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。     

Lowering induction temperature for enhanced production of polygalacturonate lyase in recombinant Pichia pastoris

Various effects of temperature on heterologous alkaline polygalacturonate lyase produced in recombinant Pichia pastoris were investigated. The results indicated that PGL activity could be improved significantly by decreasing the cultivation temperature. It was reached 931 U/mL with temperature lowered to 22 °C at the beginning of induction phase, which were 2.1-fold and 2.9-fold increase compared to that at 30 and 26 °C. The mechanisms behind the temperature effect on recombinant PGL production may be ascribed to poor cell viability, decrease of intracellular adenosine phosphate levels, of AOX activity but increase of extracellular proteases activities. Our study demonstrated that cultivation at lower temperatures resulted in higher cell viability, significant improvement of PGL stability and an increase intracellular AOX activity, but a lower activity of released host proteases which possibly caused the degradation of recombinant PGL. In addition, the evidence of higher intracellular adenosine phosphate levels but lower energy charge level was provided at a lower temperature induction.     

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。     

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.     

Improved production of alkaline polygalacturonate lyase by homologous overexpression pelA in Bacillus subtilis

A polygalacturonate lyase (PGL), PelA, was purified from the culture broth of Bacillus subtilis 7-3-3, with a molecular weight, optimal temperature, and pH of approximately 45 kDa, 55 °C, and 9.4, respectively. The PGL gene (pelA) was homologously overexpressed in B. subtilis 7-3-3 to increase the gene copies and enhance the PGL production. The resulting PGL activity was 2138 U mL^?1 at 44 h, and the productivity reached 48.58 U (mL h)^?1 through the homologous overexpression of strain B-pN-pelA in a 7.5 L fermentor, the highest PGL production compared to those reported in literature to the best of our knowledge. Crude enzyme has high PGL and PGase activity, which can remove 50.58% of pectin in unpretreatment ramie fibers at 50 °C for 4 h. Meanwhile, the enzyme system with a low level hemicellulase and almost no cellulase will further help in enhancing the efficiency of degumming besides maintaining tenacity of plant fiber. The B. subtilis B-pN-pelA shows high genetic stability and has great potential in the textile industry.     

Improving the monitoring of methanol concentration during high cell density fermentation of Pichia pastoris

The Pichia pastoris expression system is widely used for the production of recombinant proteins. A simple and efficient experimental set-up allowing on-line monitoring of the methanol concentration during the fermentation of P. pastoris based on the detection of the methanol vapor concentration in the exhaust air from fermenter by a tin dioxide (SnO2) semiconductor sensor is described. An experimental procedure to allow precise calibration of the system and to reduce methanol sensor's interferences (>95% reduction) are also presented and discussed. Accuracy and measurement error were estimated about 0.05 g x l(-1) and 6%, respectively. The efficient monitoring of methanol will help to advanced control of recombinant protein production and process optimization.     

Pathway analysis of Pichia pastoris to elucidate methanol metabolism and its regulation for production of recombinant proteins

This research rationally analyzes metabolic pathways of Pichia pastoris to study the metabolic flux responses of this yeast under methanol metabolism. A metabolic model of P. pastoris was constructed and analyzed by elementary mode analysis (EMA). EMA was used to comprehensively identify the cell's metabolic flux profiles and its underlying regulation mechanisms for the production of recombinant proteins from methanol. Change in phenotypes and flux profiles during methanol adaptation with varying feed mixture of glycerol and methanol was examined. EMA identified increasing and decreasing fluxes during the glycerol–methanol metabolic shift, which well agreed with experimental observations supporting the validity of the metabolic network model. Analysis of all the identified pathways also led to the determination of the metabolic capacities as well as the optimum metabolic pathways for recombinant protein synthesis during methanol induction. The network sensitivity analysis revealed that the production of proteins can be improved by manipulating the flux ratios at the pyruvate branch point. In addition, EMA suggested that protein synthesis is optimum under hypoxic culture conditions. The metabolic modeling and analysis presented in this study could potentially form a valuable knowledge base for future research on rational design and optimization of P. pastoris by determining target genes, pathways, and culture conditions for enhanced recombinant protein synthesis. The metabolic pathway analysis is also of considerable value for production of therapeutic proteins by P. pastoris in biopharmaceutical applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:28–37, 2014     

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.     

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.     

重组毕赤酵母产植酸酶发酵条件的优化

为研究优化毕赤酵母工程菌H311产植酸酶的发酵条件,采用单因素试验和L18(37)正交试验考察不同工艺条件对产酶活性的影响。结果表明:影响重组毕赤酵母产植酸酶的因素重要性从大到小依次为诱导时间、甲醇添加量、装液量、初始诱导p H、生长时间、接种量和初始生长p H,产酶最佳条件为接种量3%(体积分数)、装液量20 m L(250 m L摇瓶)、生长时间20 h,诱导时间120 h、甲醇添加量1.5%(体积分数)、生长p H 6.0、诱导p H 5.0,在此条件下进行诱导表达,植酸酶的比酶活可达334 U/m L。     

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。     

碱性果胶酶的生物制造及其在纺织工业清洁生产中的应用研究进展

以下综述了碱性果胶酶的生物制造及其在纺织工业清洁生产中的应用研究进展。微生物发酵法是目前生产碱性果胶酶的主要方式,枯草芽孢杆菌是碱性果胶酶工业发酵生产中效果较好的野生菌株。影响发酵法生产碱性果胶酶的主要因素有:底物浓度及其流加方式、细胞浓度、搅拌转速、通气速率、pH、温度等。构建基因工程菌为碱性果胶酶的发酵生产开辟了一条有效途径,其中重组毕赤酵母的产酶水平最高,在10吨发酵罐上酶活达1305U/mL。碱性果胶酶可用于棉织物前处理的精练工艺,与传统高温碱煮相比,具有保护纤维、提高精练效率、降低能耗和污染的优势。通过分子定向进化技术对碱性果胶酶进行分子改造,使其催化特性更加适合于纺织精练工艺,进而实现纺织工业的清洁生产是未来的研究重点和热点。     

Improvement of recombinant hirudin production by controlling NH4 + concentration in Pichia pastoris fermentation

In recombinant Pichia pastoris fermentation for hirudin production in a 5 l fermenter, a new strategy was explored to match the short fermentation time at low NH4+ concentration with decreased hirudin degradation at high NH4+ concentration. A combination of a defined medium containing initial 0.025 m NH4+ with NH4+ addition up to 0.6 m in the growth phase was achieved in both the improvement of hirudin production and the repression of hirudin degradation. Intact and total hirudin reached 2.63 g l(-1) and 4.25 g l(-1), respectively.     

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.     

Different methanol feeding strategies to recombinant Pichia pastoris cultures producing high level of dextranase

Three different strategies to control the methanol flow rate in cultures of a dextranase-producing Mut clone of the methylotrophic yeast Pichia pastoris were analysed. The cell growth and secretion of dextranase were different for each strategy. The increment of methanol-feeding rate used in the strategy based on dissolved oxygen (DO) in the culture medium gave better results for both cell growth and enzyme secretion, reaching 56.2 g dry biomass and 5.14 g of dextranase per litre of broth.     

Oxygen-limited control of methanol uptake for improved production of a single-chain antibody fragment with recombinant Pichia pastoris

The yeast Pichia pastoris is a suitable production system for recombinant proteins due to its strong methanol-inducible AOX1 promoter. A key parameter of the production process is the specific methanol uptake rate. To control the methanol uptake and simultaneously maintain a constant methanol concentration during the production phase, two strategies were developed to generate purposeful oxygen limitation and to feed-forward control the specific methanol uptake rate into the optimum range. First, the cell density at induction was adjusted by prolonged preinduction glycerol feeding. Alternatively, the airflow rate was restricted and increased in parallel with the biomass. While the product accumulation started 20 h earlier with the first approach, the specific production rate of a single-chain antibody fragment was three times higher in the latter case. After 70 h of production, both schemes yielded product concentrations in the gram-per-liter range. Moreover, they release the requirement for dosage of pure oxygen and thereby can facilitate the scale-up of the production process. The different production profiles indicate that the impact of specific methanol uptake rate on protein production by recombinant P. pastoris depends on the control mode.     

On-line monitoring and control of methanol concentration in shake-flask cultures of Pichia pastoris

The methylotrophic yeast Pichia pastoris can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Accurate regulation of the methanol concentration in P. pastoris cultures is necessary to maintain induction, while preventing accumulation of methanol to cytotoxic levels. We developed an inexpensive methanol sensor that uses a gas-permeable silicone rubber tube immersed in the culture medium and an organic solvent vapor detector. The sensor was used to monitor methanol concentration continuously throughout a fed-batch shake-flask culture of a P. pastoris clone producing the N-lobe of human transferrin. The sensor calibration was stable for the duration of the culture and the output signal accurately reflected the methanol concentration determined off-line by HPLC. A closed-loop control system utilizing this sensor was developed and used to maintain a 0.3% (v/v) methanol concentration in the culture. Use of this system resulted in a fivefold increase in volumetric protein productivity over levels obtained using the conventional fed-batch protocol. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 279-286, 1997.     

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.