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1.
Kinetics of inclusion body production in batch and high cell density fed-batch culture of Escherichia coli expressing ovine growth hormone. 总被引:3,自引:0,他引:3
A process for maximizing the volumetric productivity of recombinant ovine growth hormone (r-oGH) expressed in Escherichia coli during high cell density fermentation process has been devised. Kinetics of r-oGH expression as inclusion bodies and its effect on specific growth rates of E. coli cells were monitored during batch fermentation process. It was observed that during r-oGH expression in E. coli, the specific growth rate of the culture became an intrinsic property of the cells which reduced in a programmed manner upon induction. Nutrient feeding during protein expression phase of the fed-batch process was designed according to the reduction in specific growth rate of the culture. By feeding yeast extract along with glucose during fed-batch operation, high cell growth with very little accumulation of acetic acid was observed. Use of yeast extract helped in maintaining high specific cellular protein yield which resulted in high volumetric productivity of r-oGH. In 16 h of fed-batch fermentation, 3.2 g l-1 of r-oGH were produced at a cell OD of 124. This is the highest concentration of r-oGH reported to date using E. coli expression system. The volumetric productivity of r-oGH was 0.2 g l-1 h-1, which is also the highest value reported for any therapeutic protein using IPTG inducible expression system in a single stage fed-batch process. 相似文献
2.
Jung Hun Kim Seon-Won Kim Do Quynh Anh Nguyen He Li Sung Bae Kim Yang-Gon Seo Jae-Kyung Yang In-Young Chung Dae Hwan Kim Chang-Joon Kim 《Biotechnology and Bioprocess Engineering》2009,14(5):559-564
Recombinant Escherichia coli engineered to contain the whole mevalonate pathway and foreign genes for β-carotene biosynthesis, was utilized for production
of β-carotene in bioreactor cultures. Optimum culture conditions were established in batch and pH-stat fed-batch cultures
to determine the optimal feeding strategy thereby improving production yield. The specific growth rate and volumetric productivity
in batch cultures at 37°C were 1.7-fold and 2-fold higher, respectively, than those at 28°C. Glycerol was superior to glucose
as a carbon source. Maximum β-carotene production (titer of 663 mg/L and overall volumetric productivity of 24.6 mg/L × h)
resulted from the simultaneous addition of 500 g/L glycerol and 50 g/L yeast extract in pH-stat fed-batch culture. 相似文献
3.
Optimization of fusion proinsulin production by high cell-density fermentation of recombinantE. coli
Cheon-Soon Bae Min-Sun Hong Seung-Gu Chang Dae-Young Kim Hang-Cheol Shin 《Biotechnology and Bioprocess Engineering》1997,2(1):27-32
The optimum conditions for mass production of fusion proinsulin were studied in recombinantEscherichia coli strain BL21 (DE3) [pT7-PI] using fed-batch culture employing pH-stat method. Yeast extract was found to enhance both the growth rate of recombinantE. coli strain BL21 (DE3) [pT7-PI] and its cell mass yield. When the glucose concentration was 10 g/L in the initial medium, 10 g/L concentration of yeast extract was found to be optimal to control the acetate production and to augment both the cell mass yield and the growth rate. Optimum ratio of glucose to yeast extract to minimize the cost of the feeding medium in the fed-batch culture was calculated to be 1.225 and verified by the subsequent experiments. The appropriate inducer concentration and induction time were examined with isopropyl-β-D-thiogalactopyranoside (IPTG). Irrespective of the induction time, IPTG induction resulted in the reduction of growth rate, but the expression level of the fusion protein was maintained at the level of about 20% of the total proteins. Since the volumetric productivity was well maintained in the range between 0.15 and 0.18 g/L.hr at the inducer concentration of above 0.025 mM, the appropriate inducer concentration, in relation to the inducer cost, is considered to be about 0.025 mM. 相似文献
4.
Mixed-feed exponential feeding for fed-batch culture of recombinant methylotrophic yeast 总被引:2,自引:0,他引:2
A simple, accurate model capable of predicting cell growth and methanol utilization during the mixed substrate fed-batch fermentation of MutS recombinant Pichia pastoris was developed and was used to design an exponential feeding strategy for mixed substrate fed-batch fermentation at a constant specific growth rate. Mixed substrate feeding has been shown to boost productivity in recombinant fed-batch culture of P. pastoris, while fixed growth rate exponential feeding during fed-batch culture is a useful tool in process optimization and control. 相似文献
5.
为了评价虾青素高产菌株-法夫酵母JMU-MVP14的生产性能及建立虾青素高产发酵技术,通过测定糖、生物量、虾青素产量、总类胡萝卜素产量等发酵参数,用摇瓶试验对比了法夫酵母JMU-MVP14和出发菌株的差异,用7 L罐试验对比了pH值调控方式及补料培养基成分对发酵的影响,用1 m3罐试验评估了法夫酵母JMU-MVP14高密度发酵虾青素的产量水平。摇瓶发酵结果表明,法夫酵母JMU-MVP14虾青素及总类胡萝卜素的细胞产率分别达到6.01 mg/g及10.38 mg/g;7 L罐分批发酵试验结果表明,自动流加调 相似文献
6.
Abstract Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32°C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system. 相似文献
7.
Jeong-Woo Choi Young-Kee Kim Won Hong Lee Henrik Pedersen Chee-Kok Chin 《Biotechnology and Bioprocess Engineering》1999,4(2):138-146
Optimal substrate feeding strategy in bioreactor operation was investigated to increase the production of secondary metabolite
in a high density culture of plant cell. It was accomplished by the previously proposed structured kinetic model that describes
the cell growth and synthesis of the secondary metabolite, berberine, in a batch suspension culture ofThalictrum rugosum. Four types of operation strategies for sugar feeding intoT. rugosum culture were proposed based on the model, which were the periodic fedbatch operations to maintain the cell activity, the
cell viability, and the specific production rate, and the perfusion operation to maintain the specific production rate. From
the simulation results of these strategies, it could be found that the periodic fed-batch operation and the perfusion operation
could achieve the higher volumetric production of berberine (mg berberine/L) and specific production yield (mg berberine/g
dry cell weight) than those of batch cultures. Although the highest productivity (mg berberine/day) of berberine could be
achieved by the periodic fed-batch operation to maintain the cell activity compared with the other strategies in the periodic
fed-batch operations, the specific production yield was low due to the higher maximum dry cell weight than other cases. The
periodic fed-batch operation to maintain cell viability resulted in the highest volumetric production of berberine and specific
production yield compared with the other strategies. In the cases of maintaining the specific production rate, the per-formance
of the periodic fed-batch operation was better than that of the perfusion operation in the respect of the volumetric production
and productivity of berberine. In order to increase the volumetric production of berberine and to get the highest specific
production yield, the periodic fed-batch operation to maintain cell viability could be chosen as the optimal operating strategy
in high density, culture ofT. rugosum plant cell. 相似文献
8.
C. S. Shin M. S. Hong D. Y. Kim H. C. Shin J. Lee 《Applied microbiology and biotechnology》1998,49(4):364-370
Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at
high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment
is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch
operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed
is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant
product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased
to 15 g fusion growth hormone l−1 and 7 g fusion glucagon l−1. The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion
bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein
during the whole induction period. The stressful conditions of cultivation employed (i.e. high-cell-density cultivation at
low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency
of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more
efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity
is likely to be related to the change in cellular ribosomal content.
Received: 27 May 1997 / Received last revision: 31 October 1997 / Accepted: 21 November 1997 相似文献
9.
The DO-controlled glucose limited fed-batch technique was investigated in an E. coli process for production of a recombinant protein. The kLac* value (oxygen transfer rate at zero oxygen concentration) was calculated from on-line gas analysis data during the process. In the investigated processes with induced production of recombinant protein, the kLac* value decreased drastically several hours after induction. The reason for the decrease was found in increasing concentrations of DNA in the medium and increased viscosity due to cell lysis. The consequences of such a dramatic decrease in the volumetric oxygen transfer coefficient on the glucose feed and specific rates are described in computer simulations and experimental data. 相似文献
10.
Tai Hyun Park Sung Kwan Yoon Whan Koo Kang 《Biotechnology and Bioprocess Engineering》1996,1(1):22-25
An exponential feeding strategy has been frequently used in fed-batch fermentation of recombinantE. coli. In this feeding scheme, growth yield and initial cell concentration, which can be erroneously determined, are needed to calculate the feed rate for controlling specific growth rate at the set point. The effect of the incorrect growth yield and initial cell concentration on the control of the specific growth rate was theoretically analyzed. Insignificance of the correctness of those parameters for the control of the specific growth rate was shown theoretically and experimentally. 相似文献
11.
Robert Gustavsson Carl-Fredrik Mandenius 《Bioprocess and biosystems engineering》2013,36(10):1375-1384
A soft sensor approach is described for controlling metabolic overflow from mixed-acid fermentation and glucose overflow metabolism in a fed-batch cultivation for production of recombinant green fluorescence protein (GFP) in Escherichia coli. The hardware part of the sensor consisted of a near-infrared in situ probe that monitored the E. coli biomass and an HPLC analyzer equipped with a filtration unit that measured the overflow metabolites. The computational part of the soft sensor used basic kinetic equations and summations for estimation of specific rates and total metabolite concentrations. Two control strategies for media feeding of the fed-batch cultivation were evaluated: (1) controlling the specific rates of overflow metabolism and mixed-acid fermentation metabolites at a fixed pre-set target values, and (2) controlling the concentration of the sum of these metabolites at a set level. The results indicate that the latter strategy was more efficient for maintaining a high titer and low variability of the produced recombinant GFP protein. 相似文献
12.
Yang-Ming Lo Shang-Tian Yang David B. Min 《Applied microbiology and biotechnology》1997,47(6):689-694
Although available kinetic data provide a useful insight into the effects of medium composition on xanthan production by
Xanthomonas campestris, they cannot account for the synergetic effects of carbon (glucose) and nitrogen (yeast extract) substrates on cell growth
and xanthan production. In this work, we studied the effects of the glucose/yeast-extract ratio (G/YE) in the medium on cell
growth and xanthan production in various operating modes, including batch, two-stage batch, and fed-batch fermentations. In
general, both the xanthan yield and specific production rate increased with increasing G/YE in the medium, but the cell yield
and specific growth rate decreased as G/YE increased. A two-stage batch fermentation with a G/YE shift from an initial low
level (2.5% glucose/0.3% yeast extract) to a high level (5.0% glucose/0.3% yeast extract) at the end of the exponential growth
phase was found to be preferable for xanthan production. This two-stage fermentation design both provided fast cell growth
and gave a high xanthan yield and xanthan production rate. In contrast, fed-batch fermentation with intermittent additions
of glucose to the fermentor during the stationary phase was not favorable for xanthan production because of the relatively
low G/YE resulting in low xanthan production rate and yield. It is also important to use a moderately high yeast extract concentration
in the medium in order to reach a high cell density before the culture enters the stationary phase. A high cell density is
also important to the overall xanthan production rate.
Received: 30 September 1996 / Received revision: 21 January 1997 / Accepted: 10 February 1997 相似文献
13.
Seung-Jin Choi Doo-Hong Park Kyung-Hwan Jung 《Bioprocess and biosystems engineering》2001,24(1):51-58
The long-term process for producing human granulocyte-colony stimulating factor (hG-CSF) was developed using two-stage cyclic fed-batch culture, in which hG-CSF expressing-recombinant Escherichia coli was directed by an L-arabinose promoter system. For the optimization, the preinduction growth rate during the growth stage and the feeding strategy during the production stage were investigated. The maximum harvest volume during the production stage was predicted before long-term cyclic operation. Based on those optimized strategies, the two-stage cyclic fed-batch culture was performed for 12 cycles (86 h). The cell growths in both stages were maintained at 45-50 g/L and 71-77 g/L, respectively. hG-CSF was stably produced at a level of 8-9 g/L and the plasmid stability was maintained at more than 90%. Volumetric productivity by the two-stage cyclic fed-batch culture was 0.643 g/L/h, which was about 280% higher than that of conventional DO-stat fed-batch culture. 相似文献
14.
Dr. Dane W. Zabriskie Dave A. Wareheim Michael J. Polansky 《Journal of industrial microbiology & biotechnology》1987,2(2):87-95
Summary A variety of feeding strategies have been described for attaining high cell densities in fed-batch fermentors. Although cell density is an important component in the produtivity of recombinant fermentations, it must be achievable with high product expression levels. Experiments were conducted to study the influence of fermentation feeding strategies on the production of a recombinant malaria antigen inEscherichia coli. C-source feeding profiles were calculated to maintain specific growth rates at 0.1, 0.2, 0.35, and 0.5 l/h prior to induction in defined and complex media using an exponential growth model. Fed-batch fermentations employing these feeding profiles effectively controlled the specific growth rates prior to induction. Antigen yields per dry cell weight did not vary with specific growth rate. Antigen yields from fed-batch fermentations achieving high cell densities were similar to batch fermentations achieving low cell densities. These results show that C-feeding policies can limit growth without reducing expression levels in some systems, and suggest applications in managing oxygen demand and catabolic by-product formation during process scale-up. 相似文献
15.
A cyclic fed-batch bioprocess is designed and a significant improvement of rice alpha-amylase productivity of recombinant Yarrowia lipolytica is illustrated. A bioprocess control strategy developed and reported here entails use of a genetically stable recombinant cloned for heterologous protein, use of optimized media for cell growth and enzyme production phases, and process control strategy enabling high cell-density culture and high alpha-amylase productivity. This process control can be achieved through maintaining a constant optimal specific cell growth rate at a predetermined value (i.e., 0.1 h-1), controlling medium feed rate commensurate with the cell growth rate, and maintaining a high cell-density culture (i.e., 60-70 g/L) for high productivity of cloned heterologous protein. The volumetric enzyme productivity (1, 960 units/L. h) achieved from the cyclic fed-batch process was about 3-fold higher than that of the fed-batch culture process (630 units/L. h). 相似文献
16.
The most common strategy to produce recombinant proteins using Escherichia coli as expression vector is fed-batch culture, since high cell density cultures strategies have successfully been applied. Several methodologies to limit the specific growth rate in order to control E. coli metabolism have been defined, demonstrating that cultures can be grown under glucose limitation up to high cell densities without accumulation of acetic acid. However, under induction conditions it has been observed that E. coli metabolism is reorganized again and leads to acetic acid accumulation, causing inhibition of cell growth and decreasing protein expression efficiency.We propose a double limitation strategy (glucose and IPTG) for E. coli fed-batch cultures to avoid the deregulation of the metabolism in the induction phase. Reducing the concentration of IPTG while keeping glucose growth limitation, the accumulation of acetic acid decreased. At an IPTG concentration of 0.03 mmol/g DCW no accumulation of acetic acid was observed during the induction phase, in contraposition to what has normally been observed.Although a slight reduction of protein expression rate was observed when applying this double limitation strategy, the bioprocess volumetric productivity was enhanced due to the capability to prolong the induction phase, reaching higher levels of protein production. Another advantage of this strategy is the reduction of media cost due to the lower level of IPTG used. 相似文献
17.
K. Jagannadha Rao Chul-Ho Kim Bong Hyun Chung Jung-Hoon Sohn Sang-Ki Rhee 《Bioprocess and biosystems engineering》1998,19(5):385-388
Different feeding strategies of galactose were employed to improve the production of anticoagulant, hirudin, by fed-batch mode of cultivation from recombinant Saccharomyces cerevisiae. The structural gene coding for hirudin was harboured with GAL10 promoter for controlled expression of hirudin and the MFα 1 signal sequence for secretion into the growth medium. A step-wise feeding of galactose was found as more suitable feeding strategy of galactose which resulted in the final hirudin volumetric productivity of 6,840?μg/l?·?h, than intermittent, continuous and ethanol controlled feeding of galactose. The final volumetric productivity of hirudin obtained by step-wise feeding of galactose was 3.88 fold higher compared with simple batch fermentation. 相似文献
18.
Ana Mendes Pedro Guerra Vânia Madeira Francisco Ruano Teresa Lopes da Silva Alberto Reis 《World journal of microbiology & biotechnology》2007,23(9):1209-1215
In this work, carob pulp syrup was used as carbon source in C. cohnii fermentations for docosahexaenoic acid production. In preliminary experiments different carob pulp dilutions supplemented
with sea salt were tested. The highest biomass productivity (4 mg/lh) and specific growth rate (0.04/h) were observed at the
highest carob pulp dilution (1:10.5 (v/v), corresponding to 8.8 g/l glucose). Ammonium chloride and yeast extract were tested
as nitrogen sources using different carob pulp syrup dilutions, supplemented with sea salt as growth medium. The best results
were observed for yeast extract as nitrogen source. A C. cohnii fed-batch fermentation was carried out using diluted carob pulp syrup (1:10.5 v/v) supplemented with yeast extract and sea
salt. The biomass productivity was 420 mg/lh, and the specific growth rate 0.05/h. Under these conditions the DHA concentration
and DHA production volumetric rate attained 1.9 g/l and 18.5 mg/lh respectively after 100.4 h. The easy, clean and safe handling
of carob pulp syrup makes this feedstock a promising carbon source for large-scale DHA production from C. cohnii. In this way, this carob industry by-product could be usefully disposed of through microbial production of a high value fermentation
product. 相似文献
19.
Xu Zhinan Liu Gang Cen Peilin W. K. R. Wong 《Bioprocess and biosystems engineering》2000,23(6):669-674
Human epidermal growth factor (hEGF) was expressed and excreted into culture medium in an excretory recombinant Escherichia coli system. Conditions for the production of hEGF in this system were investigated. Results showed that the slight reduction of promoter strength improved the stability of plasmid and the production of hEGF in this recombinant system. One favorable MMBL medium for hEGF production was formulated by evaluating the effects of medium components, ampicilin addition and pH. hEGF production was affected obviously by culture conditions, especially fermentation temperature. High temperature (32 °C) was very beneficial for culture process by increasing productivity and reducing the quantity of isopropyl-#-d-thiogalactopyranoside (IPTG) for sufficient induction. High cell density and hEGF productivity could be accomplished concomitantly by inducing the culture at middle or late log phase of cell growth. In comparison with the batch process, fed-batch cultivation could improve plasmid stability from ca. 72% to ca. 83%, and increased hEGF productivity by 24.4%. Under all these circumstances, almost all expressed hEGF (%) was fully excreted into the culture medium. 相似文献
20.
K. Divakar M. Suryia Prabha G. Nandhinidevi 《Preparative biochemistry & biotechnology》2017,47(4):323-332
The simultaneous production of intracellular esterase and extracellular protease from the strain Lysinibacillus fusiformis AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking–Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820?×?103 U/L and extracellular protease activity of 172?×?103 U/L were obtained at the 16th?hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions. 相似文献