Physiological responses to mixing in large scale bioreactors

Enfors, Sven‐Olof; Jahic, Mehmedalija; Rozkov, Aleksei; Xu, Bo; Hecker, Michael; Brinckmann, Jürgen; Krüger, Elke; Schweder, Thomas; Hamer, G.; O’Beirne, D.; Noisommit-Rizzi, Naruemol; Reuß, Matthias; Boone, Lindsey R.; Hewitt, Christopher J.; McFarlane, Caroline M.; Nienow, Alvin W.; Kovács, T.; Trägårdh, Christian; Fuchs, László; Revstedt, Johan; Friberg, P. C.; Hjertager, Bjørn Helge; Blomsten, Gustav; Skogman, H.; Hjort, S.; Hoeks, Frans W. J. M. M.; Hong, Lin; Neubauer, Peter; Lans, Ralf van der; Luyben, Karel C.-H. A. M.; Vrábel, Peter; Manelius, Åsa

doi:10.1016/s0168-1656(00)00365-5

Cited by 405 publications

(361 citation statements)

References 14 publications

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“…Moreover, installing a functional phosphagen kinase system such as shown here for AK appears to be a pertinent metabolic engineering strategy to improve the biotechnological potential of microbes that are exposed to energetically stressful conditions. One such condition may be large scale processes in which cells are often exposed to fluctuating availability of nutrients such as carbon source and oxygen because of imperfect mixing (44).…”

Section: Discussionmentioning

confidence: 99%

Functional Expression of Phosphagen Kinase Systems Confers Resistance to Transient Stresses in Saccharomyces cerevisiae by Buffering the ATP Pool

Canonaco¹,

Schlattner²,

Pruett³

et al. 2002

Journal of Biological Chemistry

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Phosphagen kinase systems provide different advantages to tissues with high and fluctuating energy demands, in particular an efficient energy buffering system. In this study we show for the first time functional expression of two phosphagen kinase systems in Saccharomyces cerevisiae, which does not normally contain such systems. First, to establish the creatine kinase system, in addition to overexpressing creatine kinase isoenzymes, we had to install the biosynthesis pathway of creatine by co-overexpression of L-arginine:glycine amidinotransferase and guanidinoacetate methyltransferase. Although we could achieve considerable creatine kinase activity, together with more than 3 mM intracellular creatine, this was not sufficient to confer an obvious advantage to the yeast under the specific stress conditions examined here. Second, using arginine kinase, we successfully installed an intracellular phosphagen pool of about 5 mM phosphoarginine. Such arginine kinase-expressing yeast showed improved resistance under two stress challenges that drain cellular energy, which were transient pH reduction and starvation. Although transient starvation led to 50% reduced intracellular ATP concentrations in wild-type yeast, arginine kinase overexpression stabilized the ATP pool at the pre-stress level. Thus, our results demonstrate that temporal energy buffering is an intrinsic property of phosphagen kinases that can be transferred to phylogenetically very distant organisms.The availability of biochemical energy, with ATP as the primary energy currency, is fundamental to most cellular processes. Although ATP and its congeners are involved in literally hundreds of biochemical reactions, the intracellular concentration of ATP is generally kept very constant at about 2-5 mM, depending on organisms and tissues, with a turnover rate of the ATP pool that is in the range of a few seconds (1). Hence, metabolic ATP generation in a cell must be balanced tightly with ATP-consuming processes. Small deviations from the standard cellular concentrations of free ATP, ADP, and AMP serve important regulatory roles in fine tuning this delicate balance.This balance between energy-consuming and -producing processes is particularly challenged in tissues that experience periods of high and fluctuating energy demand, such as brain, heart, or skeletal muscle. To maintain constant ATP levels, these tissues express creatine kinase (CK 1 ; EC 2.7.3.2) that uses creatine (Cr) to create a metabolically inert pool of phosphocreatine (PCr). Among other functions, this PCr pool serves as a temporal energy buffer that can replenish ATP rapidly during phases of high energy demand, according to the following reaction (2): MgADP Ϫ ϩ PCr 2Ϫ ϩ H ϩ i MgATP 2Ϫ ϩ Cr.

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Section: Discussionmentioning

confidence: 99%

Functional Expression of Phosphagen Kinase Systems Confers Resistance to Transient Stresses in Saccharomyces cerevisiae by Buffering the ATP Pool

Canonaco¹,

Schlattner²,

Pruett³

et al. 2002

Journal of Biological Chemistry

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“…The results suggested that the production of lactic acid, as the major metabolite influencing the pH change, was also affected by the scaled up conditions in the reactor. It has been frequently reported that scaling up fails to achieve the same results concerning the biomass yield and other metabolites associated with its growth (Enfors et al 2001). Part of the mechanisms responsible for these changes are the different systems of agitation and heating responsible for the homogeneity and the heat distribution in the fermentor (Hewitt and Nienow 2007).…”

Section: Fermentation In a Laboratory Bioreactormentioning

confidence: 99%

Synbiotic functional drink from Jerusalem artichoke juice fermented by probiotic Lactobacillus plantarum PCS26

et al. 2015

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A probiotic strain Lactobacillus plantarum PCS26 was used to ferment Jerusalem artichoke juice. Growth kinetics of the bacterial strain was followed during juice fermentation both in flask and in laboratory fermentor. Jerusalem artichoke showed to be an excellent source of nutrients for L. plantarum PCS26 growth. The culture grew very well reaching more than 10 10 cfu/ml in just 12 h. The pH changed from the initial 6.5 to 4.6 at the end of fermentation. The culture hydrolyzed fructooligosaccharides present in the Jerusalem artichoke juice, yielding fructose which was presumably consumed along with the malic acid as energy and carbon source. Lactic acid was the main metabolite produced in concentration of 4.6 g/L. Acetic and succinic acid were also identified. Sensory evaluation of the fermented Jerusalem artichoke juice and its mixtures with blueberry juice showed that the 50/50 % v/v mixture would be very well accepted by the consumers. Above 80 % of the panelists would buy this drink, and over 60 % were willing to pay more for it. Culture survivability in the fermented juices during storage at 4-7°C was assayed by the Weibullian model. The product shelf-life was extended from 19.70 ± 0.50 days of pure Jerusalem artichoke juice to 35.7 ± 6.4 days of the mixture containing 30 % blueberry juice.

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“…However, after second-phase addition, the steady-state cell concentration slightly decreased and small amounts of acetate appeared in the medium (results not shown). The presence of the viscous organic phase may have led to incomplete mixing and thus to nonuniform glucose and oxygen concentrations inside the reactor causing acetate formation, as observed in large-scale bioprocesses (17,25,46,62). After the two-liquid-phase culture had attained a steady state, octane was added to the organic phase feed tank (60 mM) and directly into the culture broth to induce styAB expression.…”

Section: Continuous Two-liquid-phase Cultivation Of E Coli Jm101 (Psmentioning

confidence: 99%

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

Bühler

Park

Blank

et al. 2008

Appl Environ Microbiol

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Styrene can efficiently be oxidized to (S)-styrene oxide by recombinant Escherichia coli expressing the styrene monooxygenase genes styAB from Pseudomonas sp. strain VLB120. Targeting microbial physiology during whole-cell redox biocatalysis, we investigated the interdependency of styrene epoxidation, growth, and carbon metabolism on the basis of mass balances obtained from continuous two-liquid-phase cultures. Full induction of styAB expression led to growth inhibition, which could be attenuated by reducing expression levels. Operation at subtoxic substrate and product concentrations and variation of the epoxidation rate via the styrene feed concentration allowed a detailed analysis of carbon metabolism and bioconversion kinetics. Fine-tuned styAB expression and increasing specific epoxidation rates resulted in decreasing biomass yields, increasing specific rates for glucose uptake and the tricarboxylic acid (TCA) cycle, and finally saturation of the TCA cycle and acetate formation. Interestingly, the biocatalysis-related NAD(P)H consumption was 3.2 to 3.7 times higher than expected from the epoxidation stoichiometry. Possible reasons include uncoupling of styrene epoxidation and NADH oxidation and increased maintenance requirements during redox biocatalysis. At epoxidation rates of above 21 mol per min per g cells (dry weight), the absence of limitations by O 2 and styrene and stagnating NAD(P)H regeneration rates indicated that NADH availability limited styrene epoxidation. During glucose-limited growth, oxygenase catalysis might induce regulatory stress responses, which attenuate excessive glucose catabolism and thus limit NADH regeneration. Optimizing metabolic and/or regulatory networks for efficient redox biocatalysis instead of growth (yield) is likely to be the key for maintaining high oxygenase activities in recombinant E. coli.Oxygenases catalyze regio-and enantioselective oxyfunctionalization reactions and have a considerable potential in the area of asymmetric organic synthesis (2, 35). These enzymes typically depend on coenzymes such as NAD(P)H and are unstable outside cells, and they often consist of multiple components. Thus, for synthetic applications, their use in whole cells is favored over the use of isolated enzymes (7,18).The productivity of oxygenase-based whole-cell biocatalysts is influenced by various factors, such as maximal enzyme activity, enzyme level, substrate mass transfer, substrate and/or product inhibition/toxicity, and cofactor regeneration (7,13,54,68). Enzyme synthesis and cofactor regeneration are related to host metabolism, which in turn may be affected by the toxicity of organic substrates and products. Such toxicity can efficiently be attenuated by regulated substrate feeding (1, 11) and in situ product removal (7,38,61,66,74). Yet, microbial cells, especially in a nongrowing state, often lose biooxidation activity over time, which may be due to oxygenase inactivation and/or the metabolic burden imposed by redox-coenzyme withdrawal and reactive oxygen species formed via...

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Physiological responses to mixing in large scale bioreactors

Cited by 405 publications

References 14 publications

Functional Expression of Phosphagen Kinase Systems Confers Resistance to Transient Stresses in Saccharomyces cerevisiae by Buffering the ATP Pool

Functional Expression of Phosphagen Kinase Systems Confers Resistance to Transient Stresses in Saccharomyces cerevisiae by Buffering the ATP Pool

Synbiotic functional drink from Jerusalem artichoke juice fermented by probiotic Lactobacillus plantarum PCS26

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

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