循环利用重组大肠杆菌细胞转化合成丁二酸

研究了回收丁二酸发酵液中的大肠杆菌进行细胞转化的可行性,以转化率和生产效率为指标,考察了不同菌体浓度、底物浓度、pH调节剂对细胞转化的影响。发酵结果表明大肠杆菌可以在仅含有葡萄糖和pH调节剂的水环境中转化生产丁二酸,并确定了最佳的转化条件为:细胞浓度(OD600)50,底物浓度40g/L,缓冲盐为MgCO3。基于优化好的条件,在7L发酵罐中进行重复批次转化,第1次转化的转化率和生产效率分别达到91%和3.22g/(L·h),第2次转化的生产效率和转化率达到了86%和2.04g/(L·h),第3次转化的转化率和生产效率分别达到了83%和1.82g/(L·h)。     

Closed-loop control of fed-batch cultures of recombinant Escherichia coli using on-line HPLC

This article describes a fully automated system for the on-line monitoring and closed-loop control of a fed-batch fermentation of recombinant Escherichia coli, and presents two case studies of its used in limiting production of unwanted byproducts such as acetic in fed-batch fermentations. The system had two components. The first components, on-line monitoring, comprised an aseptic sampling device, a microcentrifuge, and HPLC System. These instruments removed a Sample from a fermentor, spun it at high speed to separate solid and liquid components, and then automatically injected the supernatant onto an HPLC column for analysis. The second component consisted of control algorithms programmed using the LabView visual programming environment in a control computer that was linked via a remote components were linked so that results from the on-line HPLC were captured and used by the control algorithm was designed to demonstrate coarse feedback control to confirm the operability of the controller. The second case study showed how the system could be used in a more sophisticated feedings strategy providing fine control and limiting acetate concentration to a low level throughout the fermentation. (c) 1994 John Wiley & Sons, Inc.     

Fermentation kinetics of recombinant yeast in batch and fed-batch cultures

Fed-batch cultures of recombinant microorganisms have attracted attention as they can separate cell growth stage from cloned-gene expression phase during fermentations. In this work, the effect of different glucose feeding strategies on cell growth and cloned gene expression was studied during aerobic fed-batch fermentations of recombinant yeast, containing the plasmid pRB58. The plasmid contains the yeast SUC2 gene, which codes for the enzyme invertase. Some feeding policies resulted in a constant glucose concentration inside the fermentor, while others deliberately introduced a cyclic variation. The cell mass yield was found to be higher at low glucose concentrations, thus indicating a shift to the more energy-efficient respiratory pathway. The SUC2 gene expression was derepressed at glucose levels below 2 g/L. The response of specific invertase activity to changes in the medium glucose concentration was found to be almost immediate.     

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.     

Heterotrophic high cell-density fed-batch cultures of the phycocyanin-producing red alga Galdieria sulphuraria

Growth and phycocyanin production in batch and fed-batch cultures of the microalga Galdieria sulphuraria 074G, which was grown heterotrophically in darkness on glucose, fructose, sucrose, and sugar beet molasses, was investigated. In batch cultures, specific growth rates and yields of biomass dry weight on the pure sugars were 1.08-1.15 day-1 and 0.48-0.50 g g-1, respectively. They were slightly higher when molasses was the carbon source. Cellular phycocyanin contents during the exponential growth phase were 3-4 mg g-1 in dry weight. G. sulphuraria was able to tolerate concentrations of glucose and fructose of up to 166 g L-1 (0.9 M) and an ammonium sulfate concentration of 22 g L-1 (0.17 M) without negative effects on the specific growth rate. When the total concentration of dissolved substances in the growth medium exceeded 1-2 M, growth was completely inhibited. In carbon-limited fed-batch cultures, biomass dry weight concentrations of 80-120 g L-1 were obtained while phycocyanin accumulated to concentrations between 250 and 400 mg L-1. These results demonstrate that G. sulphuraria is well suited for growth in heterotrophic cultures at very high cell densities, and that such cultures produce significant amounts of phycocyanin. Furthermore, the productivity of phycocyanin in the heterotrophic fed-batch cultures of G. sulphuraria was higher than is attained in outdoor cultures of Spirulina platensis, where phycocyanin is presently obtained.     

Pulsed addition of limiting-carbon during Aspergillus oryzae fermentation leads to improved productivity of a recombinant enzyme

Fungal morphology in many filamentous fungal fermentations leads to high broth viscosity which limits oxygen mass transfer, and often results in reduced productivity. The objective in this study was to determine if a simple, fed-batch, process strategy-pulsed addition of limiting-carbon source-could be used to reduce fungal broth viscosity, and increase productivity of an industrially relevant recombinant enzyme (glucoamylase). As a control, three Aspergillus oryzae fed-batch fermentations were carried out with continuous addition of limiting-carbon. To determine the effect of pulse-feeding, three additional fermentations were carried out with limiting-carbon added in 90-second pulses, during repeated five-minute cycles. In both cases, overall carbon feed-rate was used to control dissolved oxygen concentration, such that increased oxygen availability led to increased addition of limiting-carbon. Pulse-fed fermentations were found to have smaller fungal mycelia, lower broth viscosity, and improved oxygen mass transfer. As a result, more carbon was added to pulse-fed fermentations that led to increased enzyme productivity by as much as 75%. This finding has significant implications for the bioprocessing industry, as a simple process modification which is likely to cost very little to implement in most production facilities, has the potential to substantially increase productivity.     

Enhancement of monoclonal antibody yield by hybridoma fed-batch culture, resulting in extended maintenance of viable cell population

Hybridoma batch cultures were extended using feed formulations based on nutrient consumption measured during different batch culture phases when (a) growth but negligible antibody production was taking place; (b) maximum antibody production rate and declining viable cell growth rate were observed. Strategy (a) was the more successful (2.8-fold compared with 1.8-fold antibody titer increase) and maintained cell viability for longer. Analysis of the effects of omitting individual amino acids yielded results which were consistent with those from the feeding experiment (c) 1994 John Wiley & Sons, Inc.     

Performance of high intensity fed-batch mammalian cell cultures in disposable bioreactor systems

The adoption of disposable bioreactor technology as an alternate to traditional nondisposable technology is gaining momentum in the biotechnology industry. Evaluation of current disposable bioreactors systems to sustain high intensity fed-batch mammalian cell culture processes needs to be explored. In this study, an assessment was performed comparing single-use bioreactors (SUBs) systems of 50-, 250-, and 1,000-L operating scales with traditional stainless steel (SS) and glass vessels using four distinct mammalian cell culture processes. This comparison focuses on expansion and production stage performance. The SUB performance was evaluated based on three main areas: operability, process scalability, and process performance. The process performance and operability aspects were assessed over time and product quality performance was compared at the day of harvest. Expansion stage results showed disposable bioreactors mirror traditional bioreactors in terms of cellular growth and metabolism. Set-up and disposal times were dramatically reduced using the SUB systems when compared with traditional systems. Production stage runs for both Chinese hamster ovary and NS0 cell lines in the SUB system were able to model SS bioreactors runs at 100-, 200-, 2,000-, and 15,000-L scales. A single 1,000-L SUB run applying a high intensity fed-batch process was able to generate 7.5 kg of antibody with comparable product quality.     

Protein-free fed-batch culture of non-GS NS0 cell lines for production of recombinant antibodies

Presented is a novel antibody production platform based on the fed-batch culture of recombinant, NS0-derived cell lines. A standardized fed-batch cell culture process was developed for five non-GS NS0 cell lines using enriched and optimized protein-free, cholesterol-free, and chemically defined basal and feed media. The process performed reproducibly and scaled faithfully from the 2-L to the 100-L bioreactor scale achieving a volumetric productivity of > 120 mg/L per day. Fed-batch cultures for all five cell lines exhibited significant lactate consumption when the cells entered the stationary or death phase. Peak and final lactate concentrations were low relative to a previously developed fed-batch process (FBP). Such low lactate production and high lactate consumption rates were unanticipated considering the fed-batch culture basal medium has an unconventionally high initial glucose concentration of 15 g/L, and an overall glucose consumption in excess of 17 g/L. The potential of this process platform was further demonstrated through additional media optimization, which has resulted in a final antibody concentration of 2.64 +/- 0.19 g/L and volumetric productivity of > 200 mg/L per day in a 13-day FBP for one of the five production cell lines. Use of this standardized protein-free, cholesterol-free NS0 FBP platform enables consistency in development time and cost effectiveness for manufacturing of therapeutic antibodies.     

Mass production of human epidermal growth factor using fed-batch cultures of recombinant Escherichia coli

Fed-batch cultures of recombinant E. coli HB101 harboring expression plasmid pTRLBT1 or pTREBT1, with acetate concentration monitoring, are investigated to obtain high cell density and large amounts of human epidermal growth factor (hEGF). The expression plasmid pTRlBT1 contains a synthetic hEGF gene attached downstream of the N-terminal fragment of the trp L gene preceded by the trp promoter. The expression plasmid pTREBT1 contains the same coding sequence attached downstream of the N-terminal fragment of the trp E gene preceded by the trp promoter, trp L gene, and attenuator region. E. coli harboring pTREBT1 produces 0.56 mg/L hEGE and immediately degrades it. On the other hand E. coli harboring pTRLBT1 produces 6.8 mg/L hEGF and does not decompose it. Prominent inclusion bodies are observed in E. coli cells harboring pTRLBT1 using an election microscope. To Cultivate E. coli harboring pTRLBT1, a fed-batch culture system, divided into a cell growth step and an hEGF production step, is carried out. The cells grow smoothly without acetate-induced inhibition. Cell concentration and hEGF quantity reach the high values of 21 g/L and 60 mg/L, respectively.     

Autophagy and apoptosis of recombinant Chinese hamster ovary cells during fed-batch culture: effect of nutrient supplementation

Upon nutrient depletion during recombinant Chinese hamster ovary (rCHO) cell batch culture, cells are subjected to apoptosis, type I programmed cell death (PCD), and autophagy which can be type II PCD or a cell survival mechanism. To investigate the effect of nutrient supplementation on the two PCDs and protein production in rCHO cells, an antibody-producing rCHO cell line was cultivated in batch and fed-batch modes. The feed medium containing glucose, amino acids, and vitamins was determined through flask culture tests and used in bioreactor cultures. In the bioreactor cultures, the nutrient feedings extended the culture longevity and enhanced antibody production. In addition, cells in the fed-batch culture showed delayed onset of both apoptosis and autophagy, compared with those in the batch culture. The inhibition of apoptosis was demonstrated by a decreased amount of cleaved caspase-7 protein and less fragmentation of chromosomal DNA. Concurrently, reduced LC3 conversion, from LC3-I to LC3-II, was observed in cells that received the feeds. Cultivation with pharmacological autophagy inducer (rapamycin) or inhibitor (bafilomycin A1) indicated that autophagy is necessary for the cells to survive under nutrient depletion. Taken together, the delayed and relieved cell death by nutrient supplementation could improve antibody production.     

Protein engineering of toluene 4-monooxygenase of Pseudomonas mendocina KR1 for synthesizing 4-nitrocatechol from nitrobenzene

After discovering that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes nitrobenzene to 4-nitrocatechol, albeit at a very low rate, this reaction was improved using directed evolution and saturation mutagenesis. Screening 550 colonies from a random mutagenesis library generated by error-prone PCR of tmoAB using Escherichia coli TG1/pBS(Kan)T4MO on agar plates containing nitrobenzene led to the discovery of nitrocatechol-producing mutants. One mutant, NB1, contained six amino acid substitutions (TmoA Y22N, I84Y, S95T, I100S, S400C; TmoB D79N). It was believed that position I100 of the alpha subunit of the hydroxylase (TmoA) is the most significant for the change in substrate reactivity due to previous results in our lab with a similar enzyme, toluene ortho-monooxygenase of Burkholderia cepacia G4. Saturation mutagenesis at this position resulted in the generation of two more nitrocatechol mutants, I100A and I100S; the rate of 4-nitrocatechol formation by I100A was more than 16 times higher than that of wild-type T4MO at 200 microM nitrobenzene (0.13 +/- 0.01 vs. 0.008 +/- 0.001 nmol/min.mg protein). HPLC and mass spectrometry analysis revealed that variants NB1, I100A, and I100S produce 4-nitrocatechol via m-nitrophenol, while the wild-type produces primarily p-nitrophenol and negligible amounts of nitrocatechol. Relative to wild-type T4MO, whole cells expressing variant I100A convert nitrobenzene into m-nitrophenol with a Vmax of 0.61 +/- 0.037 vs. 0.16 +/- 0.071 nmol/min.mg protein and convert m-nitrophenol into nitrocatechol with a Vmax of 3.93 +/- 0.26 vs. 0.58 +/- 0.033 nmol/min.mg protein. Hence, the regiospecificity of nitrobenzene oxidation was changed by the random mutagenesis, and this led to a significant increase in 4-nitrocatechol production. The regiospecificity of toluene oxidation was also altered, and all of the mutants produced 20% m-cresol and 80% p-cresol, while the wild-type produces 96% p-cresol. Interestingly, the rate of toluene oxidation (the natural substrate of the enzyme) by I100A was also higher by 65% (7.2 +/- 1.2 vs. 4.4 +/- 0.3 nmol/min mg protein). Homology-based modeling of TmoA suggests reducing the size of the side chain of I100 leads to an increase in the width of the active site channel, which facilitates access of substrates and promotes more flexible orientations.     

Inhibition of glutamine-dependent autophagy increases t-PA production in CHO cell fed-batch processes

Understanding the cellular responses caused by metabolic stress is crucial for the design of robust fed-batch bioprocesses that maximize the expression of recombinant proteins. Chinese hamster ovary cells were investigated in chemically defined, serum-free cultures yielding 10(7) cells/mL and up to 500 mg/L recombinant tissue-plasminogen activator (t-PA). Upon glutamine depletion increased autophagosome formation and autophagic flux were observed, along with decreased proliferation and high viability. Higher lysosomal levels correlated with decreased productivity. Chemical inhibition of autophagy with 3-methyl adenine (3-MA) increased the t-PA yield by 2.8-fold. Autophagy-related MAP1LC3 and LAMP2 mRNA levels increased continuously in all cultures. Analysis of protein quality revealed that 3-MA treatment did not alter glycan antennarity while increasing fucosylation, galactosylation, and sialylation. Taken together, these findings indicate that inhibition of autophagy can considerably increase the yield of biotechnology fed-batch processes, without compromising the glycosylation capacity of cells. Monitoring or genetic engineering of autophagy provides novel avenues to improve the performance of cell culture-based recombinant protein production.     

Metabolic load of recombinant protein production: inhibition of cellular capacities for glucose uptake and respiration after induction of a heterologous gene in Escherichia coli

The strong expression of recombinant proteins in bacteria affects the primary carbon and energy metabolism resulting in growth inhibition and acetate formation. By applying glucose pulses to fed-batch fermentations performed for production of a heterologous (alpha-glucosidase in Escherichia coli, we show that the induction of the recombinant gene strongly inhibits the maximum specific uptake capacities for glucose and the respiration capacity. The accumulation of glucose in the fermentation medium promotes the growth of plasmid-free cells. These inhibition effects are well described by including the kinetics of product formation into a recently published dynamic model (Lin et al. [2001] Biotechnol Bioeng 73:349-357). The new model also includes the population characteristics and gives a good fit to the measured data describing growth, production, substrate consumption, by-product formation, and respiration.     

Simplified modeling of fed-batch alcoholic fermentation of sugarcane blackstrap molasses

Simplified modeling based on material balances for biomass, ethanol and substrate was used to describe the kinetics of fed-batch alcohol fermentation of sugarcane blackstrap molasses. Maintenance requirements were previously shown to be of particular significance in this system, owing to the use of massive inoculum to minimize inhibitions; therefore, they were taken into consideration for kinetic modeling. Average values of biomass and ethanol yields, productivities, and substrate consumption rates, calculated at the end of runs performed either at constant or exponentially varying flow rates, demonstrated that all of these parameters were influenced by the initial sugar-feeding rate, F(o)S(o). Under conditions of substrate shortage (F(o)S(o) 相似文献   

High-titer n-butanol production by clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping

Acetone–butanol–ethanol (ABE) fermentation with a hyper‐butanol producing Clostridium acetobutylicum JB200 was studied for its potential to produce a high titer of butanol that can be readily recovered with gas stripping. In batch fermentation without gas stripping, a final butanol concentration of 19.1 g/L was produced from 86.4 g/L glucose consumed in 78 h, and butanol productivity and yield were 0.24 g/L h and 0.21 g/g, respectively. In contrast, when gas stripping was applied intermittently in fed‐batch fermentation, 172 g/L ABE (113.3 g/L butanol, 49.2 g/L acetone, 9.7 g/L ethanol) were produced from 474.9 g/L glucose in six feeding cycles over 326 h. The overall productivity and yield were 0.53 g/L h and 0.36 g/g for ABE and 0.35 g/L h and 0.24 g/g for butanol, respectively. The higher productivity was attributed to the reduced butanol concentration in the fermentation broth by gas stripping that alleviated butanol inhibition, whereas the increased butanol yield could be attributed to the reduced acids accumulation as most acids produced in acidogenesis were reassimilated by cells for ABE production. The intermittent gas stripping produced a highly concentrated condensate containing 195.9 g/L ABE or 150.5 g/L butanol that far exceeded butanol solubility in water. After liquid–liquid demixing or phase separation, a final product containing ～610 g/L butanol, ～40 g/L acetone, ～10 g/L ethanol, and no acids was obtained. Compared to conventional ABE fermentation, the fed‐batch fermentation with intermittent gas stripping has the potential to reduce at least 90% of energy consumption and water usage in n‐butanol production from glucose. Biotechnol. Bioeng. 2012; 109: 2746–2756. © 2012 Wiley Periodicals, Inc.     

Proteome analysis to assess physiological changes in Escherichia coli grown under glucose-limited fed-batch conditions

Proteome analysis was used to compare global protein expression changes in Escherichia coli fermentation between exponential and glucose-limited fed-batch phase. Two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry were used to separate and identify 49 proteins showing >2-fold difference in expression. Proteins upregulated during exponential phase include ribonucleotide biosynthesis enzymes and ribosomal recycling factor. Proteins upregulated during fed-batch phase include those involved in high-affinity glucose uptake, transport and degradation of alternate carbon sources and TCA cycle, suggesting an enhanced role of the cycle under glucose- and energy-limited conditions. We report the upregulation of several putative proteins (ytfQ, ygiS, ynaF, yggX, yfeX), not identified in any previous study under carbon-limited conditions.     

Optimization of a feed medium for fed-batch culture of insect cells using a genetic algorithm

Insect cells have been cultured for over 30 years, but their application is still hampered by low cell densities in batch fermentations and expensive culture media. With respect to the culture method, the fed-batch culture mode is often found to give the best yields. However, optimization of the feed composition is usually a laborious task. In this report, the successful use of genetic algorithms (GAs) to optimize the growth of insect cells is described. A feed was developed from 11 different medium components, each used at a wide range of concentrations. The feed was optimized within four sets of 20 experiments. The optimized feed was tested in bioreactors and the addition scheme was further improved. The viable-cell density of HzAm1 (Helicoverpa zea) insect cells improved 550% to 19.5 x 10(6) cells/mL compared to a control fermentation in an optimized commercial medium. No accumulation of waste products was found, and none of the amino acids was depleted. Glucose was depleted, which suggests that even further improvement is possible. We show that GAs are a successful method to optimize a complex fermentation in a relatively short time frame and without the need of detailed information concerning the cellular physiology or metabolism.     

Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures

As we pursue the means to improve yields to meet growing therapy demands, it is important to examine the impact of process control on glycosylation patterns to ensure product efficacy and consistency. In this study, we describe a dynamic on-line fed-batch strategy based on low glutamine/glucose concentrations and its impact on cellular metabolism and, more importantly, the productivity and N-glycosylation quality of a model recombinant glycoprotein, interferon gamma (IFN-gamma). We found that low glutamine fed-batch strategy enabled up to 10-fold improvement in IFN-gamma yields, which can be attributed to reduced specific productivity of ammonia and lactate. Furthermore, the low glutamine concentration (0.3 mM) used in this fed-batch strategy could maintain both the N-glycosylation macro- and microheterogeneity of IFN-gamma. However, very low glutamine (<0.1 mM) or glucose (<0.70 mM) concentrations can lead to decreased sialylation and increased presence of minor glycan species consisting of hybrid and high-mannose types. This shows that glycan chain extension and sialylation can be affected by nutrient limitation. In addition to nutrient limitation, we also found that N-glycosylation quality can be detrimentally affected by low culture viability. IFN-gamma purified at low culture viability had both lower sialylation as well as glycans of lower molecular masses, which can be attributed to extensive degradation by intracellular glycosidases released by cytolysis. Therefore, in order to maintain good N-glycosylation quality, there is a need to consider both culture viability and nutrient control setpoint in a nutrient-limiting fed-batch culture strategy. A greater understanding of these major factors that affect N-glycosylation quality would surely facilitate future development of effective process controls.