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This work aimed to assess, following upstream optimization in Schott flasks, the scalability from this culture platform to a stirred-tank bioreactor in order to yield rabies-recombinant baculovirus, bearing genes of G (BVG) and M (BVM) proteins, and to obtain rabies virus-like particles (VLP) from them, using Sf9 insect cells as a host. Equivalent assays in Schott flasks and a bioreactor were performed to compare both systems and a multivariate statistical approach was also carried out to maximize VLP production as a function of BVG and BVM’s multiplicity of infection (MOI) and harvest time (HT). Viable cell density, cell viability, virus titer, BVG and BVM quantification by dot-blot, and BVG quantification by Enzyme-Linked Immunosorbent Assay (ELISA) were monitored throughout the assays. Furthermore, transmission electron microscopy was used to characterize rabies VLP. The optimal combination for maximum VLP expression was BVG and BVM MOI of 2.3 pfu/cell and 5.1 pfu/cell, respectively, and 108 h of harvest time. The current study confirmed that the utilization of Schott flasks and a benchtop bioreactor under the conditions applied herein are equivalent regarding the cell death kinetics corresponding to the recombinant baculovirus infection process in Sf9 cells. According to the results, the hydrodynamic and chemical differences in both systems seem to greatly affect the virus and VLP integrity after release.

Recently, in Brazil, corn ethanol industries are being installed and the integration with sugar/energy-cane has been proposed, using bagasse for cogeneration and the juice to dilute the corn. However, this integration may have some limitations, such as the quality of the cane juice and potential contamination by microorganisms brought with the cane from the field. In this article, we first tested the effects of mixing energy cane juice with corn on fermentative parameters. We also assessed the effects of Lactobacilli. contamination on organic acids produced during the fermentation and fermentation parameters and proposed the use of ionizing radiation to replace antibiotics as a disinfection control method. Our results showed that mixing energy cane juice with corn does not have any negative effect on fermentation parameters, including ethanol production. The contamination with Lactobacilli. considerably increased the production of acetic, lactic, and succinic acid, reducing the pH and ethanol content from 89.2 g L−1 in the sterilized treatment to 72.9 g L−1 in the contaminated treatment. Therefore, for the integration between corn and cane to be applied on an industrial scale, it is essential to have effective disinfection before fermentation. Ionizing radiation (20 kGy) virtually disinfected the wort, showing itself to be a promising technology; however, an economic viability study for adopting it in the industry should be carried out.

This work aimed to quantify growth and biochemical parameters (viable cell density, Xv; cell viability, CV; glucose, lactate, glutamine, glutamate, ammonium, and potassium concentrations) in upstream stages to obtain rabies virus-like particles (rabies VLP) from insect cell-baculovirus system using on-line and off-line Raman spectra to calibrate global models with minimal experimental data. Five cultivations in bioreactor were performed. The first one comprised the growth of uninfected Spodoptera frugiperda (Sf9) cells, the second and third runs to obtain recombinant baculovirus (rBV) bearing Rabies G glycoprotein and matrix protein. The fourth one involved the generation of rabies VLP from rBVs and the last one was a repetition of the third one with cell inoculum infected by rBV. The spectra were acquired through a Raman spectrometer with a 785-nm laser source. The fitted Partial Least Square models for nutrients and metabolites were comparable with those previously reported for mammalian cell lines (Relative error15%). However, the use of this chemometrics approach for Xv and CV was not as accurate as it was for other parameters. The findings from this work established the basis for bioprocess Raman spectroscopical monitoring using insect cells for VLP manufacturing, which are gaining ground in the pharmaceutical industry.

This work aimed to describe the dynamics of the Sf9 insect cells death and primary metabolism when this host is infected simultaneously by two recombinant baculoviruses (BV) expressing rabies glycoprotein (BVG) and matrix protein (BVM) genes to produce rabies virus-like particles (VLP) at different multiplicities of infection (MOI). Schott flasks essays covering a wide range of MOI for both BV were performed. Viable cell density, cell viability, glucose, glutamine, glutamate, lactate, ammonium, and rabies proteins concentrations were monitored over the infection phase. The expression of both recombinant proteins was not limited by glucose, glutamine, and glutamate in a broad MOI (pfu/cell) range of BVG (0.15-12.5) and BVM (0.1-5.0) using SF900 III serum free culture medium. Death phase initiation and the specific death rate depend on BV MOI. The wave pattern of nutrient/metabolite profiles throughout the viral infection phase is related to the baculovirus lytic cycle. The optimal MOIs ratio between BVG (2.5-4.5) and BVM (1.0-3.0) for maximum protein expression was defined. The produced rabies VLP sizes are close to 78 nm. In general, these work outputs bring a better understanding of the metabolic performance of Sf9 cells when infected by BV for producing VLP, and specifically, for progressing in a rabies VLP vaccine development.

The importance of studying aeration in industrial bioprocesses is because most of these processes are aerobic, for example, for the production of different biomolecules such as amino acids, vitamins, organic acids, antibiotics, bacteriocins, enzymes, microbial and animal cells and recombinant proteins. Mixing is the process of achieving uniformity, i.e., it is a process by which substrates are intimately dispersed throughout the reactor to afford cells that move through the reactor volume with, as far as possible, the same environment at every point. The response of the specific growth rate to dissolved oxygen concentration is analogous to other limiting substrates. In order to avoid shear stress in the cells, some systems without mechanical agitation were developed. Bubble columns systems utilize spargers in different configurations that release small diameter bubbles of gas into the liquid, from the bottom to the top of the bioreactor, promoting the mixture of the culture media and the dissolution of oxygen.

Currently, only the biopharmaceutical l -asparaginase (ASNase), a chemotherapy agent, from bacteria is approved for clinical use. Although it is efficient, it causes severe side effects due to its origin. Therefore, new sources are investigated to reduce immunogenicity. Moreover, it is urgent to enhance protein stability, folding and kinetics with post-translational modifications, such as glycosylation. Aiming at these attributes, this study focused on the production of a glycosylated l -asparaginase of Dickeya chrysanthemi expressed by Pichia pastoris Glycoswitch®. Producer strains of Pichia, one with auxotrophy for histidine and another prototrophic were compared by using a complex medium in flasks and a synthetic medium in a bioreactor. The prototrophic strain showed higher productivity than the auxotrophic in both scales. Following, two induction strategies with the prototrophic strain were executed: methanol pulses and a DO-stat. The latter resulted in 2-fold more maximum volumetric activity. Lastly, the glycosylation analysis of the final product showed that the site Asn170, which is associated with allergies in patients, was glycosylated, thus, reducing potential immunogenic effects. Therefore, this study showed that the prototrophic strain was the most suitable l -asparaginase producer and that methanol–oxygen control in bioreactor enhanced the production of a glycosylated ASNase.

L-asparaginase (ASNase) is an essential drug in the treatment of acute lymphoblastic leukemia (ALL). Commercial bacterial ASNases increase patient survival, but the consequent immunological reactions remain a challenge. Yeasts ASNase is closer to human congeners and could lead to lower side effects. Among 134 yeast strains isolated from marine-sediments in King George Island, Antarctica, nine were L-asparaginase producing yeasts and glutaminase-free.

Objective Cell growth curves constitute one of the primary assays employed to analyze cell proliferation dynamics of in vitro cultured cells under specific culture conditions. From the cell growth curve, it is possible to assess the behavior of proliferating cells under different conditions, such as drug treatment and genomic editions. Traditionally, growth curves for adherent cells are obtained by seeding the cells in multiple-well plates and counting the total number of cells at different time points. Here, we compare this traditional method to the fluorescence-based method, which is based on the CFSE fluorescence decay over time. Results The fluorescence-based method is not dependent on the determination of the total number of cells, but rather is approached by assessing the fluorescence of a sample of single cells from a cell population at different time points after plating. Therefore, this method is not biased due to either cell loss during harvesting or to the presence of cellular debris and cell clumps. Moreover, the fluorescence-based method displays lower variation among different measurements of the same time point, which increases the reliability on the determination of lag, log and stationary phase transitions.

Made available in DSpace on 2020-12-12T02:49:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 L-asparaginase (ASNase) is an essential drug in the treatment of acute lymphoblastic leukemia (ALL). Commercial bacterial ASNases increase patient survival, but the consequent immunological reactions remain a challenge. Yeasts ASNase is closer to human congeners and could lead to lower side effects. Among 134 yeast strains isolated from marine-sediments in King George Island, Antarctica, nine were L-asparaginase producing yeasts and glutaminase-free. Leucosporidium muscorum CRM 1648 yielded the highest ASNase activity (490.41 U.L−1) and volumetric productivity (5.12 U.L−1 h−1). Sucrose, yeast extract and proline were the best carbon and nitrogen sources to support growth and ASNase production. A full factorial design analysis pointed the optimum media condition for yeast growth and ASNase yield: 20 g L−1 sucrose, 15 g L−1 yeast extract and 20 g L−1 proline, which resulted in 4582.5 U L−1 and 63.64 U L−1 h−1 of ASNase and volumetric productivity, respectively. Analysis of temperature, pH, inoculum and addition of seawater indicated the best condition for ASNase production by this yeast: 12–15 °C, pH 5.5–6.5 and seawater >25% (v/v). Inoculum concentration seems not to interfere. This work is pioneer on the production of ASNase by cold-adapted yeasts, highlighting the potential of these microbial resources as a source of glutaminase-free L-asparaginase for commercial purposes. Department of Biochemical and Pharmaceutical Technology School of Pharmaceutical Sciences University of Sao Paulo Department of Chemical Engineering Polytechnic School University of Sao Paulo Department of Pharmacy School of Pharmaceutical Sciences University of Sao Paulo Department of General and Applied Biology Institute of Biosciences State University Julio de Mesquita Filho (UNESP) Department of General and Applied Biology Institute of Biosciences State University Julio de Mesquita Filho (UNESP)

Made available in DSpace on 2019-10-06T15:55:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Adipose-derived mesenchymal stromal/stem cells (ASC) have acquired a prominent role in tissue engineering and regenerative medicine. However, the standardization of basic culture procedures in this cellular type is still not well established according to the main qualitative cellular attributes. We evaluate the cell growth profile of human ASC in a different culture medium volumes and their nutritional composition utilizing static cultivation. Culture medium volumes (5, 10 and 15 mL/25 cm2) in T-flasks were evaluated by kinetic parameters and the metabolic composition was determined by biochemical analysis and Fourier transform infrared (FT-IR) absorption spectroscopy. 50% renewal of culture medium volume every 48 h was adopted. Immunophenotypic characterization and cell differentiation were performed. There was no difference (p > 0.05) in the kinetic parameters of cell proliferation between the culture medium volumes or in FT-IR composition. However, the concentrations of glucose, glutamine, lactate, and glutamate varied significantly during the cultivation process as a function of the medium volume. ASC presented specific antigens and differentiation potential of mesenchymal stromal/stem cells. It was concluded that the minimal culture medium volume (5 mL/25 cm2 in static culture) was sufficient to maintain the stability, potency, and growth of ASC, representing an economic and safe standardization for this cell culture process. School of Medicine University of São Paulo School of Sciences Humanities and Languages São Paulo State University (UNESP) Biomedical Science Institute University of São Paulo School of Medicine of Marilia Hemocenter Polytechnic School University of São Paulo School of Sciences Humanities and Languages São Paulo State University (UNESP) FAPESP: #2014/03324–4

Made available in DSpace on 2019-10-04T12:30:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-04-01. Added 1 bitstream(s) on 2021-07-15T15:04:48Z : No. of bitstreams: 1 S0104-66322018000200441.pdf: 2141133 bytes, checksum: e5b5d899a88b91892ec1b6542baf7d2a (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Baby Hamster Kidney cells (BHK-21) are commonly used in research and the biopharmaceutical industry. This work aimed to model the kinetic performance in batch operation mode of BHK-21 cells cultured in two stirred tank configurations using different dissolved oxygen concentrations and pH control strategies. Viable and dead cell concentrations, as well as glucose, glutamine, lactate and ammonium concentrations, were monitored. Statistical multiple linear regression, logistic equation and multiplicative Monod kinetic models were fitted. Statistical models for viable cells concentration as a function of nutrient and metabolite concentrations were significant (R-2 > 0.91). Logistic model parameters: intrinsic growth rate, cell density level in the medium and time for reaching maximum cell concentrations were within 0.061-0.083 h(-1), 1.85-5.39 x 10(9) cell L-1 and 52-90 h ranges, respectively. A Monod-type model was satisfactorily fitted to the experimental data. Relative errors were lower than 10% for six monitored state variables in most of the assessed experimental conditions. The three models developed in this work can be used in bioprocesses involving BHK-21 with good fitting. Univ Estadual Paulista, Dept Ciencias Biol, Campus Assis,Ave Dom Antonio 2100, BR-19806900 Assis, SP, Brazil Inst Butantan, Lab Especial Pesquisa & Desenvolvimento Imunol Ve, Av Vital Brasil 1500, BR-05503900 Sao Paulo, SP, Brazil Univ Sao Paulo, Dept Engn Quim, Escola Politecn, Lab Celulas Anim, Av Prof Luciano Gualberto,Trav 3,380, BR-05508900 Sao Paulo, SP, Brazil Univ Fed ABC, CCNH, Ave Estados 5001, BR-09210580 Santo Andre, SP, Brazil Univ Estadual Paulista, Dept Ciencias Biol, Campus Assis,Ave Dom Antonio 2100, BR-19806900 Assis, SP, Brazil FAPESP: 2010/52521-6 CNPq: CNPq: 483009/2010-5

Currently, mammalian cells are the most utilized hosts for biopharmaceutical production. The culture media for these cell lines include commonly in their composition a pH indicator. Spectroscopic techniques are used for biopharmaceutical process monitoring, among them, UV-Vis spectroscopy has found scarce applications. This work aimed to define artificial neural networks architecture and fit its parameters to predict some nutrients and metabolites, as well as viable cell concentration based on UV-Vis spectral data of mammalian cell bioprocess using phenol red in culture medium. The BHK-21 cell line was used as a mammalian cell model. Off-line spectra of supernatant samples taken from batches performed at different dissolved oxygen concentrations in two bioreactor configurations and with two pH control strategies were used to define two artificial neural networks. According to absolute errors, glutamine (0.13 ± 0.14 mM), glutamate (0.02 ± 0.02 mM), glucose (1.11 ± 1.70 mM), lactate (0.84 ± 0.68 mM) and viable cell concentrations (1.89 10(5) ± 1.90 10(5) cell/mL) were suitably predicted. The prediction error averages for monitored variables were lower than those previously reported using different spectroscopic techniques in combination with partial least squares or artificial neural network. The present work allows for UV-VIS sensor development, and decreases cost related to nutrients and metabolite quantifications.

Insect cells are used for the expression of complex proteins in products such as vaccines and biopharmaceuticals. Physiology of a Drosophila melanogaster (lineage S2), transfected to stably express rabies virus glycoprotein (RVGP), was studied in batch culture and in a chemostat with serum-free medium. In batch mode, the system reached 3 × 10(7) cells mL(-1) with specific growth rate of 1.5 d(-1) with RVGP at 2.50 µg L(-1). When operated continuously, three well-defined steady states were achieved at dilution rates (D) of 0.8, 0.5 and 0.2 d(-1). The residual glucose and glutamine concentrations and the cell yields on glucose and glutamine decreased at lower D. High values of substrate consumption for maintenance may explain this variation in yields. The results indicated that glucose is not the limiting substrate of this process.

Made available in DSpace on 2015-08-21T17:53:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2014 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação para o Desenvolvimento Tecnológico da Engenharia (FDTE) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10–30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 109 BHK-21 cells from 4 × 106 cells in 13 day with 1,051 mL culture medium. Universidade Estadual Paulista Júlio de Mesquita Filho, Assis, Unesp - Campus Assis, Parque Universitário, CEP 19806900, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, Assis, Unesp - Campus Assis, Parque Universitário, CEP 19806900, SP, Brasil FAPESP: 2010/52521-6 CNPq: 483009/2010-5

Made available in DSpace on 2014-12-03T13:11:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundacao para o Desenvolvimento Tecnologico da Engenharia (FDTE) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Monitoring mammalian cell culture with UV-vis spectroscopy has not been widely explored. The aim of this work was to calibrate Partial Least Squares (PLS) models from off-line UV-vis spectral data in order to predict some nutrients and metabolites, as well as viable cell concentrations for mammalian cell bioprocess using phenol red in culture medium. The BHK-21 cell line was used as a mammalian cell model. Spectra of samples taken from batches performed at different dissolved oxygen concentrations (10, 30, 50, and 70% air saturation), in two bioreactor configurations and with two strategies to control pH were used to calibrate and validate PLS models. Glutamine, glutamate, glucose, and lactate concentrations were suitably predicted by means of this strategy. Especially for glutamine and glucose concentrations, the prediction error averages were lower than 0.50 +/- 0.10 mM and 2.21 +/- 0.16 mM, respectively. These values are comparable with those previously reported using near infrared and Raman spectroscopy in conjunction with PLS. However, viable cell concentration models need to be improved. The present work allows for UV-vis at-line sensor development, decrease cost related to nutrients and metabolite quantifications and establishment of fed-batch feeding schemes. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:241-248, 2014 Inst Butantan, Lab Especial Pesquisa & Desenvolvimento Imunol V, BR-05503900 Sao Paulo, Brazil Univ Sao Paulo, Escola Politecn, Dept Engn Quim, Lab Celulas Anim, BR-05508900 Sao Paulo, Brazil Univ Estadual Paulista, Dept Ciencias Biol, BR-19806900 Assis, SP, Brazil Inst Butantan, Lab Imunol Viral, BR-05503900 Sao Paulo, Brazil Inst Pesquisas Tecnol Estado Sao Paulo SA, Nucleo Bionanomanufatura, Lab Biotecnol Ind, BR-05508901 Sao Paulo, Brazil Univ Estadual Paulista, Dept Ciencias Biol, BR-19806900 Assis, SP, Brazil FAPESP: 10/52521-6 CNPq: CNPq: 483009/2010-5

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained approximately 1.5-3 x 10(7)cells/mL after 3-4 days of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 microg/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 microg/10(7) cells at days 4-5. RVGP values in supernatant as a result of cell lysis were always very low (

A combination of chemostat cultivation and a defined medium was used to demonstrate that uracil limitation leads to a drastic alteration in the physiology of auxotrophic cells of Saccharomyces cerevisiae. Under this condition, the carbon source is dissimilated mainly to ethanol and acetate, even in fully aerobic cultures grown at 0.1 h(-1), which is far below the critical dilution rate. Differently from nitrogen-, sulphur-, or phosphate-limited cultures, uracil limitation leads to residual sugar (either glucose or sucrose) concentrations below 2 mM, which characterizes a situation of double-limitation: by the carbon source and by uracil. Furthermore, the specific rates of CO2 production and O2 consumption are increased when compared to the corresponding prototrophic strain. We conclude that when auxotrophic strains are to be used for quantitative physiological studies, special attention must be paid to the cultivation conditions, mainly regarding medium formulation, in order to avoid limitation of growth by the auxotrophic nutrient.

Studies of a bioprocess optimization and monitoring for protein synthesis in animal cells face a challenge on how to express in quantitative terms the system performance. It is possible to have a panel of calculated variables that fits more or less appropriately the intended goal. Each mathematical expression approach translates different quantitative aspects. We can basically separate them into two categories: those used for the evaluation of cell physiology in terms of product synthesis, which can be for bioprocess improvement or optimization, and those used for production unit sizing and for bioprocess operation. With these perspectives and based on our own data of kinetic S2 cells growth and metabolism, as well as on their synthesis of the transmembrane recombinant rabies virus glycoprotein, here indicated as P, we show and discuss the main characteristics of calculated variables and their recommended use. Mainly applied to a bioprocess improvement/optimization and that mainly used for operation definition and to design the production unit, we expect these definitions/recommendations would improve the quality of data produced in this field and lead to more standardized procedures. In turn, it would allow a better and easier comprehension of scientific and technological communications for specialized readers.

The present study investigated the effects of agitation speed, N (200, 500, 600 or 700 rpm), and dissolved oxygen concentration, C (120, >70, 70, 60, 10 or < 10%), on red pigment and citrinin production by Monascus purpureus ATCC 36928, cultivated in liquid medium by a batch process. The gas flow rate was the same for all runs with C controlled by means of the incoming gas composition control (air/N2 or air/O2). From the response surface plots it can be verified that the effect of C was greater than that of N on the production of both metabolites. The absorbance for red pigments varied from 1.6 U (C< 10%; N=200 rpm) up to 3.3 U (C=60%; N=600 rpm), an increase of 106%, while citrinin concentration increased 257%, from 14.2 to 50.7 mg.L-1. The most appropriate conditions were C=60% and N=600rpm, under which the highest red pigment absorbance (3.3U) and half of the highest citrinin concentration were obtained.

Viral antigens can be obtained from infected mammalian cells cultivated on microcarriers. We have worked out parameters for the production of bovine parainfluenza 3 (PI-3) virus by Mandin-Darby Bovine Kidney (MDBK) cells cultivated on Cytodex 1 microcarriers (MCs) in spinners flasks and bioreactor using fetal bovine serum (FBS) supplemented Eagle minimal essential medium (Eagle-MEM). Medium renewal during the cell culture was shown to be crucial for optimal MCs loading (>90% MCs with confluent cell monolayers) and cell growth (2.5 x 10(6)cells/mL and a micro(x) (h(-1)) 0.05). Since cell cultures performed with lower amount of MCs (1g/L), showed good performances in terms of cell loading, we designed batch experiments with a lower concentration of MCs in view of optimizing the cell growth and virus production. Studies of cell growth with lower concentrations of MCs (0.85 g/L) showed that an increase in the initial cell seeding (from 7 to 40 cells/MC) led to a different kinetic of initial cell growth but to comparable final cell concentrations ((8-10)x10(5)cells/mL at 120 h) and cell loading (210-270 cells/MC). Upon infection with PI-3 virus, cultures showed a decrease in cell growth and MC loading directly related to the multiplicity of infection (moi) used for virus infection. Infected cultures showed also a higher consumption of glucose and production of lactate. The PI-3 virus and PI-3 antigen production among the cultures was not significantly different and attained values ranging from, respectively, 7-9 log(10) TCID(50)/mL and 1.5-2.2 OD. The kinetics of PI-3 virus production showed a sharp increase during the first 24h and those of PI-3 antigen increased after 24h. The differential kinetics of PI-3 virus and PI-3 antigen can be explained by the virus sensitivity to temperature. In view of establishing a protocol of virus production and based on the previous experiments, MDBK cell cultures performed under medium perfusion in a bioreactor of 1.2L were infected and the PI-3 virus production in 12L attained 12 log(10) TCID(50). Other than establishing a protocol for PI-3 production in MDBK cell cultures on Cytodex 1, the experiments are proposed as a basis for approaching the development of a virus production protocol in mammalian cells cultivated on microcarriers in bioreactors.

Os processos biotecnológicos com células animais tem despertado interesse crescente devido à sua capacidade de sintetizar moléculas complexas de altíssimo valor agregado, mesmo exigindo condições especiais de cultivo. Torna-se portanto fundamental o correto monitoramento de tais processos, para se manter seus parâmetros dentro de limites desejados. O objetivo deste trabalho foi efetuar cultivos de células animais em processo de perfusão, aos quais se acoplavam sistemas de monitoramento visando melhor controle, de modo a contribuir para o aumento da produtividade. Realizaram-se ensaios com células BHK e hibridoma em biorreator de 12L em alta densidade celular (20E6 cel/mL), para produção de fator de coagulação FVIII e anticorpo anti-TNF. O processo de perfusão foi analisado, buscando-se melhoria em seu controle através da correta estimativa da concentração celular. O sistema de transferência de oxigênio através de membranas de silicone foi estudado, tendo-se elaborado uma simplificação para a estimativa da velocidade de consumo de oxigênio, baseando-a na pressão média da membrana de silicone, e tendo-se determinado a influência da concentração de oxigênio no crescimento e na produção e seu valor limitante (entre 5 e 10%). Por fim, estudou-se o efeito da osmolalidade do meio no processo, tanto com relação ao crescimento e consumo de substratos, como na produtividade. Observou-se o aumento do tamanho da célula com o aumento da osmolalidade (até 470mmol/kg) e como este fato influencia o cálculo da velocidade específica de produção. Tentou-se sem sucesso fazer um monitoramento on-line deste aumento de tamanho, com o uso da sonda Lasentec FBRM. No entanto esta se mostrou capaz de estimar a concentração celular. O trabalho demonstra a importância de se efetuar um adequado monitoramento do processo para garantir a correta operação, em especial em cultivos com células animais em perfusão. Animal cell culture technology has become apparent, due to its capacity to synthesize highly valuable complex molecules, even requiring special attention. In this scenario, adequate process monitoring becomes essential to keep its parameters well controlled. The aim of this work is to perform perfusion cultures with animal cells, and couple them to monitoring systems, in order to improve the efficiency of the control and consequently productivity. Runs were realized with BHK and hibridoma cells in a 12-L bioreactor at high cell density (20E6 cel/mL), to produce clotting factor FVIII and antibody anti-TNF. The perfusion process was analyzed, to enhance its control through reliable cell density estimate. Oxygen transfer system through silicone tubing was studied, resulting in a simple way to estimate oxygen uptake rate and establishing the influence of oxygen partial pressure in the growth and production. At last, medium osmolality effect was investigated related to growth, substrate consumption, and productivity. Cell enlargement was observed at higher osmolalities (till 470mmol/kg) and its results over the specific production rate were analyzed. The cell size change was measured off-line. Lasentec FBRM probe could not follow the size change, but showed capable to estimate cell density This work makes evident the importance in having adequate monitoring systems, in order to assure correct process operation, and specially cell culture perfusion processes.

This work aimed to compare the predictive capacity of empirical models, based on the uniform design utilization combined to artificial neural networks with respect to classical factorial designs in bioprocess, using as example the rabies virus replication in BHK-21 cells. The viral infection process parameters under study were temperature (34°C, 37°C), multiplicity of infection (0.04, 0.07, 0.1), times of infection, and harvest (24, 48, 72 hours) and the monitored output parameter was viral production. A multilevel factorial experimental design was performed for the study of this system. Fractions of this experimental approach (18, 24, 30, 36 and 42 runs), defined according uniform designs, were used as alternative for modelling through artificial neural network and thereafter an output variable optimization was carried out by means of genetic algorithm methodology. Model prediction capacities for all uniform design approaches under study were better than that found for classical factorial design approach. It was demonstrated that uniform design in combination with artificial neural network could be an efficient experimental approach for modelling complex bioprocess like viral production. For the present study case, 67% of experimental resources were saved when compared to a classical factorial design approach. In the near future, this strategy could replace the established factorial designs used in the bioprocess development activities performed within biopharmaceutical organizations because of the improvements gained in the economics of experimentation that do not sacrifice the quality of decisions.

To assess the expression of rabies virus G-glycoprotein (RVGP) expression using Semliki Forest virus as a vector in combination with BHK-21 cells cultured in suspension.A multilevel factorial design was used to quantify effects of temperature (33-37 °C), fresh medium addition after the viral adsorption step (100-200 % with respect to the initial cell suspension volume before infection) and harvest time (8-40 h) on RVGP production. Experimental runs were performed in 24-well cell culture plates at a multiplicity of infection (MOI) of 16. An additional experiment in spinner-flask was performed at MOI of 9, using the optimal conditions determined in cell culture plates. Values for temperature, fresh medium addition and harvest time of 33 °C, 100 % and 16 h, respectively, ensured the optimal RVGP production in culture plates. The volumetric yield (239 ng ml(-1)) in these conditions was higher than that reported previously for adherent cell culture. In spinner-flasks, the volumetric yield was improved (559 ng ml(-1)).These results establish the basis for designing bioprocess to produce RVGP.

We have studied parameters for optimizing the Spodoptera frugiperda (Sf9) cell culture and viral infection for the production of Anticarsia gemmatalis multiple nucleopolyhedrosis virus (AgMNPV) polyhedra inclusion bodies (PIBs) in shaker-Schott or spinner bottles and bioreactors. We have assayed the k(L)a of the systems, initial cell seeding, cell culture volume, dissolved oxygen (DO), multiplicity of infection (MOI), nutrients consumption, and metabolites production. The medium surface oxygen transfer was shown to be higher in shaker bottles than in spinner ones, which was in direct correlation to the higher cell density obtained. Best quantitative performances of PIBs production were obtained with a SF900II medium volume/shaker-bottle volume ratio of 15% and MOI of 0.5 to 1 performed at a cell concentration at infection (CCI) of 1 to 2.5x10(6) cells/ml in a medium containing enough glucose and glutamine. Upon infection, a decrease in the cell multiplication was observed to be dependent on the MOI used, and the muX at the exponential growth phase in infected and non-infected cultures were, respectively, of 0.2832 and 0.3914 (day(-1)). The glucose consumption and lactate production were higher in the infected cultures (muGlucose and muLactate of, respectively, 0.0248 and 0.0089x10(-8) g/cellxday in infected cultures and 0.0151 and 0.0046x10(-8) g/cellxday in non infected ones). The glutamine consumption did not differ in both cultures (muGlutamine of 0.0034 and 0.0037x10(-8) g/cellxday in, respectively, infected and non infected cultures). When a virus MOI of 0.1 to 1 was used for infection, a higher concentration of PIBs/ml was obtained. This was in direct correlation to a higher cell concentration present in these cultures, where a decrease in cell multiplication due to virus infection is minimized. When a MOI of 1 was used, a more effective decrease in cell multiplication was observed and a lower concentration of PIBs/ml was obtained, but with the best performance of PIBs/cell. Correlations between MOI and CCI indicate that a MOI 0.1 to 1.4 and a CCI of 10(6) to 2x10(6) cells/ml led to the best PIBs production performances. The virulence of PIBs produced in cultures infected at low or high MOI showed comparable DL(50). Culture and infection in scaling-up conditions, performed in a bioreactor, were shown to provide the cells with a better environment and be capable of potentially improving the shaker-Schott findings. For an accurate qualitative control of PIB virulence, hemolymph from AgMNPV infected Anticarsia gemmatalis was used as starting material for passages in Sf9 cells. These led to a loss of virulence among the PIBs with an increase in the DL(50). The loss of virulence was accompanied by a loss in budded virus titer, a decreased number of PIBs produced and an altered DNA restriction pattern, suggesting the generation of defective interference particles (DIPs). Transmission electron microscopy (TEM) studies revealed that after cell passages, PIBs lacking virions were progressively synthesized. The study described here point out the biological constraints and bioprocess issues for the preparation of AgMNPV PIBs for biological control.

Cell growth and retamycin production in Streptomyces olindensis So20 were studied at different dissolved oxygen levels during fermentation. The profiles for cell growth and retamycin production in an experiment with dissolved oxygen control at 5% air saturation during production were similar to those in an experiment without dissolved oxygen control. However, despite slower cell growth, a twofold increase in the final retamycin concentration was achieved when dissolved oxygen was controlled at 100% during the growth phase, compared to a base experiment (no DO control). Therefore, the results revealed that retamycin production was dependent on dissolved oxygen concentration during the growth phase, even with sufficient oxygen. On the other hand, a large oxygen supply during the production phase did not contribute to an improvement in retamycin production.

Made available in DSpace on 2014-12-03T13:11:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-09-01Bitstream added on 2014-12-03T13:22:33Z : No. of bitstreams: 1 S1516-89132013000500018.pdf: 107013 bytes, checksum: 0a4b1e7c2d2c2bac93af970e5ee6fbc7 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale. Univ Sao Paulo, Escola Politecn, Dept Engn Quim, Lab Celulas Anim, Sao Paulo, Brazil Univ Estadual Paulista Julho de Mesquita Filho, Fac Ciencia & Letras, Dept Ciencias Biol, Assis, SP, Brazil Inst Butantan, Lab Imunol Viral, Sao Paulo, Brazil Inst Pesquisas Tecnol Estado Sao Paulo SA, Nucleo Bionanomanufatura, Lab Biotecnol Ind, Sao Paulo, Brazil Univ Estadual Paulista Julho de Mesquita Filho, Fac Ciencia & Letras, Dept Ciencias Biol, Assis, SP, Brazil FAPESP: 10/52521-6

This work focused on determining the effect of dissolved oxygen concentration (DO) on growth and metabolism of BHK-21 cell line (host cell for recombinant proteins manufacturing and viral vaccines) cultured in two stirred tank bioreactors with different aeration-homogenization systems, as well as pH control mode. BHK-21 cell line adapted to single-cell suspension was cultured in Celligen without aeration cage (rotating gas-sparger) and Bioflo 110, at 10, 30 and 50 % air saturation (impeller for gas dispersion from sparger-ring). The pH was controlled at 7.2 as far as it was possible with gas mixtures. In other runs, at 30 and 50 % (DO) in Bioflo 110, the cells grew at pH controlled with CO2 and NaHCO3 solution. Glucose, lactate, glutamine, and ammonium were quantified by enzymatic methods. Cell concentration, size and specific oxygen consumption were also determined. When NaHCO3 solution was not used, the optimal DOs were 10 and 50 % air saturation for Celligen and Bioflo 110, respectively. In this condition maximum cell concentrations were higher than 4 × 10(6) cell/mL. An increase in maximum cell concentration of 36 % was observed in batch carried out at 30 % air saturation in a classical stirred tank bioreactor (Bioflo 110) with base solution addition. The optimal parameters defined in this work allow for bioprocess developing of viral vaccines, transient protein expression and viral vector for gene therapy based on BHK-21 cell line in two stirred tank bioreactors with different agitation-aeration systems.

Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-proton symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9% higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5' coding sequences of SUC2, resulting in predominant (94%) cytosolic localization of invertase. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 90 generations of laboratory evolution in anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11% higher ethanol yield on sucrose in chemostat cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. After deletion of both copies of the duplicated AGT1, growth characteristics reverted to that of the unevolved SUC2 and iSUC2 strains. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.

In the present review we discuss strategies that have been used for heterologous gene expression in Drosophila melanogaster Schneider 2 (S2) cells using plasmid vectors. Since the growth of S2 cells is not dependent on anchorage to solid substrates, these cells can be easily cultured in suspension in large volumes. The factors that most affect the growth and gene expression of S2 cells, namely cell line, cell passage, inoculum concentration, culture medium, temperature, dissolved oxygen concentration, pH, hydrodynamic forces and toxic metabolites, are discussed by comparison with other insect and mammalian cells. Gene expression, cell metabolism, culture medium formulation and parameters involved in cellular respiration are particularly emphasized. The experience of the authors with the successful expression of a biologically functional protein, the rabies virus glycoprotein (RVGP), by recombinant S2 cells is presented in the topics covered.

Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type1 Diabetes, however, it is severely limited by the shortage of organ donors. Ex-vivo islet cell cultures appear as an attractive but still elusive approach for curing type 1 Diabetes. It has recently been shown that, even in the absence of fibrotic overgrowth, several factors, such as insufficient nutrition of the islet core, represent a major barrier for long-term survival of islets grafts. The use of immobilized dispersed cells may contribute to solve this problem due to conceivably easier nutritional and oxygen support to the cells. Therefore, we set out to establish an immobilization method for primary cultures of human pancreatic cells by adsorption onto microcarriers (MCs). Dispersed human islets cells were seeded onto Cytodex1 microcarriers and cultured in bioreactors for up to eight days. The cell number increased and islet cells maintained their insulin secretion levels throughout the time period studied. Moreover, the cells also presented a tendency to cluster upon five days culturing. Therefore, this procedure represents a useful tool for controlled studies on islet cells physiology and, also, for biotechnological applications.

Monitoring the specific respiration rate (Q(O2)) is a valuable tool to evaluate cell growth and physiology. However, for low Q(O2) values the accuracy may depend on the measurement methodology, as it is the case in animal cell culture. The widely used "Dynamic Method" imposes serious difficulties concerning oxygen transfer cancellation, especially through membrane oxygenation. This paper presents an improved procedure to this method, through an automated control of the gas inlet composition that can minimize the residual oxygen transfer driving force during the Q(O2) measurement phase. The improved technique was applied to animal cell cultivation, particularly three recombinant S2 (Drosophila melanogaster) insect cell lines grown in a membrane aeration bioreactor. The average measurements of the proposed method reached 98% of stationary liquid phase balance method, taken as a reference, compared to 21% when the traditional method was used. Furthermore, this methodology does not require knowledge of the volumetric transfer coefficient k(L)a, which may vary during growth.

Since the recombinant thyroid-stimulating hormone (rhTSH) is secreted by stably transfected Chinese hamster ovary (CHO-hTSH) cells, a bioprocess consisting of immobilizing the cells on a substrate allowing their multiplication is very suitable for rhTSH recovering from supernatants at relative high degree of purity. In addition, such a system has also the advantage of easily allowing delicate manipulations of culture medium replacement. In the present study, we show the development of a laboratory scale bioprocess protocol of CHO-hTSH cell cultures on cytodex microcarriers (MCs) in a 1 L bioreactor, for the preparation of rhTSH batches in view of structure/function studies. CHO-hTSH cells were cultivated on a fetal bovine serum supplemented medium during cell growth phase. For rhTSH synthesis phase, 75% of supernatant was replaced by animal protein-free medium every 24 h. Cell cultures were monitored for agitation (rpm), temperature (°C), dissolved oxygen (% DO), pH, cell concentration, MCs coverage, glucose consumption, lactate production, and rhTSH expression. The results indicate that the amount of MCs in the culture and the cell concentration at the beginning of rhTSH synthesis phase were crucial parameters for improving the final rhTSH production. By cultivating the CHO-hTSH cells with an initial cell seeding of four cells/MC on 4 g/L of MCs with a repeated fed batch mode of operation at 40 rpm, 37 °C, 20% DO, and pH 7.2 and starting the rhTSH synthesis phase with 3 × 10(6) cells/mL, we were able to supply the cultures with enough glucose, to maintain low levels of lactate, and to provide high percent (∼80%) of fully covered MCs for a long period (5 days) and attain a high cell concentration (∼9 × 10(5) cells/mL). The novelty of the present study is represented by the establishment of cell culture conditions allowing us to produce ∼1.6 mg/L of rhTSH in an already suitable degree of purity. Batches of produced rhTSH were purified and showed biological activity.

Specific respiration rate ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ Q_{{{\text{O}}_{2} }} $$\end{document}) is a key parameter to understand cell metabolism and physiological state, providing useful information for process supervision and control. In this work, we cultivated different insect cells in a very controlled environment, being able to measure \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ Q_{{{\text{O}}_{2} }} $$\end{document}. Spodoptera frugiperda (Sf9) cells have been used through virus infection as host for foreign protein expression and bioinsecticide production. Transfected Drosophila melanogaster (S2) cells can be used to produce different proteins. The objective of this work is to investigate respiratory activity and oxygen transfer during the growth of different insect cells lines as Spodoptera frugiperda (Sf9), Drosophila melanogaster (S2) wild and transfected for the expression of GPV and EGFP. All experiments were performed in a well-controlled 1-L bioreactor, with SF900II serum free medium. Spodoptera frugiperda (Sf9) cells reached 10.7 × 106 cells/mL and maximum specific respiration rate (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ Q_{{{\text{O}}_{2} \max }} $$\end{document}) of 7.3 × 10−17 molO2/cell s. Drosophila melanogaster (S2) cells achieved 51.2 × 106 cells/mL and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ Q_{{{\text{O}}_{2} \max }} $$\end{document} of 3.1 × 10–18 molO2/cell s. S2AcGPV (expressing with rabies virus glycoprotein) reached 24.9 × 106 cells/mL and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ Q_{{{\text{O}}_{2} \max }} $$\end{document} of 1.7 × 10–17 molO2/cell s, while S2MtEGFP (expressing green fluorescent protein) achieved 15.5 × 106 cells/mL and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ Q_{{{\text{O}}_{2} \max }} $$\end{document} = 1.9 × 10−17 molO2/cell s. Relating to the Sf9, S2 cells reached higher maximum cell concentrations and lower specific respiration rate, which can be explained by its smaller size. These results presented useful information for scale-up and process control of insect cells.

International audience; Recombinant rabies virus glycoprotein (rRVGP) was expressed in Drosophila melanogaster Schneider 2 (S2) cells. The cDNA encoding the entire RVGP gene was cloned in an expression plasmid under the control of the constitutive actin promoter (Ac), which was co-transfected into S2 cells together with a hygromycin selection plasmid. Selected S2 cell populations (S2AcRVGP) had a decreased ability to grow and consume substrates, when compared to the non-transfected cells (S2). They were shown, by PCR, to express the RVGP gene and mRNA and, by immunoblotting, to synthesize the rRVGP in its expected molecular mass of 65 kDa. ELISA kinetic studies showed the rRVGP expression in cell lysates and supernatants attaining concentrations of 300 microg/L. By flow cytometry analysis, about 30% of the cells in the co-transfected populations were shown to express the rRVGP. Cell populations selected by limiting dilution expressed higher rRVGP yields. Mice immunized with rRVGP were shown to synthesize antibodies against rabies virus and be protected against experimental infection with rabies virus. The data presented here show that S2 cells can be suitable hosts for the rRVGP expression, allowing its synthesis in a high degree of physical and biological integrity.

Address: 1Laboratorio de Imunologia Viral, Instituto Butantan, 05503-900 Sao Paulo, Brasil, 2Laboratorio de Imunopatologia, Instituto Butantan, 05503-900 Sao Paulo, Brasil, 3Laboratorio de Imunogenetica, Instituto Butantan, 05503-900 Sao Paulo, Brasil, 4Servico de Controle de Qualidade, Instituto Butantan, 05503-900 Sao Paulo, Brasil and 5Departamento de Engenharia Quimica, Escola Politecnica, Universidade de Sao Paulo, Brasil * Corresponding author

The filamentous fungus Trichoderma reesei is adapted to nutrient-poor environments, in which it uses extracellular cellulases to obtain glucose from the available cellulose biomass. We have isolated and characterized Trhxt1, a putative glucose transporter gene, as judged by the glucose accumulation phenotype of a DeltaTrhxt1 mutant. This gene is repressed at high glucose concentrations and expressed at micromolar levels and in the absence of glucose. The gene is also induced during the growth of T. reesei on cellulose when the glucose concentration generated from the hydrolysis of cellulose present in the culture medium is in the micromolar range. We also show that oxygen availability controls the expression of the Trxht1 gene. In this regard, the gene is down-regulated by hypoxia and also by the inhibition of the flow of electrons through the respiratory chain using antimycin A. Intriguingly, anoxia but not hypoxia strongly induces the expression of the gene in the presence of an otherwise repressive concentration of glucose. These results indicate that although the absence of repressing concentrations of glucose and an active respiratory chain are required for Trhxt1 expression under normoxic conditions these physiological processes have no effect on the expression of this gene under an anoxic state. Thus, our results highlight the presence of a novel coordinated interaction between oxygen and the regulatory circuit for glucose repression under anoxic conditions.

Oxygen is essential for the survival of obligatorily aerobic eukaryotic microorganisms, such as the multicellular fungus Trichoderma reesei. However, the molecular basis for the inability of such cells to survive for extended periods under anoxic conditions is not fully understood. Using cDNA microarray analysis, we show that changes in oxygen availability have a drastic effect on gene expression in T. reesei. The expression levels of 392 (19.6%) out of 2000 genes examined changed significantly in response to hypoxia, transient anoxia, and reoxygenation. In addition to modulating many genes with no previously assigned function, cells respond to hypoxia by readjusting the balance of expression between genes required for energy production and consumption, and altering the expression of genes involved in protective mechanisms and signaling pathways. Moreover, we show that transient anoxia strongly represses genes for enzymes that are critical for glycolysis, and are essential for energy production under anaerobic conditions. Our study thus reveals crucial differences between the facultative anaerobe Saccharomyces cerevisiae and T. reesei with regard to the oxygen-dependent transcriptional control of the glycolytic pathway, which can account for the differential survival of the two species in the absence of oxygen.