶活达到72.34 U/mL,较优化前提高了5.8倍;进一步选择12个伴侣蛋白分别与脂肪酶Lip605进行共表达,其中共表达伴侣蛋白Rpl10(pPICZA-RPL10)效果最佳,可使Lip605表达量进一步提高46.8%;在此基础上,经过10 L发酵罐分批补料的高密度发酵,工程菌株发酵142 h,胞外脂肪酶酶活最高达到680 U/mL,蛋白浓度为15.89 g/L。【结论】应用复合策略有效提高了脂肪酶Lip605在毕赤酵母中的发酵产量,为其进一步工业化生产奠定了良好的基础。     

以葡萄糖为生长相基质的重组毕赤酵母表达植酸酶发酵

甲醇营养型毕赤酵母表达外源蛋白是在醇氧化酶（alcohol oxidase,AOX）启动子（PAOXI）严格调控下进行的,然而这种启动子在转录水平受到葡萄糖的阻遏。本文研究了毕赤酵母在葡萄糖替代甘油为生长相碳源时表达重组植酸酶蛋白的发酵特征。结果表明：初始葡萄糖浓度为20dL的细胞得率高,为0．39g[DCW]／g。通过基于实时参数（溶氧和呼吸商）调控的葡萄糖补料策略,生长相40h后细胞密度达到100g[DCW]／L,甲醇诱导100h后植酸酶产量达到2200FTUphytase／mL,甲醇得率系数为0．25FTU phytase／gmethnol。因此,在毕赤酵母高表达重组蛋白培养中葡萄糖能够用作生长相基质,并能实现重组蛋白的高效表达。