共查询到20条相似文献,搜索用时 31 毫秒
1.
Tobias Ladner David Flitsch Mihaly Lukacs Michaela Sieben Jochen Büchs 《Biotechnology and bioengineering》2019,116(12):3215-3227
Oxygen supply is one of the most critical process parameters in aerobic cultivations. To assure sufficient oxygen supply, shake flasks are usually used in combination with orbital shaking machines. In this study, a measurement technique for the dissolved oxygen tension (DOT) in shake flask cultures with viscosity changes is presented. The movement of the shaker table is monitored by means of a Hall effect sensor. For DOT measurements, infrared fluorescent oxygen-sensitive nanoparticles are added to the culture broth. The position of the rotating bulk liquid needs to be determined to assure measurements inside the liquid. The leading edge of the bulk liquid is detected based on the fluorescence signal intensity of the oxygen-sensitive nanoparticles. Furthermore, online information about the viscosity of the culture broth is acquired due to the detection of the position of the leading edge of the bulk liquid relative to the direction of the centrifugal force, as described by Sieben et al. (2019. Sci. Rep., 9, 8335). The DOT measurement is combined with a respiration activity monitoring system which allows for the determination of the oxygen transfer rate (OTR) in eight parallel shake flasks. Based on DOT and OTR, the volumetric oxygen transfer coefficient (kLa) is calculated during cultivation. The new system was successfully applied in cultivations of Escherichia coli, Bacillus licheniformis, and Xanthomonas campestris. 相似文献
2.
Konstantin Schneider Verena Schütz Gernot Thomas John Elmar Heinzle 《Bioprocess and biosystems engineering》2010,33(5):541-547
We describe a new device with parallel optical measurement of dissolved oxygen (DO) and pH in up to nine shake flasks applicable
in any conventional shaking incubator. Measurement ranges are 0–500% of air saturation for oxygen and 5.5–8.5 for pH. It was
used to characterize growth profiles of different l-lysine producing strains of Corynebacterium glutamicum, of Saccharomyces cerevisiae and of Escherichia coli. Cultures in unbaffled flasks were highly reproducible. Oxygen limitation was indicated online which is particularly important
when cultivating fast growing cells as E. coli. C. glutamicum strains showed distinct characteristic patterns of DO and pH indicating biological events. During the cultivation of S. cerevisiae on glucose, fructose and galactose, oxygen uptake rate was determined using the predetermined value of k
L
a. pH measurement was used to determine the minimum buffer requirement for a culture of C. glutamicum. 相似文献
3.
Tobias Habicher Vroni Czotscher Tobias Klein Andreas Daub Timm Keil Jochen Büchs 《Biotechnology and bioengineering》2019,116(9):2250-2262
Bioprocesses operated in batch mode can induce adverse effects like overflow metabolism, substrate inhibition, osmotic inhibition, oxygen limitation, and catabolite repression. To avoid these adverse effects, fed-batch is the predominant operation mode in industrial production. Nevertheless, screening for optimal production strains is usually performed in microtiter plates and shake flasks operated in batch mode without any online monitoring. Recently, a polymer-based controlled-release fed-batch microtiter plate with stable glucose release characteristics was described. In this study, a glucose-containing polymer matrix was used to manufacture polymer rings that were placed at the bottom of a 48-well microtiter plate. Thereby, the liquid content of the well became accessible for optical measurement by the BioLector device. Reflections caused by the polymer ring were minimized by adjusting the scattered-light measurement position. Influences on the measurement of the dissolved oxygen tension and pH could be avoided by choosing appropriate polymer-ring geometries. These adjustments enabled parallel online measurement of scattered light, fluorescence, dissolved oxygen tension, and pH of Escherichia coli BL21 (DE3) fed-batch cultivations. The online monitoring and fed-batch operation capabilities of the fed-batch microtiter plate presented in this study finds optimal application in screenings and initial process development. 相似文献
4.
A new online monitoring technique to measure the physiological parameters, dissolved oxygen (DO) and pH of microbial cultures
in continuously shaken 24-well microtiter plates (MTP) is introduced. The new technology is based on immobilised fluorophores
at the bottom of standard 24-well MTPs. The sensor MTP is installed in a sensor dish reader, which can be fixed on an orbital
shaker. This approach allows real online measurements of physiological parameters during continuous shaking of cultures without
interrupting mixing and mass transfer like currently available technologies do. The oxygen transfer conditions at one constant
shaking frequency (250 1/min) and diameter (25 mm) was examined with the chemical sulphite oxidation method. Varied filling
volumes (600–1,200 μL) of Escherichia coli cultures demonstrated the importance of sufficient oxygen transfer to the culture. Cultures with higher filling volumes were
subjected to an oxygen limitation, which influenced the cell metabolism and prolongated the cultivation time. The effects
could be clearly monitored by online DO and pH measurements. A further study of different media in an E. coli fermentation elucidated the different growth behaviour in response to the medium composition. The MTP fermentations correlated
very well with parallel fermentations in shake flasks. The new technique gives valuable new insights into biological processes
at a very small scale, thus enabling parallel experimentation and shorter development times in bioprocessing. 相似文献
5.
Seletzky JM Noack U Hahn S Knoll A Amoabediny G Büchs J 《Journal of industrial microbiology & biotechnology》2007,34(2):123-130
Respiration measurement is applied as a universal tool to determine the activity of biological systems. The measurement techniques
are difficult to compare, due to the vast variety of devices and analytical procedures commonly in use. They are used in fields
as different as microbiology, gene engineering, toxicology, and industrial process monitoring to observe the physiological
activity of living systems in environments as diverse as fermenters, shake flasks, lakes and sewage plants. A method is introduced
to determine accuracy, quantitation limit, range and precision of different respiration measurement devices. Corynebacterium
glutamicum cultures were used to compare an exhaust gas analyzer (EGA), a RAMOS device (respiration measurement in shake flasks) and
a respirometer. With all measuring devices it was possible to determine the general culture characteristics. The EGA and the
RAMOS device produced almost identical results. The scatter of the respirometer was noticeably higher. The EGA is the technique
of choice, if the reaction volume is high or a short reaction time is required. The possibility to monitor cultures simultaneously
makes the RAMOS device an indispensable tool for media and strain development. If online monitoring is not compulsive, the
respiration of the investigated microbial system extremely low, or the sample size small, a respirometer is recommended. 相似文献
6.
Johanna S. Rehfeld Louis M. Kuhnke Christian Ude Gernot T. John Sascha Beutel 《Engineering in Life Science》2023,23(9):e2300204
In the field of bioprocess development miniaturization, parallelization and flexibility play a key role reducing costs and time. To precisely meet these requirements, additive manufacturing (3D-printing) is an ideal technology. 3D-printing enables rapid prototyping and cost-effective fabrication of individually designed devices with complex geometries on demand. For successful bioprocess development, monitoring of process-relevant parameters, such as pH, dissolved oxygen (DO), and biomass, is crucial. Online monitoring is preferred as offline sampling is time-consuming and leads to loss of information. In this study, 3D-printed cultivation vessels with optical prisms are evaluated for the use in upstream processes of different industrially relevant microorganisms and cell lines. It was shown, that the 3D-printed optically modified well (OMW) is of benefit for a wide range of biotechnologically relevant microorganisms and even for mammalian suspension cells. Evaluation tests with Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Chinese hamster ovary (CHO) cells were performed, providing highly reproducible results. Growth behavior of OMW cultures was comparable to behavior of shake flask (SF) cultivations and the signal to noise ratio in online biomass measurement was shown to be reduced up to 95.8% by using the OMW. Especially the cultivation phases with low turbidity respective optical densities below 1.0 rel.AU could be monitored accurately for the first time. Furthermore, it was demonstrated that the 3D-printed optics are transferable to different well geometries and sizes, enabling efficient biomass monitoring for individual requirements with tailor-made 3D-printed cultivation vessels in small scale. 相似文献
7.
Background
Dissolved oxygen tension (DOT) is an important parameter for evaluating a bioprocess. Conventional means to measure DOT in shake flasks using fixed Clark-type electrodes immersed in the bulk liquid are problematic, because they inherently alter the hydrodynamics of the systems. Other approaches to measure DOT that apply fluorescing sensor spots fixed at the inside wall of a shake flask are also suboptimal. At low filling volumes for cultivating microorganisms with a high oxygen demand, the measured DOT signal may be erroneous. Here, the sensor spot is sometimes exposed to gas in the head space of the flask. Merely repositioning the sensor spot elsewhere in the flask does not address this problem, since there is no location in the shake flask that is always covered by the rotating bulk liquid. Thus, the aim of this prospective study is first, to verify the systemic error of Clark-type electrodes for measuring DOT in shake flasks. The second principle aim is to use the newly built "flexitube optical sensor" to verify potential errors in conventional optical DOT measurements based on fixed sensor spots. 相似文献8.
Christoph Ries Gernot John Corinne John Regine Eibl Dieter Eibl 《Engineering in Life Science》2010,10(1):75-79
While wave‐mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culture‐based production at liter‐scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter‐scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250 mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (kLa‐values) for PreSens 250 mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation‐free growth of insect cells up to high cell densities in batch mode (1.6×107 cells/mL) and for the efficient expression of an intracellular model protein. 相似文献
9.
Louis Maximilian Kuhnke Johanna Sophie Rehfeld Christian Ude Sascha Beutel 《Engineering in Life Science》2022,22(6):440
Integrating optical sensors and 3D‐printed optics into single‐use (SU) cultivation vessels for customized, tailor‐made equipment could be a next big step in the bioreactor and screening platform development enabling online bioprocess monitoring. Many different parameters such as pH, oxygen, carbon dioxide and optical density (OD) can be monitored more easily using online measuring instruments compared to offline sampling. Space‐saving integrated sensors in combination with adapted optics such as prisms open up vastly new possibilities to precisely guide light through SU vessels. This study examines how optical prisms can be 3D‐printed with a 3D‐inkjet printer, modified and then evaluated in a custom made optical bench. The prisms are coated or bonded with thin cover glasses. For the examination of reflectance performance and conformity prisms are compared on the basis of measured characteristics of a conventional glass prism. In addition, the most efficient and reproducible prism geometry and modification technique is applied to a customized 3D‐printed cultivation vessel. The vessel is evaluated on a commercial sensor‐platform, a shake flask reader (SFR) vario, to investigate its sensing‐characteristics while monitoring scattered light with the turbidity standard formazine and a cell suspension of Saccharomyces cerevisiae as model organism. It is demonstrated that 3D‐printed prisms can be used in combination with commercial scattered light sensor‐platforms to determine OD of a microbial culture and that a 3D‐printed unibody design with integrated optics in a cultivation vessel is feasible. In the range of OD600 0–1.16 rel.AU a linear correlation between sensor amplitude and offline determined OD can be achieved. Thus, enabling for the first time a measurement of low cell densities with the SFR vario platform. Moreover, sensitivity is also at least three times higher compared to the commonly used method. 相似文献
10.
Kusterer A Krause C Kaufmann K Arnold M Weuster-Botz D 《Bioprocess and biosystems engineering》2008,31(3):207-215
Single-use stirred tank bioreactors on a 10-mL scale operated in a magnetic-inductive bioreaction block for 48 bioreactors
were equipped with individual stirrer-speed tracing, as well as individual DO- and pH-monitoring and control. A Hall-effect
sensor system was integrated into the bioreaction block to measure individually the changes in magnetic field density caused
by the rotating permanent magnets. A restart of the magnetic inductive drive was initiated automatically each time a Hall-effect
sensor indicates one non-rotating gas-inducing stirrer. Individual DO and pH were monitored online by measuring the fluorescence
decay time of two chemical sensors immobilized at the bottom of each single-use bioreactor. Parallel DO measurements were
shown to be very reliable and independently from the fermentation media applied in this study for the cultivation of Escherichia coli and Saccharomyces cerevisiae. The standard deviation of parallel pH measurements was pH 0.1 at pH 7.0 at the minimum and increased to a standard deviation
of pH 0.2 at pH 6.0 or at pH 8.5 with the complex medium applied for fermentations with S. cerevisiae. Parallel pH-control was thus shown to be meaningful with a tolerance band around the pH set-point of ± pH 0.2 if the set-point
is pH 6.0 or lower. 相似文献
11.
We show the application of a novel optical on-line sensor fixed in spinner flasks for the online monitoring of dissolved O2 concentrations during mammalian cell growth. Using this sensor that requires only minute changes to the flask to be made,
we could determine the volumetric O2 transfer coefficient as well as O2 consumption rates. Under normal growth conditions the cells did not undergo O2 limitation. Also, the transfer of O2 from the atmosphere to the spinner flasks is influenced by the use of screw caps. The on-line measurement was further applied
to determine the O2 uptake rates which can then be used to monitor the metabolic state of the cells and also for online process monitoring. 相似文献
12.
U. Brand L. Brandes V. Koch T. Kullik B. Reinhardt F. Rüther T. Scheper K. Schügerl S. Wang X. Wu R. Ferretti S. Prasad D. Wilhelm 《Applied microbiology and biotechnology》1991,36(2):167-172
Summary Single and multisensor field effect transistors (FET) with a pH-sensitive Si/SiO2/Si3N4/Ta2O5-gate and reference electrode (for single sensor) were developed and used for manufacturing the following biological (Bio)-FETs: for glucose analysis, glucose oxidase-FET (GOD-FET); for urea analysis, urease-FET; and for cephalosporin C analysis, cephalosporinase-FET. The GOD-FETs were integrated into flow injection analysis (FIA) of the Eppendorf variables analyser (EVA) system and used for monitoring the glucose concentration in microbial cultivation and production processes with recombinant Escherichia coli K12 MF, recombinant E. coli JM103, Saccharomyces cerevisiae H620, and Candida boidinii. Urease-FET-FIA was used to monitor the urea concentration in a simulated cultivation of Cephalosporium acremonium and urease-FET-FIA and GOD-FET-FIA for the monitoring of urea and glucose concentrations in simulated S. cerevisiae cultivations. 相似文献
13.
Kyra Hoffmann Birthe Halmschlag Simon Briel Michaela Sieben Sastia Putri Eiichiro Fukusaki Lars M. Blank Jochen Büchs 《Biotechnology progress》2023,39(1):e3293
Poly-γ-glutamic acid (γ-PGA) is a biopolymer with a wide range of applications, mainly produced using Bacillus strains. The formation and concomitant secretion of γ-PGA increases the culture broth viscosity, while enzymatic depolymerisation and degradation of γ-PGA decreases the culture broth viscosity. In this study, the recently published ViMOS (V iscosity M onitoring O nline S ystem) is applied for optical online measurements of broth viscosity in eight parallel shake flasks. It is shown that the ViMOS is suitable to monitor γ-PGA production and degradation online in shake flasks. This online monitoring enables the detailed analysis of the Ppst promoter and γ-PGA depolymerase knockout mutants in genetically modified Bacillus subtilis 168. The Ppst promoter becomes active under phosphate starvation. The different single depolymerase knockout mutants are ∆ggt, ∆pgdS, ∆cwlO and a triple knockout mutant. An increase in γ-PGA yield in gγ-PGA/gglucose of 190% could be achieved with the triple knockout mutant compared to the Ppst reference strain. The single cwlO knockout also increased γ-PGA production, while the other single knockouts of ggt and pgdS showed no impact. Partial depolymerisation of γ-PGA occurred despite the triple knockout. The online measured data are confirmed with offline measurements. The online viscosity system directly reflects γ-PGA synthesis, γ-PGA depolymerisation, and changes in the molecular weight. Thus, the ViMOS has great potential to rapidly gain detailed and reliable information about new strains and cultivation conditions. The broadened knowledge will facilitate the further optimization of γ-PGA production. 相似文献
14.
David?Flitsch Sebastian?Krabbe Tobias?Ladner Mario?Beckers Jana?Schilling Stefan?Mahr Uwe?Conrath Werner?K.?Schomburg Jochen?Büchs
Background
Small-scale micro-bioreactors have become the cultivation vessel of choice during the first steps of bioprocess development. They combine high cultivation throughput with enhanced cost efficiency per cultivation. To gain the most possible information in the early phases of process development, online monitoring of important process parameters is highly advantageous. One of these important process parameters is the oxygen transfer rate (OTR). Measurement of the OTR, however, is only available for small-scale fermentations in shake flasks via the established RAMOS technology until now. A microtiter plate-based (MTP) μRAMOS device would enable significantly increased cultivation throughput and reduced resource consumption. Still, the requirements of miniaturization for valve and sensor solutions have prevented this transfer so far. This study reports the successful transfer of the established RAMOS technology from shake flasks to 48-well microtiter plates. The introduced μRAMOS device was validated by means of one bacterial, one plant cell suspension culture and two yeast cultures.Results
A technical solution for the required miniaturized valve and sensor implementation for an MTP-based μRAMOS device is presented. A microfluidic cover contains in total 96 pneumatic valves and 48 optical fibers, providing two valves and one optical fiber for each well. To reduce costs, an optical multiplexer for eight oxygen measuring instruments and 48 optical fibers is introduced. This configuration still provides a reasonable number of measurements per time and well. The well-to-well deviation is investigated by 48 identical Escherichia coli cultivations showing standard deviations comparable to those of the shake flask RAMOS system. The yeast Hansenula polymorpha and parsley suspension culture were also investigated.Conclusions
The introduced MTP-based μRAMOS device enables a sound and well resolved OTR monitoring for fast- and slow-growing organisms. It offers a quality similar to standard RAMOS in OTR determination combined with an easier handling. The experimental throughput is increased 6-fold and the media consumption per cultivation is decreased roughly 12.5-fold compared to the established eight shake flask RAMOS device.15.
Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains
下载免费PDF全文
![点击此处可从《Biotechnology progress》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Nina Ihling Natalie Bittner Sylvia Diederichs Maximilian Schelden Anna Korona Georg Theo Höfler Alexander Fulton Karl‐Erich Jaeger Kohsuke Honda Hisao Ohtake Jochen Büchs 《Biotechnology progress》2018,34(2):315-327
Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7‐RNA polymerase‐dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3‐hydroxybutyryl‐CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315–327, 2018 相似文献
16.
Jian Li Jennifer Jaitzig Friederike Hillig Roderich Süssmuth Peter Neubauer 《Applied microbiology and biotechnology》2014,98(2):591-601
Nonribosomal peptides (NRPs), a large family of natural products, possess numerous pharmaceutically significant bioactivities. However, many native microbial producers of NRPs are not cultivable or have low production yields making mass production infeasible. The recombinant production of natural products in a surrogate host has emerged as a strategy to overcome these limitations. De novo recombinant production of the NRP antibiotic valinomycin in an engineered Escherichia coli host strain was established with the necessary biosynthetic pathway constituents from Streptomyces tsusimaensis. In the present study, the initially modest valinomycin yields could be significantly increased from 0.3 up to 2.4 mg L?1 by switching from a batch to an enzyme-based fed-batch mode in shake flasks. A subsequent design of experiment-driven optimization of parallel fed-batch cultivations in 24-well plates with online monitoring of dissolved oxygen and pH led to valinomycin yields up to 6.4 mg L?1. Finally, repeated glucose polymer feeding to enzyme-based high cell density cultivations in shake flasks resulted in cell densities of OD600 >50 and a valinomycin titer of appr. 10 mg L?1. This represents a 33-fold improvement compared to the initial batch cultivations and is the highest concentration of a nonribosomal peptide which has been produced in E. coli without feeding of specific precursors so far to our knowledge. Also, such a small-scale optimization under fed-batch conditions may be generally applicable for the development and scale-up of natural product production processes in E. coli. 相似文献
17.
Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and
hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos
of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass;
however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo
cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture
where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from
SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with
both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
18.
Viki R. Chopda Timothy Holzberg Xudong Ge Brandon Folio Lynn Wong Michael Tolosa Yordan Kostov Leah Tolosa Govind Rao 《Biotechnology and bioengineering》2020,117(4):992-998
Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O2 (DO) and dissolved CO2 (dCO2) is of paramount importance. At bioreactor level these parameters can be monitored online and can be controlled by sparging air/oxygen or stirrer speed. However, traditional small-scale systems such as shake flasks lack real time monitoring and also employ only surface aeration with additional diffusion limitations imposed by the culture plug. Here we present implementation of intensifying surface aeration by sparging air in the headspace of the reaction vessel and real-time monitoring of DO and dCO2 in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO2 at low level, which significantly improved not only biomass growth but also protein yield. We expect that implementing such controlled smart shake flasks can minimize the process development gap which currently exists in shake flask level and bioreactor level results. 相似文献
19.
Reenu Sanil Vishwanathgouda Maralingannavar Mugdha Gadgil 《Journal of industrial microbiology & biotechnology》2014,41(4):647-655
Shake flasks are widely used to culture microorganisms, but they do not allow for pH control without additional infrastructure. In the presence of a carbon source like glucose, culture pH typically decreases due to overflow metabolism and can limit the growth of microorganisms in shake flasks. In this study, we demonstrate the use of magnesium hydroxide-loaded pH managing hydrogels (m-pHmH) for in situ base release to counter the decrease in culture pH in shake flasks using Escherichia coli as a model organism, in both complex and mineral salts medium. Base release from m-pHmH is shown to increase with decreasing pH (22-fold increase in release rate from pH 8 to 5), thus providing feedback from culture pH. The addition of m-pHmH resulted in better pH maintenance and higher biomass yields of E. coli K12 in media containing glucose as a carbon source. The use of m-pHmH with additional buffer resulted in pH being maintained above 6.9 while pH decreases below 5 without m-pHmH. We demonstrate one application of such in situ pH management to increase the volumetric plasmid yield from E. coli in shake flask culture. In situ glucose release through a hydrogel to mimic fed-batch culture along with the addition of m-pHmH resulted in a 395 % increase in volumetric plasmid yield to 38 μg/ml in shake flask culture. 相似文献
20.
Huayi Liu Yujuan Tian Yi Zhou Yeyi Kan Tingting Wu Wenhai Xiao Yunzi Luo 《Microbial biotechnology》2021,14(6):2605-2616
Tyrosol and its glycosylated product salidroside are important ingredients in pharmaceuticals, nutraceuticals and cosmetics. Despite the ability of Saccharomyces cerevisiae to naturally synthesize tyrosol, high yield from de novo synthesis remains a challenge. Here, we used metabolic engineering strategies to construct S. cerevisiae strains for high-level production of tyrosol and salidroside from glucose. First, tyrosol production was unlocked from feedback inhibition. Then, transketolase and ribose-5-phosphate ketol-isomerase were overexpressed to balance the supply of precursors. Next, chorismate synthase and chorismate mutase were overexpressed to maximize the aromatic amino acid flux towards tyrosol synthesis. Finally, the competing pathway was knocked out to further direct the carbon flux into tyrosol synthesis. Through a combination of these interventions, tyrosol titres reached 702.30 ± 0.41 mg l−1 in shake flasks, which were approximately 26-fold greater than that of the WT strain. RrU8GT33 from Rhodiola rosea was also applied to cells and maximized salidroside production from tyrosol in S. cerevisiae. Salidroside titres of 1575.45 ± 19.35 mg l−1 were accomplished in shake flasks. Furthermore, titres of 9.90 ± 0.06 g l−1 of tyrosol and 26.55 ± 0.43 g l−1 of salidroside were achieved in 5 l bioreactors, both are the highest titres reported to date. The synergistic engineering strategies presented in this study could be further applied to increase the production of high value-added aromatic compounds derived from the aromatic amino acid biosynthesis pathway in S. cerevisiae. 相似文献