共查询到20条相似文献,搜索用时 31 毫秒
1.
To improve the specific production rate of Rhizopus oryzae lipase (ROL) in Pichia pastoris, a protein that triggers the unfolded protein response in P. pastoris, the effect of sorbitol/methanol mixed substrates was tested in batch and fed-batch cultures. Remarkably, a different substrate consumption behaviour was observed depending on the host's phenotype (Mut(+) or Mut(s)) in batch cultures: when the methanol assimilation capacity is genetically reduced (Mut(s) phenotype), both substrates were consumed simultaneously, allowing not only a higher specific growth rate but also higher lipase levels (8.7-fold) compared to those obtained by cells growing on methanol as a sole carbon source in batch culture. This effect was not observed in Mut(+) phenotype, where the two substrates were consumed sequentially and the levels of heterologous product were only slightly higher (1.7-fold). A mixed substrate strategy was also applied to a Mut(s) fed-batch culture at a low methanol concentration set-point (0.5 gl(-1)). This resulted in a 2.2-fold increase in the heterologous protein level achieved, compared with the methanol-only feeding strategy. In addition, sorbitol co-feeding permitted the achievement of higher specific growth rates, and avoided the drastic decrease of the specific production rate observed after the start of the induction phase when methanol was used as sole carbon source This resulted in a significant increase in the overall bioprocess volumetric productivity (2.2-fold) and specific productivity (1.7-fold). Moreover, whereas increased ROL gene dosage in Mut(s) strains have been previously reported to be deleterious for P. pastoris cells growing on methanol, sorbitol co-feeding allowed for sustained cell growth and lipase production. 相似文献
2.
Polygalacturonate lyase (PGL) production by Pichia pastoris GS115 was used as a model to study the mechanism and strategy for enhancing heterologous protein production. It was found that the ratio of methanol to cell concentration had a significant influence on PGL production. In this study, an advanced glycerol exponential feeding strategy was developed for biomass accumulation in cell growth phase, by which cell concentration reached 140 g L(-1) after 19 h glycerol feeding. In subsequent production phase, a methanol feeding profile was proposed according to the optimal ratio of methanol to cell concentration at a range of 0.163-0.171 g g(-1), and PGL activity and productivity reached 430 U mL(-1) and 4.34 U mL(-1)h(-1), respectively. The strategy for enhancing PGL production by controlling the optimal ratio may provide an alternative approach to enhance heterologous protein production with P. pastoris. 相似文献
3.
Effect of dilution rate and methanol‐glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastoris Mut+ phenotype in continuous culture
下载免费PDF全文
![点击此处可从《Biotechnology progress》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The induction using substrate mixtures is an operational strategy for improving the productivity of heterologous protein production with Pichia pastoris. Glycerol as a cosubstrate allows for growth at a higher specific growth rate, but also has been reported to be repressor of the expression from the AOX1 promoter. Thus, further insights about the effects of glycerol are required for designing the induction stage with mixed substrates. The production of Rhizopus oryzae lipase (ROL) was used as a model system to investigate the application of methanol‐glycerol feeding mixtures in fast metabolizing methanol phenotype. Cultures were performed in a simple chemostat system and the response surface methodology was used for the evaluation of both dilution rate and methanol‐glycerol feeding composition as experimental factors. Our results indicate that productivity and yield of ROL are strongly affected by dilution rate, with no interaction effect between the involved factors. Productivity showed the highest value around 0.04–0.06 h?1, while ROL yield decreased along the whole dilution rate range evaluated (0.03–0.1 h?1). Compared to production level achieved with methanol‐only feeding, the highest specific productivity was similar in mixed feeding (0.9 UA g‐biomass?1 h?1), but volumetric productivity was 70% higher. Kinetic analysis showed that these results are explained by the effects of dilution rate on specific methanol uptake rate, instead of a repressor effect caused by glycerol feeding. It is concluded that despite the effect of dilution rate on ROL yield, mixed feeding strategy is a proper process option to be applied to P. pastoris Mut+ phenotype for heterologous protein production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:707–714, 2015 相似文献
4.
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 总被引:4,自引:0,他引:4
为进行高密度发酵并实现外源基因的高表达,在表型为MutS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。 相似文献
5.
Sang-Min Jung Yong-Cheol Park Kyungmoon Park 《Biotechnology and Bioprocess Engineering》2010,15(4):614-619
Methanol is a commonly used acyl acceptor for lipase-driven biodiesel production, but a high concentration of methanol is
detrimental for lipase activity. To overcome this drawback, a simple fed-batch process was developed by optimization of the
methanol feeding strategy and reaction conditions. For the feeding strategy, an equal volume of pure methanol was fed twice
with specified time intervals into a reactor initially containing a 1:1 molar ratio of soybean oil to methanol in order to
adjust the net molar ratio of the oil to methanol to 1:3. In contrast with the batch reaction, a higher agitation speed in
the fed-batch process elevated the conversion yield of soybean oil to biodiesel. An agitation speed of 600 rpm and a reaction
temperature of 70°C were chosen as the optimal environmental conditions. Residual lipase activities for the fed-batch operation
at 40 ∼ 70°C and 600 rpm were 7.1 ± 1.4 times higher than that of the batch method at 40°C with the same agitation speed,
indicating that methanol feeding can prevent significant deactivation of lipase. Finally, two times feeding methanol at 2
and 6 hr resulted in a biodiesel productivity of 10.7%/h and 94.9% final conversion yield under the optimal conditions. 相似文献
6.
A rational approach to improving productivity in recombinant Pichia pastoris fermentation 总被引:6,自引:0,他引:6
A Mut(S) Pichia pastoris strain that had been genetically modified to produce and secrete sea raven antifreeze protein was used as a model system to demonstrate the implementation of a rational, model-based approach to improve process productivity. A set of glycerol/methanol mixed-feed continuous stirred-tank reactor (CSTR) experiments was performed at the 5-L scale to characterize the relationship between the specific growth rate and the cell yield on methanol, the specific methanol consumption rate, the specific recombinant protein formation rate, and the productivity based on secreted protein levels. The range of dilution rates studied was 0. 01 to 0.10 h(-1), and the residual methanol concentration was kept constant at approximately 2 g/L (below the inhibitory level). With the assumption that the cell yield on glycerol was constant, the cell yield on methanol increased from approximately 0.5 to 1.5 over the range studied. A maximum specific methanol consumption rate of 20 mg/g. h was achieved at a dilution rate of 0.06 h(-1). The specific product formation rate and the volumetric productivity based on product continued to increase over the range of dilution rates studied, and the maximum values were 0.06 mg/g. h and 1.7 mg/L. h, respectively. Therefore, no evidence of repression by glycerol was observed over this range, and operating at the highest dilution rate studied maximized productivity. Fed-batch mass balance equations, based on Monod-type kinetics and parameters derived from data collected during the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential feeding strategy using two carbon sources. Two exponential fed-batch fermentations were conducted according to the predicted feeding strategy at specific growth rates of 0.03 h(-1) and 0.07 h(-1) to verify the accuracy of the model. Cell growth was accurately predicted in both fed-batch runs; however, the model underestimated recombinant product concentration. The overall volumetric productivity of both runs was approximately 2.2 mg/L. h, representing a tenfold increase in the productivity compared with a heuristic feeding strategy. 相似文献
7.
A predictive control algorithm coupled with a PI feedback controller has been satisfactorily implemented in the heterologous Rhizopus oryzae lipase production by Pichia pastoris methanol utilization slow (Mut(s)) phenotype. This control algorithm has allowed the study of the effect of methanol concentration, ranging from 0.5 to 1.75 g/L, on heterologous protein production. The maximal lipolytic activity (490 UA/mL), specific yield (11,236 UA/g(biomass)), productivity (4,901 UA/L . h), and specific productivity (112 UA/g(biomass)h were reached for a methanol concentration of 1 g/L. These parameters are almost double than those obtained with a manual control at a similar methanol set-point. The study of the specific growth, consumption, and production rates showed different patterns for these rates depending on the methanol concentration set-point. Results obtained have shown the need of implementing a robust control scheme when reproducible quality and productivity are sought. It has been demonstrated that the model-based control proposed here is a very efficient, robust, and easy-to-implement strategy from an industrial application point of view. 相似文献
8.
Calado CR Ferreira BS da Fonseca MM Cabral JM Fonseca LP 《Journal of biotechnology》2004,109(1-2):147-158
By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterologous cutinase directly from the crude feedstock. However, it was observed that in a highly condensed and consequently economically advantageous purification process as EBA, the cultivation step highly influences the following purification step. Thus, the yeast cultivation and cutinase purification by EBA cannot be considered as independent entities, and the understanding of the interactions between them are crucial for the development of a highly cost effective overall cutinase production process. From the cultivation strategies studied, one batch, one continuous and two fed-batch cultivations, the strategy that resulted in a more economical cutinase overall production process was a fed-batch mode with a feeding in galactose. This last cultivation strategy, exhibited the highest culture cutinase activity and bioreactor productivity, being obtained 3.8-fold higher cutinase activity and 3.0-fold higher productivity that could compensate the 40% higher cultivation medium costs when compared with a fed-batch culture with a feeding on glucose and galactose. Moreover, a 3.8-fold higher effective cutinase dynamic adsorption capacity and 3.8-fold higher effective purification productivity were obtained in relation to the fed-batch culture with the feeding on glucose and galactose. The cultivation strategy with a feeding on galactose, that presented 5.6-fold higher effective purification productivity, could also compensate the 32% effective adsorption capacity obtained with a continuous cultivation broth. Furthermore, a 205-fold higher cutinase activity, 24-fold higher bioreactor productivity and 6% of the cultivation medium costs were obtained in relation to the continuous culture. 相似文献
9.
A highly efficient bioprocessing strategy was developed for enhancing the production of plant secondary metabolites by repeatedly eliciting a fed-batch culture with a newly synthesized powerful jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA). In suspension cultures of a high taxuyunnanine C (Tc)-producing cell line of Taxus chinensis, 100 microM DHPJA was added on day 7 to fed-batch cultures with feeding of 20 g L(-1) sucrose on the same day. The synergistic effect of elicitation and substrate feeding on Tc biosynthesis was observed, which resulted in higher Tc accumulation than that by elicitation or sucrose feeding alone. More interestingly, both specific Tc yield (i.e., Tc content) and volumetric yield was further improved by a second addition of 100 microM DHPJA (on day 12) to the fed-batch cultures. In particular, with repeated elicitation and sucrose feeding the Tc volumetric yield was increased to 827 +/- 29 mg L(-1), which was 5.4-fold higher than that of the nonelicited batch culture. Furthermore, the above novel strategy was successfully applied from shake flask to a 1-L airlift bioreactor. A high Tc production and productivity of 738 +/- 41 mg L(-1) and 33.2 +/- 1.9 mg L(-1) d(-1), respectively, was achieved, which is higher than previous reports on Tc production in bioreactors. The results suggest that the aforementioned bioprocessing strategy may potentially be applied to other cell culture systems for efficient production of plant secondary metabolites. 相似文献
10.
Production of a Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies
Surribas A Stahn R Montesinos JL Enfors SO Valero F Jahic M 《Journal of biotechnology》2007,130(3):291-299
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. 相似文献
11.
Effects of temperature and glycerol and methanol‐feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris
下载免费PDF全文
![点击此处可从《Biotechnology progress》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Optimization of protein production from methanol‐induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol‐feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut+ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5‐fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. © 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728–735, 2014 相似文献
12.
Cos O Serrano A Montesinos JL Ferrer P Cregg JM Valero F 《Journal of biotechnology》2005,116(4):321-335
An important number of heterologous proteins have been produced in the methylotrophic yeast Pichia pastoris using the alcohol oxidase promoter. Two factors that drastically influence protein production and cultivation process development in this system are gene dosage and methanol assimilation capacity of the host strain (Mut phenotype). Using a battery of four strains which secrete a Rhizopus oryzae lipase (ROL), the combined effects of gene dosage and Mut phenotype on recombinant protein production in Pichia pastoris was studied in fed-batch cultures. Regarding the effect of phenotype, the specific productivity and the Y(P/X) were 1.29- and 2.34-fold higher for Mut(s)ROL single copy strain than for Mut+ROL single copy strain. On the contrary, the productivity of Mut+ROL single copy strain was 1.34-fold higher than Mut(s)ROL single copy strain. An increase in ROL gene dosage seems to negatively affect cell's performance in bioreactor cultures, particularly in Mut(s) strains. Overall, the Mut(s) strain may be still advantageous to use because it allows for easier process control strategies. 相似文献
13.
AIMS: Investigation of the phosphate effect and feeding strategy, i.e. linear and exponential feeding, to improve beta-lactamase production by Bacillus licheniformis considering the viability of the cells. METHODS AND RESULTS: Effect of phosphate enrichment on beta-lactamase production was investigated and resulted in 1.2-fold increase in beta-lactamase activity. Thereafter, exponential and linear feed profiles were established, after an initial batch phase for t = 0-7.5 h. The highest beta-lactamase activity was obtained at fed-batch operation with exponential feeding (FBO1) condition as A = 106 U cm(-3), which is c. 1.7-fold higher than that of the phosphate-enriched batch operation (PE-BO). CONCLUSIONS: Biphasic variations in beta-lactamase production was enhanced to monophasic variation with the exponential feeding strategy where the activity was obtained as A = 106 U cm(-3) at t = 16 h. SIGNIFICANCE AND IMPACT OF THE STUDY: Phosphate enrichment decreases the intracellular ammonium concentration and organic acid excretion, but increrases beta-lactamase production. When batch operation (BO) and PE-BO are compared, it is seen that succinic acid formation decreased with the phosphate enrichment as a result of smooth operation of the tricarboxylic acid cycle. At FBO1 despite the increased lactic and acetic acid formation, beta-lactamase production increased 1.7-fold, and 92% of the cells were alive at the end of the fermentation. 相似文献
14.
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。 相似文献
15.
Dissolved-oxygen-stat controlling two variables for methanol induction of rGuamerin in Pichia pastoris and its application to repeated fed-batch 总被引:3,自引:0,他引:3
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. 相似文献
16.
Stationary phase cell suspension cultures of Agrobacterium tumefaciens transformed Lithospermum erythrorhizon respond to additions of sucrose-rich (C-rich) medium with a 2-3-fold increase in the accumulation of shikonin derivatives and a 3-3.5-fold increase in the accumulation of soluble phenolics while showing a modest (10-30%) increase in cell concentration. Conversely, the addition of nitrate-rich (N-rich) medium resulted in 25-35% increase in biomass concentration but only 2-9% increase in shikonin production and approximately 3% increase in the yield of soluble phenolics. Repeated additions of C-rich medium resulted in only a modest (less than 10%) improvement in shikonin production over the levels obtained after the first application. No obvious correlation could be discerned between intracellular ATP levels or protein synthesis patterns and the pattern of shikonin accumulation following the addition of C-rich medium, suggesting that the precursor diversion mechanism is not generally applicable in our cell line. It was found that alternating feeding of N-rich and C-rich media could be used as an effective strategy for enhancing the productivity of plant secondary metabolite. (c) 1993 John Wiley & Sons, Inc. 相似文献
17.
Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris 总被引:8,自引:0,他引:8
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. 相似文献
18.
Jin-li Tao Xue-dong Wang Ya-ling Shen Dong-zhi Wei 《World journal of microbiology & biotechnology》2005,21(6-7):969-972
A Serratia marcescens mutant for prodigiosin production was obtained by u.v. mutation with rational screening methods and a two-step feeding strategy
was used to increase its productivity. In flasks, the mutant strain B6 gave a 2.8-fold higher prodigiosin production than
that of the parent strain with glycerol as a carbon source. In a 5-l bioreactor, with a two-step feeding strategy in which
glucose was selected as the initial carbon source in the fermentation media and glycerol was fed as a ‘prodigiosin inducer’,
it gave a 7.8 times higher prodigiosin production (583 mg/l) than the parent stain with the original cultivation mode. 相似文献
19.
Mohammad Reza Kazemali Keivan Majidzadeh Soroush Sardari Amir Hossein Saadati Farzaneh Barkhordari Ahmad Adeli Fereidoun Mahboudi Amir Maghsoudi 《Bioprocess and biosystems engineering》2016,39(4):565-573
Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µ set glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µ set glycerol and that of methanol to be 0.05 and 0.03 h?1, respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained. 相似文献
20.
Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process 总被引:3,自引:0,他引:3
Alfenore S Molina-Jouve C Guillouet SE Uribelarrea JL Goma G Benbadis L 《Applied microbiology and biotechnology》2002,60(1-2):67-72
Several bottlenecks in the alcoholic fermentation process must be overcome to reach a very high and competitive performance of bioethanol production by the yeast Saccharomyces cerevisiae. In this paper, a nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30 degrees C. This performance was achieved by implementing exponential feeding of vitamins throughout the fermentation process. In comparison to an initial addition of a vitamin cocktail, an increase in the amount of vitamins and an exponential vitamin feeding strategy improved the final ethanol titre from 126 g l(-1) to 135 g l(-1) and 147 g l(-1), respectively. A maximum instantaneous productivity of 9.5 g l(-1) h(-1) was reached in the best fermentation. These performances resulted from improvements in growth, the specific ethanol production rate, and the concentration of viable cells in response to the nutritional strategy. 相似文献