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1.
Chinese Journal of Biotechnology ; (12): 3364-3378, 2023.
Article in Chinese | WPRIM | ID: wpr-1007963

ABSTRACT

With various diseases ravaging internationally, the demands for recombinant adenoviral vector (Adv) vaccines have increased dramatically. To meet the demand for Adv vaccine, development of a new cell culture process is an effective strategy. Applying hyperosmotic stress in cells before virus infection could increase the yield of Adv in batch culture mode. Emerging perfusion culture can significantly increase the yield of Adv as well. Therefore, combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Adv at high cell density. In this study, a shake flask combined with a semi-perfusion culture was used as a scaled-down model for bioreactor perfusion culture. Media with osmotic pressure ranging from 300 to 405 mOsm were used to study the effect of hyperosmotic stress on cell growth and Adv production. The results showed that using a perfusion culture process with a hyperosmotic pressure medium (370 mOsm) during the cell growth phase and an isosmotic pressure medium (300 mOsm) during the virus production phase effectively increased the yield of Adv. This might be due to the increased expression of HSP70 protein during the late phases of virus replication. The Adv titer in a bioreactor with such a process reached 3.2×1010 IFU/mL, three times higher than that of the traditional perfusion culture process. More importantly, this is the first time that a strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Adv in HEK 293 cells. It also reveals the reason why the hyperosmotic stress process increased the yield of Adv, which may facilitate the process optimization of for producing other Adv in HEK 293 cells.


Subject(s)
Humans , HEK293 Cells , Genetic Vectors/genetics , Batch Cell Culture Techniques , Bioreactors , Perfusion
2.
Electron. j. biotechnol ; 50: 45-52, Mar. 2021. tab, graf
Article in English | LILACS | ID: biblio-1292328

ABSTRACT

BACKGROUND: Lawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vaccinated pigs. Also, protection was demonstrated after the challenge. In this study, we established a production process to increase the yields of the three antigens as a vaccine candidate. RESULTS: Batch and fed-batch fermentations were evaluated in different culture conditions using a 2 L bioreactor. A fed-batch culture with a modified Terrific broth medium containing glucose instead of glycerol, and induced with 0.75 mM IPTG at 8 h of culture (11 g/L of biomass) raised the volumetric yield to 627.1 mg/L. Under these culture conditions, plasmid-bearing cells increased by 10% at the induction time. High efficiency in cell disruption was obtained at passage six using a high-pressure homogenizer and a bead mill. The total antigen recovery was 64% (400 mg/L), with a purity degree of 70%. The antigens retained their immunogenicity in pigs, inducing high antibody titers. CONCLUSIONS: Considering that the antigen production process allowed an increment of more than 70-fold, this methodology constitutes a crucial step in the production of this vaccine candidate against L. intracellularis.


Subject(s)
Animals , Swine Diseases/immunology , Bacterial Vaccines/immunology , Lawsonia Bacteria/immunology , Desulfovibrionaceae Infections/prevention & control , Swine , Swine Diseases/prevention & control , Bacterial Vaccines/administration & dosage , Vaccines, Synthetic , Cell Survival , Vaccination , Fermentation , Batch Cell Culture Techniques , Immunity
3.
Braz. J. Pharm. Sci. (Online) ; 57: e181083, 2021. tab
Article in English | LILACS | ID: biblio-1350228

ABSTRACT

Among the methods described for determining the solubility, shake-flask is suitable to evaluate the equilibrium solubility according to the BCS. Nevertheless, experimental conditions related to the shake-flask method are not well described. Evaluating the effects of experimental conditions on solubility measurements by shake-flask method is important and contributes in biowaiver decision. For this work, propranolol hydrochloride and nimesulide were used as model compound of high and low solubility, respectively. Equilibrium solubility was evaluated at 37 ºC, 100 rpm during 48 hours in buffer media. Effects of the rotation speed, temperature, substance in excess and aliquot withdrawn were evaluated. Small variations of temperature caused significant differences in the solubility and then this parameter must be controlled. Excess of raw material influenced the results of the nimesulide, then, little excess is recommended. Rotation speed did not cause differences in the equilibrium solubilities, but at 150 rpm the equilibrium was reached faster. Aliquot did not present significant differences, but excessive withdrawn should be avoided. Therefore, the evaluation of equilibrium solubility using shake-flask method must be performed in physiological pH conditions, 37 ± 1 ºC, substance in excess 10% above saturation, 50, 100 or 150 rpm and aliquot withdrawn not more than 10% of the media volume.


Subject(s)
Solubility , Batch Cell Culture Techniques/methods , Pharmaceutical Preparations/administration & dosage
4.
Chinese Journal of Biotechnology ; (12): 1041-1050, 2020.
Article in Chinese | WPRIM | ID: wpr-826872

ABSTRACT

In recent years, the demand of biologics has increased rapidly. Cell culture process with perfusion mode has become more and more popular due to its high productivity, good quality and high efficiency. In this paper, the unique operation and the details of process optimization for perfusion culture mode are discussed by comparing with traditional batch culture process. Meanwhile, the progress and strategies in the development and optimization of perfusion culture process in recent years are summarized to provide reference for the future development of mammalian cell perfusion culture technology.


Subject(s)
Animals , Batch Cell Culture Techniques , Bioreactors , Reference Standards , CHO Cells , Cricetulus , Mammals , Perfusion
5.
Chinese Journal of Biotechnology ; (12): 1209-1215, 2020.
Article in Chinese | WPRIM | ID: wpr-826857

ABSTRACT

Bioreactors have been central in monoclonal antibodies and vaccines manufacturing by mammalian cells in suspension culture. Numerical simulation of five impeller combinations in a stirred bioreactor was conducted, and characteristics of velocity vectors, distributions of gas hold-up, distributions of shear rate in the bioreactor using 5 impeller combinations were numerically elucidated. In addition, genetically engineered CHO cells were cultivated in bioreactor installed with 5 different impeller combinations in fed-batch culture mode. The cell growth and antibody level were directly related to the maximum shear rate in the bioreactor, and the highest viable cell density and the peak antibody level were achieved in FBMI3 impeller combination, indicating that CHO cells are sensitive to shear force produced by impeller movement when cells were cultivated in bioreactor at large scale, and the maximum shear rate would play key roles in scaling-up of bioreactor at industrial scale.


Subject(s)
Animals , Cricetinae , Batch Cell Culture Techniques , Bioreactors , Reference Standards , CHO Cells , Cell Count , Computer Simulation , Cricetulus , Industrial Microbiology , Methods
6.
Electron. j. biotechnol ; 41: 56-59, sept. 2019. tab, graf
Article in English | LILACS | ID: biblio-1087166

ABSTRACT

Background: Chinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells. Results: In this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15 g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95­100% of productivity after 40 days of continuous culture (~48­56 generations). Conclusions: Suspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.


Subject(s)
Retroviridae , Recombinant Proteins/metabolism , CHO Cells/metabolism , Immunoglobulin Fc Fragments , Cell Line , Chromatography, Gel/methods , Disease Vectors , Glucagon-Like Peptide 1 , Tandem Mass Spectrometry , Batch Cell Culture Techniques
7.
Electron. j. biotechnol ; 38: 32-39, Mar. 2019. ilus, graf, tab
Article in English | LILACS | ID: biblio-1051317

ABSTRACT

BACKGROUND: Eugenol is an economically favorable substrate for the microbial biotransformation of aromatic compounds. Coniferyl aldehyde is one kind of aromatic compound that is widely used in condiment and medical industries; it is also an important raw material for producing other valuable products such as vanillin and protocatechuic acid. However, in most eugenol biotransformation processes, only a trace amount of coniferyl aldehyde is detected, thus making these processes economically unattractive. As a result, an investigation of new strains with the capability of producing more coniferyl aldehyde from eugenol is required. RESULTS: We screened a novel strain of Gibberella fujikuroi, labeled as ZH-34, which was capable of transforming eugenol to coniferyl aldehyde. The metabolic pathway was analyzed by high-performance liquid chromatography­mass spectrometry and transformation kinetics. The culture medium and biotransformation conditions were optimized. At a 6 h time interval of eugenol fed-batch strategy, 3.76 ± 0.22 g/L coniferyl aldehyde was obtained, with the corresponding yield of 57.3%. CONCLUSIONS: This work improves the yield of coniferyl aldehyde with a biotechnological approach. Moreover, the fed-batch strategy offers possibility for controlling the target product and accumulating different metabolites


Subject(s)
Acrolein/analogs & derivatives , Eugenol/metabolism , Biotransformation , Gibberella/metabolism , Biodegradation, Environmental , Acrolein/metabolism , Biotechnology , Chromatography, High Pressure Liquid , Renewable Resources , Batch Cell Culture Techniques
8.
Braz. j. microbiol ; 49(3): 621-631, July-Sept. 2018. tab, graf
Article in English | LILACS | ID: biblio-951813

ABSTRACT

Abstract Cordyceps militaris 202 is a potential fungus for biotransformation zein, due to its various proteases, high tolerance and viability in nature. In this article, single factor experiment and response surface methodology were applied to optimize the liquid fermentation conditions and improve the ability of biotransformation zein. The optimized fermentation conditions were as follows: inoculum concentration of 19%, volume of liquor of 130 mL/500 mL and pH of 4.7. Under this condition, the degree of hydrolysis (DH) was 27.31%. The zein hydrolysates from fungi fermentation maintained a high thermal stability. Compared to the original zein, the zein hydrolysates were found to have high solubility, which most likely results in improved foaming and emulsifying properties. Overall, this research demonstrates that hydrolysis of zein by C. militaris 202 is a potential method for improving the functional properties of zein, and the zein hydrolysates can be used as functional ingredients with an increased antioxidant effect in both food and non-food applications.


Subject(s)
Zein/metabolism , Zein/chemistry , Cordyceps/metabolism , Batch Cell Culture Techniques/methods , Protein Hydrolysates/metabolism , Protein Hydrolysates/chemistry , Solubility , Biotransformation , Fermentation , Hydrolysis
9.
Braz. j. microbiol ; 48(1): 180-185, Jan.-Mar. 2017. tab, graf
Article in English | LILACS | ID: biblio-839351

ABSTRACT

Abstract Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullulan is more costly than other exopolysaccharides, which limits its effective application. The purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullulan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mixtures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose = 80:20) was 0.212 h−1, which was significantly higher than those of potato starch hydrolysate alone (0.146 h−1) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch hydrolysate:glucose:fructose = 80:10:10, 0.166 h−1) with 100 g L−1 total carbon source. The results suggest that mixtures of potato starch hydrolysate and sucrose could promote pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme responsible for pullulan synthesis and promoted effective potato starch hydrolysate conversion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan.


Subject(s)
Ascomycota/metabolism , Starch/metabolism , Sucrose/metabolism , Solanum tuberosum/chemistry , Fermentation , Glucans/biosynthesis , Starch/chemistry , Carbon/metabolism , Kinetics , Biomass , Bioreactors , Batch Cell Culture Techniques
10.
Electron. j. biotechnol ; 26: 84-92, Mar. 2017. graf, tab
Article in English | LILACS | ID: biblio-1008992

ABSTRACT

Background: Fermentation process development has been very important for efficient ethanol production. Improvement of ethanol production efficiency from sweet sorghum juice (SSJ) under normal gravity (NG, 160 g/L of sugar), high gravity (HG, 200 and 240 g/L of sugar) and very high gravity (VHG, 280 and 320 g/L of sugar) conditions by nutrient supplementation and alternative feeding regimes (batch and fed-batch systems) was investigated using a highly ethanol-tolerant strain, Saccharomyces cerevisiae NP01. Results: In the batch fermentations without yeast extract, HG fermentation at 200 g/L of sugar showed the highest ethanol concentration (PE, 90.0 g/L) and ethanol productivity (QE, 1.25 g/L·h). With yeast extract supplementation (9 g/L), the ethanol production efficiency increased at all sugar concentrations. The highest PE (112.5 g/L) and QE (1.56 g/L·h) were observed with the VHG fermentation at 280 g/L of sugar. In the fed-batch fermentations, two feeding regimes, i.e., stepwise and continuous feedings, were studied at sugar concentrations of 280 g/L. Continuous feeding gave better results with the highest PE and QE of 112.9 g/L and 2.35 g/L·h, respectively, at a feeding time of 9 h and feeding rate of 40 g sugar/h. Conclusions: In the batch fermentation, nitrogen supplementation resulted in 4 to 32 g/L increases in ethanol production, depending on the initial sugar level in the SSJ. Under the VHG condition, with sufficient nitrogen, the fed-batch fermentation with continuous feeding resulted in a similar PE and increased QP by 51% compared to those in the batch fermentation.


Subject(s)
Sorghum/metabolism , Ethanol/metabolism , Biofuels , Fermentation , Saccharomyces cerevisiae/metabolism , Dietary Supplements , Sorghum/chemistry , Batch Cell Culture Techniques , Gravitation , Nitrogen
11.
Electron. j. biotechnol ; 19(5): 2-11, Sept. 2016. ilus
Article in English | LILACS | ID: lil-797331

ABSTRACT

The buffering effect of acetate on hydrogen production during glucose fermentation by Ethanoligenens harbinense B49 was investigated compared to phosphate, a widely used fermentative hydrogen production buffer. Specific concentrations of sodium acetate or phosphate were added to batch cultures, and the effects on hydrogen production were comparatively analyzed using a modified Gompertz model. Adding 50 mM acetate or phosphate suppressed the hydrogen production peak and slightly extended the lag phase. However, the overall hydrogen yields were 113.5 and 108.5 mmol/L, respectively, and the final pH was effectively controlled. Acetate buffered against hydrogen production more effectively than did phosphate, promoting cell growth and preventing decreased pH. At buffer concentrations 100-250 mM, the maximum hydrogen production was barely suppressed, and the lag phase extended past 7 h. Therefore, although acetate inhibits hydrogen production, using acetate as a buffer (like phosphate) effectively prevented pH drops and increased substrate consumption, enhancing hydrogen production.


Subject(s)
Gram-Positive Bacteria/metabolism , Hydrogen/metabolism , Acetates/analysis , Phosphates/analysis , Buffers , Biofuels , Fermentation , Batch Cell Culture Techniques , Glucose , Hydrogen-Ion Concentration
12.
Braz. j. microbiol ; 47(3): 640-646, July-Sept. 2016. tab, graf
Article in English | LILACS | ID: lil-788973

ABSTRACT

ABSTRACT Lactic acid, which can be obtained through fermentation, is an interesting compound because it can be utilized in different fields, such as in the food, pharmaceutical and chemical industries as a bio-based molecule for bio-refinery. In addition, lactic acid has recently gained more interest due to the possibility of manufacturing poly(lactic acid), a green polymer that can replace petroleum-derived plastics and be applied in medicine for the regeneration of tissues and in sutures, repairs and implants. One of the great advantages of fermentation is the possibility of using agribusiness wastes to obtain optically pure lactic acid. The conventional batch process of fermentation has some disadvantages such as inhibition by the substrate or the final product. To avoid these problems, this study was focused on improving the production of lactic acid through different feeding strategies using whey, a residue of agribusiness. The downstream process is a significant bottleneck because cost-effective methods of producing high-purity lactic acid are lacking. Thus, the investigation of different methods for the purification of lactic acid was one of the aims of this work. The pH-stat strategy showed the maximum production of lactic acid of 143.7 g/L. Following purification of the lactic acid sample, recovery of reducing sugars and protein and color removal were 0.28%, 100% and 100%, respectively.


Subject(s)
Lactic Acid/biosynthesis , Lacticaseibacillus rhamnosus/metabolism , Industrial Waste , Temperature , Biodegradation, Environmental , Biotransformation , Adsorption , Fermentation , Batch Cell Culture Techniques , Hydrogen-Ion Concentration
13.
Electron. j. biotechnol ; 19(4): 62-67, July 2016. ilus
Article in English | LILACS | ID: lil-793954

ABSTRACT

Background: A thermostable lipase MAS1 from marine Streptomyces sp. strain was considered as a potential biocatalyst for industrial application, but its production level was relatively low. Here, the effect of chaperones co-expression on the secretory expression of lipase MAS1 in Pichia pastoris was investigated. Result: Co-expression of protein disulfide isomerase (PDI), HAC1 and immunoglobulin binding protein could increase the expression level of lipase MAS1, whereas co-expression of Vitreoscilla hemoglobin showed a negative effect to the lipase MAS1 production. Among them, PDI co-expression increased lipase MAS1 expression level by 1.7-fold compared to the control strain harboring only the MAS1 gene. Furthermore, optimizing production of lipase MAS1 with Pichia pastoris strain X-33/MAS1-PDI in a 30-L bioreactor were conducted. Lower induction temperature was found to have a benefit effect for lipase MAS1 production. Lipase activity at 24 and 22°C showed 1.7 and 2.1-fold to that at 30°C, respectively. Among the induction pH tested, the highest lipase activity was obtained at pH 6.0 with activity of 440 U/mL after 144 h fermentation. Conclusion: Our work showed a good example for improving the production of recombinant enzymes in Pichia pastoris via chaperon co-expression and fermentation condition optimization.


Subject(s)
Streptomyces/metabolism , Lipase/biosynthesis , Pichia , Temperature , Cell Survival , Molecular Chaperones , Bioreactors , Protein Disulfide-Isomerases , Vitreoscilla , Fermentation , Batch Cell Culture Techniques , Hydrogen-Ion Concentration
14.
Braz. j. microbiol ; 46(3): 893-902, July-Sept. 2015. tab, ilus
Article in English | LILACS | ID: lil-755836

ABSTRACT

The production of lactic acid from date juice by Lactobacillus caseisubsp. rhamnosus in batch and fed-batch cultures has been investigated. The fed-batch culture system gave better results for lactic acid production and volumetric productivity. The aim of this work is to determine the effects of the feeding rate and the concentration of the feeding medium containing date juice glucose on the cell growth, the consumption of glucose and the lactic acid production by Lactobacillus casei subsp. rhamnosus in fed-batch cultures. For this study, two concentrations of the feeding medium (62 and 100 g/L of date juice glucose) were tested at different feeding rates (18, 22, 33, 75 and 150 mL/h). The highest volumetric productivity (1.3 g/L.h) and lactic acid yield (1.7 g/g) were obtained at a feeding rate of 33 mL/h and a date juice glucose concentration of 62 g/L in the feeding medium. As a result, most of the date juice glucose was completely utilised (residual glucose 1 g/L), and a maximum lactic acid production level (89.2 g/L) was obtained.

.


Subject(s)
Batch Cell Culture Techniques , Lactic Acid/metabolism , Lacticaseibacillus casei/metabolism , Lacticaseibacillus rhamnosus/metabolism , Phoeniceae/metabolism , Fermentation , Plant Extracts/metabolism
15.
Electron. j. biotechnol ; 18(4): 273-280, July 2015. ilus, graf, tab
Article in English | LILACS | ID: lil-757863

ABSTRACT

Background In the field of microbial fermentation technology, how to optimize the fermentation conditions is of great crucial for practical applications. Here, we use artificial neural networks (ANNs) and support vector machine (SVM) to offer a series of effective optimization methods for the production of iturin A. The concentration levels of asparagine (Asn), glutamic acid (Glu) and proline (Pro) (mg/L) were set as independent variables, while the iturin A titer (U/mL) was set as dependent variable. General regression neural network (GRNN), multilayer feed-forward neural networks (MLFNs) and the SVM were developed. Comparisons were made among different ANNs and the SVM. Results The GRNN has the lowest RMS error (457.88) and the shortest training time (1 s), with a steady fluctuation during repeated experiments, whereas the MLFNs have comparatively higher RMS errors and longer training times, which have a significant fluctuation with the change of nodes. In terms of the SVM, it also has a relatively low RMS error (466.13), with a short training time (1 s). Conclusion According to the modeling results, the GRNN is considered as the most suitable ANN model for the design of the fed-batch fermentation conditions for the production of iturin A because of its high robustness and precision, and the SVM is also considered as a very suitable alternative model. Under the tolerance of 30%, the prediction accuracies of the GRNN and SVM are both 100% respectively in repeated experiments.


Subject(s)
Peptides, Cyclic , Neural Networks, Computer , Algorithms , Fermentation , Batch Cell Culture Techniques , Support Vector Machine
16.
Electron. j. biotechnol ; 18(2): 128-133, Mar. 2015. ilus, graf, tab
Article in English | LILACS | ID: lil-745581

ABSTRACT

Background The production of biofuels from renewable energy sources is one of the most important issues in biotechnology today. The process is known to generate various by-products, for example glycerol that is obtained in the making of biodiesel from rapeseed oil. Crude glycerol may be utilized in many ways, including microbial conversion to 1,3-propanediol. The main drawback of that technology is the use of high concentrations of glycerol, which inhibits the growth of bacterial cells. Results This study investigated the impact of crude glycerol on Clostridium butyricum DSP1 and its ability to adapt to an environment of high osmotic pressure. It was found that a crude glycerol concentration of up to 70 g/L did not have an inhibitory effect on C. butyricum DSP1. Adaptation procedures involving the passage of metabolically active biomass from a fermentation medium with a lower concentration of crude glycerol to one with a greater substrate concentration allowed breaking the barrier of high osmotic pressure (150 g/L crude glycerol) and receiving a 1,3-PD concentration of 74 g/L in a batch culture operation. The work looked into intracellular modifications shown by proteomic profiling in order to explain the mechanisms underlying the response and adaptation of bacterial cells exposed to unfavorable environmental conditions. Conclusions This study of the effect of glycerol on the growth and metabolism of C. butyricum DSP1 demonstrated that the maximum substrate concentrations that do not inhibit the metabolic activity of bacterial cells are 90 g/L and 70 g/L for pure and crude glycerol, respectively.


Subject(s)
Adaptation, Physiological , Clostridium butyricum/growth & development , Clostridium butyricum/metabolism , Glycerol/metabolism , Osmotic Pressure , Propylene Glycols , Stress, Physiological , Proteins/analysis , Environment , Biofuels , Fermentation , Batch Cell Culture Techniques , Glycerol/analysis
17.
Chinese Journal of Biotechnology ; (12): 206-219, 2015.
Article in Chinese | WPRIM | ID: wpr-345513

ABSTRACT

Coenzyme Q10 (CoQ10) is a lipophilic antioxidant that improves human immunity, delays senility and enhances the vitality of the human body and has wide applications in pharmaceutical and cosmetic industries. Microbial fermentation is a sustainable way to produce CoQ10, and attracts increased interest. In this work, the native CoQ8 synthetic pathway of Escherichia coli was replaced by the CoQ10 synthetic pathway through integrating decaprenyl diphosphate synthase gene (dps) from Rhodobacter sphaeroides into chromosome of E. coli ATCC 8739, followed by deletion of the native octaprenyl diphosphate synthase gene (ispB). The resulting strain GD-14 produced 0.68 mg/L CoQ10 with a yield of 0.54 mg/g DCW. Modulation of dxs and idi genes of the MEP pathway and ubiCA genes in combination led to 2.46-fold increase of CoQ10 production (from 0.54 to 1.87 mg/g DCW). Recruiting glucose facilitator protein of Zymomonas mobilis to replace the native phosphoenolpyruvate: carbohydrate phosphotransferase systems (PTS) further led to a 16% increase of CoQ10 yield. Finally, fed-batch fermentation of the best strain GD-51 was performed, which produced 433 mg/L CoQ10 with a yield of 11.7 mg/g DCW. To the best of our knowledge, this was the highest CoQ10 titer and yield obtained for engineered E. coli.


Subject(s)
Alkyl and Aryl Transferases , Genetics , Bacterial Proteins , Genetics , Batch Cell Culture Techniques , Escherichia coli , Genetics , Metabolism , Fermentation , Gene Deletion , Industrial Microbiology , Metabolic Engineering , Rhodobacter sphaeroides , Genetics , Ubiquinone , Zymomonas , Genetics
18.
Chinese Journal of Biotechnology ; (12): 431-435, 2015.
Article in Chinese | WPRIM | ID: wpr-240630

ABSTRACT

To enhance the production of ε-poly-L-lysine (ε-PL) by improving dissolved oxygen level of the fermentation system, different oxygen-vectors were added to broth and n-dodecane was screened as the best oxygen-vector. The best amount of n-dodecane was 0.5% (V/V) and the best time was at start of the fermentation. In a fed-batch fermentation in a 5 L bioreactor, ε-PL concentration reached a maximum of (30.8 ± 0.46) g/L and the dry cell weight obtained was (33.8 ± 0.29) g/L, increasing by 31.6% and 20.7% compared with the control group, respectively. This improvement can be related to 0.5% n-dodecane could maintain dissolved oxygen concentration > 32% of air concentration compared with 23.8% in ε-PL production phase, and the production of a main by-product, poly-L-diaminopropionic acid, fell by 31%. These results indicated that the dissolved oxygen level in the broth was improved by adding n-dodecane, which can inhibit the by-product production and improve the biosynthesis of ε-PL.


Subject(s)
Alkanes , Chemistry , Batch Cell Culture Techniques , Bioreactors , Fermentation , Oxygen , Chemistry , Polylysine
19.
Chinese Journal of Biotechnology ; (12): 752-756, 2015.
Article in Chinese | WPRIM | ID: wpr-240602

ABSTRACT

During the production of ε-poly-L-lysine (ε-PL) in fed-batch fermentation, the decline of ε-PL synthesis often occurs at middle or late phase of the fermentation. To solve the problem, we adopted two strategies, namely pH shift and feeding yeast extract, to improve the productivity of ε-PL. ε-PL productivity in fermentation by pH shift and feeding yeast extract achieved 4.62 g/(L x d) and 5.16 g/(L x d), which were increased by 27.3% and 42.2% compared with the control ε-PL fed-batch fermentation, respectively. Meanwhile, ε-PL production enhanced 36.95 g/L and 41.32 g/L in 192 h with these two strategies, increased by 27.4% and 42.48% compared to the control, respectively. ε-PL production could be improved at middle or late phase of fed-batch fermentation by pH shift or feeding yeast extract.


Subject(s)
Batch Cell Culture Techniques , Fermentation , Industrial Microbiology , Nitrogen , Chemistry , Polylysine
20.
Chinese Journal of Biotechnology ; (12): 1050-1062, 2015.
Article in Chinese | WPRIM | ID: wpr-240597

ABSTRACT

Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suitable for future industrial development. In order to develop a relatively safe microbial strain for the production of rhamnolipid biosurfactant, we constructed engineered Escherichia coli strains for rhamnolipid production by expressing different copy numbers of rhamnosyltransferase (rhlAB) gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhlAB gene and rhaBDAC gene cluster for dTDP-1-rhamnose biosynthesis with different synthetic promoters, and obtained the best engineered strain-E. coli TIB-RAB226. Through the optimization of culture temperature, the titer of rhamnolipd reached 124.3 mg/L, 1.17 fold higher than that under the original condition. Fed-batch fermentation further improved the production of rhamnolipid and the titer reached the highest 209.2 mg/L within 12 h. High performance liquid chromatography-mass spectrometry (LC-MS) analysis showed that there are total 5 mono-rhamnolipid congeners with different nuclear mass ratio and relative abundance. This study laid foundation for heterologous biosynthesis of rhanomilipd.


Subject(s)
Bacterial Proteins , Genetics , Batch Cell Culture Techniques , Decanoates , Escherichia coli , Metabolism , Fermentation , Glycolipids , Hexosyltransferases , Genetics , Industrial Microbiology , Methods , Multigene Family , Promoter Regions, Genetic , Pseudomonas aeruginosa , Rhamnose , Surface-Active Agents , Metabolism
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