Shake Cultures Research Articles

High Yield of Recombinant Protein in Shaken E. coli Cultures with Enzymatic Glucose Release Medium EnPresso B.

Expression of recombinant proteins in sufficient quantities is essential for protein structure-function studies. The most commonly used method for recombinant protein production is overexpression in E. coli cultures. However, producing high yields of functional proteins in E. coli can be a challenge in conventional shaken cultures. This is often due to nonoptimal growth conditions, which result in low cell yields and insoluble or incorrectly folded target protein. To overcome the shortcomings of shake flask cultivation, we present a cultivation method based on enzymatic glucose delivery. This system mimics the fed-batch principle used in bioreactor cultivations and provides high yields of biomass and recombinant proteins in shaken cultivations.

Three-Dimensional Construction of a Rabbit Anterior Corneal Replacement for Lamellar Keratoplasty.

The aim of this study was to construct a rabbit anterior corneal replacement for transplantation using acellular porcine corneal matrix (APCM) and rabbit epithelial or stromal cells. APCM was prepared from fresh porcine cornea treated with 0.5% (wt./vol.) sodium dodecyl sulfate (SDS) solution. The expanded stromal cells were first injected into APCM parallel to its surface and were cultured in a shaking culture system for 7 days to obtain the stromal construct. Next, corneal epithelial cells were cultured on the stromal construct surface for another 7 days to obtain rabbit anterior corneal lamella. The construct had a phenotype similar to that of normal cornea, with high expression of cytokeratin 3 in the epithelial cell layer and vimentin in the stromal cells. More importantly, the construct integrated well with the implanted host corneal tissue, and the implant cornea maintained transparency in the 6-month follow-up, although there was a slight haze in the central corneal area. The endothelium in the surgery cornea had a similar cell density and mosaic pattern with normal cornea as shown by confocal laser corneal microscopy, and the regenerated corneal epithelial cells on the implant surface showed a similar morphology to that of natural epithelial cells. These results demonstrate that the constructed anterior corneal replacement exhibits an excellent biological property for lamellar keratoplasty and might be a possible alternative to human corneal tissue in the future.

Production of flavor compounds from olive mill waste by Rhizopus oryzae and Candida tropicalis.

The purpose of this study was to investigate the production of flavor compounds from olive mill waste by microbial fermentation of Rhizopus oryzae and Candida tropicalis. Olive mill waste fermentations were performed in shake and bioreactor cultures. Production of flavor compounds from olive mill waste was followed by Gas Chromatography–Mass spectrometry, Gas chromatography- olfactometry and Spectrum Sensory Analysis®. As a result, 1.73-log and 3.23-log cfu/mL increases were observed in the microbial populations of R. oryzae and C. tropicalis during shake cultures, respectively. C. tropicalis can produce a higher concentration of d-limonene from olive mill waste than R. oryzae in shake cultures. The concentration of d-limonene was determined as 185.56 and 249.54 μg/kg in the fermented olive mill waste by R. oryzae and C. tropicalis in shake cultures respectively. In contrast, R. oryzae can produce a higher concentration of d-limonene (87.73 μg/kg) d-limonene than C. tropicalis (11.95 μg/kg) in bioreactor cultures. Based on sensory analysis, unripe olive, wet towel, sweet aromatic, fermented aromas were determined at high intensity in olive mill waste fermented with R. oryzae meanwhile olive mill waste fermented with C. tropicalis had only a high intensity of unripe olive and oily aroma.

Bioconversion of soybean and rice hull hydrolysates into ethanol and xylitol by furaldehyde-tolerant strains of Saccharomyces cerevisiae, Wickerhamomyces anomalus, and their cofermentations

The aims of this work were to evaluate the ability of furaldehyde-tolerant yeast strains Saccharomyces cerevisiae P6H9 and Wickerhamomyces anomalus WA-HF5.5 and their cofermentations and to convert soybean and rice hull hydrolysates into ethanol and xylitol. In batch shaker cultures, the strains showed the ability to tolerate high osmotic pressure (1918 mOsmkg−1), completely depleting furaldehyde in the first 12 h of cultivations, while converting the hydrolysate sugars into ethanol. Highest ethanol yields of 0.37 g g−1 and productivity of 0.31 g L−1 h−1 were obtained in the cofermentation using rice hull hydrolysate as substrate. The concentration of sugars in soybean hull hydrolysate proved to be inadequate as substrate for the cultivation of these strains, showing a low ethanol productivity of 0.08 g L−1 h−1. Bioreactor cultivations of S. cerevisiae on rice hull hydrolysate under anaerobiosis showed a relatively high ethanol productivity of 6.7 g L−1 h−1, whereas the bioreactor cofermentation produced xylitol to yields of 0.86 g g−1 under conditions of oxygen limitation.

The production and antiprotozoal activity of abietane diterpenes in Salvia austriaca hairy roots grown in shake flasks and bioreactor

ABSTRACTThe biomass and concentration of bioactive quinone methide-type diterpenes in hairy roots of Salvia austriaca were determined and compared with levels of these metabolites in roots of field-grown plants. The cultures were maintained in shake flasks and a nutrient sprinkle bioreactor. Diterpene production was more efficient in the shake flask root culture than the bioreactor one. Biomass and diterpene production within the shake flask culture was evaluated using Schenk and Hildebrandt (SH), Gamborg (B5), and woody plant medium (WPM), with both full- and half-strength macro and micronutrient concentrations (1/2 SH, 1/2 B5, and 1/2 WPM). Among the tested media, SH medium proved to be most effective for biomass and diterpene production. In this medium, the transformed roots accumulated the levels of taxodone (3.89 mg g−1 DW; equivalent to 63.3 mg L−1), taxodione (1.15 mg g−1 DW; equivalent to 17.4 mg L−1), 15-deoxy-fuerstione (2.15 mg g−1 DW; equivalent to 32.5 mg L−1), and 7-(2′-oxohexyl)-taxodione (0.076 mg g−1 DW; equivalent to 1.1 mg L−1). Three diterpenes were also detected in the roots of S. austriaca intact plants, but their concentrations were lower than those in hairy root culture. No 7-(2′-oxohexyl)-taxodione was found in the roots of field-grown plants. The hairy roots were able to maintain high metabolite levels even for 6 years of cultivation. Taxodone, taxodione, 15-deoxy-fuerstione, and 7-(2′-oxohexyl)-taxodione were tested for in vitro activity against Trypanosoma brucei rhodesiense, T. cruzi, and Plasmodium falciparum and their cytotoxicity was determined using L6 cells. Among these compounds, taxodione was the most active against T. brucei rhodesiense [IC50 = 0.05 µM with high selectivity, selectivity index (SI) = 38]. Taxodione was found to inhibit the growth of P. falciparum and T. cruzi by 50% at respective concentrations of 1.9 and 7.1 µM (SI values of 1.0 and 0.27). Other diterpenoids demonstrated weaker activity against tested parasites (IC50 values ranging from 0.62 to 194.7 µM) and lower selectivity (SI value ranged from 0.4 to 5.0).

Shoot Growth and Corm Enlargement of Freesia ‘Shiny Gold’ by Liquid Shaking Culture

Shoot Growth and Corm Enlargement of Freesia ‘Shiny Gold’ by Liquid Shaking Culture

Genome sequencing and biodegradation characteristics of the n-butyl benzyl phthalate degrading bacterium-Rhodococcus sp. HS-D2

The n-butyl benzyl phthalate (BBP) is an environmental pollutant used extensively in the manufacturing of plastics. For the bioremediation of phthalic acid ester pollutants in water, sediment, and soil, a BBP degrading bacterium Rhodococcus sp. HS-D2 was isolated from contaminated river sediment and characterized. The HS-D2 strain is capable of utilizing BBP as the sole source of carbon. A shaken culture containing 500 mgL−1 of BBP produced complete degradation in 96 h. To study the metabolic characteristics and potential strategies for enhancing the biodegradation rates of BBP, a genome-scale metabolic model (GEM) of Rhodococcus sp. iYZ1601 was reconstructed based on the genome sequence of strain HS-D2. It included several sequential transporters and hydrolases were involved in the biodegradation process of BBP. Monoethylhexylphthalate (MEHP) and phthalic acid ester (PAE) hydrolases were confirmed as the key enzymes in phthalate degradation. The MEHP and PAE hydrolases catalyzed the conversion of BBP to butanal, phenylcarbinol, and phthalate as verified by HPLC analysis. The growth rate of HS-D2 and BBP consumption rate were analyzed in silico simulation, and were found to be consistent with the rates of HS-D2 growth and BBP consumption the in vitro experiment. In summary, Rhodococcus sp. HS-D2 biodegrades BBP, but the metabolic pathway of this bacterium needs further exploration to improve the biodegradation efficiency.

Enhancing fatty acid production in Escherichia coli by Vitreoscilla hemoglobin overexpression.

Our recent 13 C-metabolic flux analysis (13 C-MFA) study indicates that energy metabolism becomes a rate-limiting factor for fatty acid overproduction in E. coli strains (after "Push-Pull-Block" based genetic modifications). To resolve this bottleneck, Vitreoscilla hemoglobin (VHb, a membrane protein facilitating O2 transport) was introduced into a fatty-acid-producing strain to promote oxygen supply and energy metabolism. The resulting strain, FAV50, achieved 70% percent higher fatty acid titer than the parent strain in micro-aerobic shake tube cultures. In high cell-density bioreactor fermentations, FAV50 achieved free fatty acids at a titer of 7.02 g/L (51% of the theoretical yield). In addition to "Push-Pull-Block-Power" strategies, our experiments and flux balance analysis also revealed the fatty acid over-producing strain is sensitive to metabolic burden and oxygen influx, and thus a careful evaluation of the cost-benefit tradeoff with the guidance of fluxome analysis will be fundamental for the rational design of synthetic biology strains. Biotechnol. Bioeng. 2017;114: 463-467. © 2016 Wiley Periodicals, Inc.

Optimization of cellulase production by Penicillium sp.

The production of cellulolytic enzymes (β-exoglucanase, β-endoglucanase and β-glucosidase) by Penicillium sp. on three different media in liquid shake culture conditions was compared. The organism exhibited relatively highest activity of endoglucanase among three enzymes measured at 7-day interval during the course of its growth on Czapek-Dox medium supplemented with 0.5 % (w/v) cellulose. Cellulose at 0.5 %, lactose at 0.5 %, sawdust at 0.5 %, yeast extract at 0.2 % as a nitrogen source, pH 5.0 and 30 °C temperature were found to be optimal for growth and cellulase production by Penicillium sp. Yields of Fpase, CMCase and β-glucosidase, attained on optimized medium with Penicillium sp. were 8.7, 25 and 9.52 U/ml, respectively with increment of 9.2, 5.9 and 43.8-folds over titers of the respective enzyme on unoptimised medium. Cellulase of the fungal culture with the ratio of β-glucosidase to Fpase greater than one will hold potential for biotechnological applications.

Preparation of an inoculum of Gluconacetobacter xylinus without mutants in shaken culture.

A high-quality inoculum of Gluconacetobacter xylinus is important to produce bacterial cellulose (BC), a versatile biomaterial. This work aims to develop a method of preparing an inoculum of this bacterium with high cell density and without mutants. Inocula of G.xylinus ACCC 10220 without and with cellulase or carboxymethyl cellulose (CMC) were prepared in shaken culture. BC pellets and BC-negative mutants were present in the inoculum without additives but absent in the inoculum with additives. Based on BC weights statically produced in fresh BC-producing media initiated by different seed culture, the 24-h-shaken inoculum with 1·50% (w/v) CMC was the best because of high biomass and absence of mutants. The BC weights in fresh media inoculated by the 96-h-static inoculum and 24-h-shaken CMC inoculum at 7% (v/v) were 0·70 and 1·05gl(-1) , respectively, implying significant difference (P<0·01) in BC weights. However, structure properties of the two BC samples, including the crystallinity index, mass fraction of cellulose Iα , degree of polymerization (DP) and micromorphology were slightly different. The 24-h-shaken CMC inoculum was the most suitable for a starter culture of BC. A novel method of preparing G.xylinus inoculum in shaken culture was developed, featuring high biomass, absence of mutants and no BC entanglements. Cellulase or CMC added into the medium completely suppressed mutation of G.xylinus, and CMC facilitated to form colloidal BC with the low DP in shaken culture, indicating less BC stress to cells. These findings suggested the mutation could be induced by BC stress, and not by shear stress commonly accepted.

Efficient very-high-gravity fermentation of sugarcane molasses by industrial yeast strains

In this study industrial strains were inoculated, in successive cycles, at high cell density into a sugarcane-based juice containing normal (22%, w/v) and very high sucrose (30%, w/v) levels and supplemented with peptone as a nitrogen source. At 30 °C, in shaken cultures, in the normal gravity situation, efficient sucrose utilization occurred in both the supplemented and unsupplemented medium. At higher sugar concentrations, supplementation with peptone induced a more efficient fermentation compared with the unsupplemented medium, with higher biomass accumulation and maintenance of cell viability, and ethanol levels as high as 16% (v/v). Trehalose was also high during the cycles, probably as a necessary response of the yeast to the high stress fermentation conditions. This suggests that it is possible to increase ethanol production by using very-high-fermentation technology and that nutritional supplementation would have a positive effect on the fermentation, allowing for efficient sugar consumption and cell viability maintenance. Copyright © 2016 The Institute of Brewing & Distilling

The method used to culture host cells (Sf9 cells) can affect the qualities of baculovirus budding particles expressing recombinant proteins.

Budded virus (BV) particles of baculovirus (Autographa californica nucleopolyhedrovirus, AcNPV) are harvested from the supernatant of liquid culture of Sf9 host cells by ultracentrifugation. Using polyacrylamide gel electrophoresis, Western blot and transmission electron microscopy (TEM) of BV samples fractionated closely by sucrose density gradient centrifugation, we observed that BVs exhibited different qualities depending on whether they had been harvested from the supernatant from a standing (static), shaking (suspension), or standing/shaking (pre-/post-infection) culture of Sf9 cells. The amount of BV protein apparently increased in the order of standing, standing/shaking, and shaking procedure, and the yield of intact particles showed an opposite trend. TEM observation clearly showed that appropriate fractions of the standing and standing/shaking cultures contained more intact BV particles than those from the shaking culture. These results suggest that the qualities of recombinant BV particles may be related to the culture conditions of the host cells.

Fabrication of bone marrow-like tissue invitro from dispersed-state bone marrow cells.

A three-dimensional (3D) bone marrow (BM) culture system may facilitate research into the molecular mechanisms involved in hematopoiesis and BM diseases. However, because >90% of BM cells are composed of non-adherent blood cells, it is difficult to organize the dispersed BM cells into 3D multicellular spheroids using conventional aggregation methods such as hanging drop, and rotary shaking culture. The objective of this study was to reproduce BM-like tissue. We reported successful formation of BM aggregates using a 3% methylcellulose (MC) medium. This medium could aggregate even non-adherent materials. In MC medium, BM cells formed tissue-like aggregates within 24 h. Although the cell density of the BM-like tissue is slightly low, sections of the organoids resembled those of intact BM tissue. Cells of the BM-like tissue were approximately 70% viable after 7 days in culture. Staining for CD68, PDGFRα, and CXCL12 indicated that the BM-like tissue contained macrophages, and mesenchymal cells including CXCL12-abundant reticular cells. These results indicated that the method using MC medium effectively reconstitutes the BM-like tissue.

Diversity in yeast-mycelium dimorphism response of the Dutch elm disease pathogens: the inoculum size effect.

Dutch elm disease (DED) is caused by the dimorphic fungi Ophiostoma ulmi, Ophiostoma novo-ulmi, and Ophiostoma himal-ulmi. A cell population density-dependent phenomenon related to quorum sensing was previously shown to affect the reversible transition from yeast-like to mycelial growth in liquid shake cultures of O. novo-ulmi NRRL 6404. Since the response to external stimuli often varies among DED fungal strains, we evaluated the effect of inoculum size on 8 strains of the 3 species of DED agents by determining the proportion of yeast and mycelium produced at different spore inoculum concentrations in defined liquid shake medium. The results show that not all DED fungi strains respond similarly to inoculum size effect, since variations were observed among strains. It is thus possible that the different strains belonging to phylogenetically close species use different signalling molecules or molecular signalling pathways to regulate their growth mode via quorum-sensing mechanisms.

Functions of VPA1418 and VPA0305 Catalase Genes in Growth of Vibrio parahaemolyticus under Oxidative Stress.

The marine foodborne enteropathogen Vibrio parahaemolyticus has four putative catalase genes. The functions of two katE-homologous genes, katE1 (VPA1418) and katE2 (VPA0305), in the growth of this bacterium were examined using gene deletion mutants with or without complementary genes. The growth of the mutant strains in static or shaken cultures in a rich medium at 37°C or at low temperatures (12 and 4°C), with or without competition from Escherichia coli, did not differ from that of the parent strain. When 175 μM extrinsic H2O2 was added to the culture medium, bacterial growth of the ΔkatE1 strain was delayed and growth of the ΔkatE1 ΔkatE2 and ΔkatE1 ΔahpC1 double mutant strains was completely inhibited at 37°C for 8 h. The sensitivity of the ΔkatE1 strain to the inhibition of growth by H2O2 was higher at low incubation temperatures (12 and 22°C) than at 37°C. The determined gene expression of these catalase and ahpC genes revealed that katE1 was highly expressed in the wild-type strain at 22°C under H2O2 stress, while the katE2 and ahpC genes may play an alternate or compensatory role in the ΔkatE1 strain. This study demonstrated that katE1 encodes the chief functional catalase for detoxifying extrinsic H2O2 during logarithmic growth and that the function of these genes was influenced by incubation temperature.

Chemical Mutagenesis for Improvement of Production of a Biologically Active Extracellular Polymeric Substance by Halomonas xianhensis SUR308

A moderately halophilic bacterium, Halomonas xianhensis SUR308 (Genbank Accession No. KJ933394) isolated from solar saltern of Surala, Odisha, India was found to produce significant amount of a biologically active extracellular polymeric substances (EPS). Under optimized cultural conditions, the strain was able to produce 7.05 g L-1 of EPS after 8 days of incubation under shake culture. To enhance the EPS production efficiency, the isolate SUR308 was subjected to chemical mutagenesis following the use of hydroxylamine (HA), acridine orange (AO) and ethyl methanesulfonate (EMS). Evaluation of the toxicity of these mutagens indicated that EMS was comparatively toxic but effective for mutagenesis. Mutagenic treatments have resulted in the isolation of 58 amoxycillin resistant morphologically distinct phenotypes. Three of these mutant phenotypes (EM3, EM13 and HM6) were capable of producing 12.98, 11.97 and 11.21 g L-1 of EPS respectively in malt extract-yeast extract medium supplemented with casein hydrolysate with significant reduction of incubation period. Compared to wild type strain, mutant strains were found to produce 1.6-1.8 fold more EPS. However, the physico-chemical nature of the EPS derived from the mutants remained unaltered when compared with that of the wild type strain. These findings, therefore, have more impact on production economy of EPS for biotechnological applications.

Effect of linoleic acid on reproduction and yeast–mycelium dimorphism in the Dutch elm disease pathogens

Elm populations from North America and Europe were devastated by Dutch elm disease (DED), which is a vascular disease caused by fungi from the genus Ophiostoma (Ascomycota). These pathogens feature a yeast–mycelium dimorphism that may be related to virulence by facilitating colonization of the host xylem. Cyclooxygenases (COX) have been proposed to modulate yeast–mycelium dimorphism of DED fungi, and homologs of cox genes have been found in the nuclear genome of O. novo-ulmi subsp. novo-ulmi. Linoleic acid, a substrate for COX, was reported to stimulate the formation of asexual and sexual reproduction structures in DED strains grown on complex media. We hypothesized that linoleic acid also induced mycelium production in liquid shake culture conditions. Linoleic acid was found to enhance the production of reproductive structures in sexual crosses conducted on a complex medium (elm sapwood agar), but was not sufficient for these structures to form on a minimal medium. In liquid shake cultures grown in a minimal medium, the addition of linoleic acid stimulated mycelial formation. Our results suggest that linoleic acid plays a role in reproduction and dimorphism in the DED pathogens.

Survival of probiotic lactic acid bacteria immobilized in different forms of bacterial cellulose in simulated gastric juices and bile salt solution

Abstract In this work, we discuss the possibilities of using bacterial cellulose (BC) produced by Gluconacetobacter xylinus as carrier support for the immobilization of probiotic strains of Lactobacillus spp. In our study, immobilization of the microorganisms was performed by the adsorption of bacterial cells on the surface of the synthetized BC and by a simultaneous cultivation of the probiotic bacteria with cellulose-synthetizing G. xylinus . Co-cultures were conducted in stationary cultures, in which BC was synthesized as pellicles on the medium surface and in shaken cultures in which BC was synthetized in the forms of beads. The experiments carried out also included the analysis of the survival of immobilized probiotic bacteria in the presence of gastric juices and bile salts solution. The results showed that immobilization of probiotic Lactobacillus in BC during co-culture with G. xylinus was the most effective immobilization method, providing high-level protection of the microorganisms against the influence of gastric juices and bile salts.

Engineering selection stringency on expression vector for the production of recombinant human alpha1-antitrypsin using Chinese Hamster Ovary cells

Background: Expression vector engineering technology is one of the most convenient and timely method for cell line development to meet the rising demand of novel production cell line with high productivity. Destabilization of dihydrofolate reductase (dhfr) selection marker by addition of AU-rich elements and murine ornithine decarboxylase PEST region was previously shown to improve the specific productivities of recombinant human interferon gamma in CHO-DG44 cells. In this study, we evaluated novel combinations of engineered motifs for further selection marker attenuation to improve recombinant human alpha-1-antitrypsin (rhA1AT) production. Motifs tested include tandem PEST elements to promote protein degradation, internal ribosome entry site (IRES) mutations to impede translation initiation, and codon-deoptimized dhfr selection marker to reduce translation efficiency. Results: After a 2-step methotrexate (MTX) amplification to 50 nM that took less than 3 months, the expression vector with IRES point mutation and dhfr-PEST gave a maximum titer of 1.05 g/l with the top producer cell pool. Further MTX amplification to 300 nM MTX gave a maximum titer of 1.15 g/l. Relative transcript copy numbers and dhfr protein expression in the cell pools were also analysed to demonstrate that the transcription of rhA1AT and dhfr genes were correlated due to the IRES linkage, and that the strategies of further attenuating dhfr protein expression with the use of a mutated IRES and tandem PEST, but not codon deoptimization, were effective in reducing dhfr protein levels in suspension serum free culture. Conclusions: Novel combinations of engineered motifs for further selection marker attenuation were studied to result in the highest reported recombinant protein titer to our knowledge in shake flask batch culture of stable mammalian cell pools at 1.15 g/l, highlighting applicability of expression vector optimization in generating high producing stable cells essential for recombinant protein therapeutics production. Our results also suggest that codon usage of the selection marker should be considered for applications that may involve gene amplification and serum free suspension culture, since the overall codon usage and thus the general expression and regulation of host cell proteins may be affected in the surviving cells.

Lipase production by a Tunisian Fusarium solani strain cultivated on olive oil wastewater-based media and a biotreatment assay

The ability of a lipolytic fungus, Fusarium solani, to grow on olive mill wastewater (OMW)-based medium and to produce a high value compound, while degrading this waste was tested. OMW with high values of chemical oxygen demand (COD) was supplemented with yeast extract to ensure the growth of this fungus. In shake flask batch cultures, OMW led to a lipase activity of about 14 U/ml. F. solani growth on OMW showed high oil and COD removal efficiencies of 100 and 24.1%, respectively. A maximum phenolic compounds removal, about 46% of the initial content, was achieved and a decrease in 68% of total sugars was registered at the second day of culture.