In Situ Product Removal (ISPR) in Whole Cell Biotechnology During the Last Twenty Years

Stark, D.; Stockar, Urs von

doi:10.1007/3-540-36782-9_5

Cited by 114 publications

(110 citation statements)

References 244 publications

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“…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 uncoupled oxygen reduction.…”

mentioning

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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mentioning

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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“…In terms of separating and recycling of the biocatalyst, if required, size differences are often exploited, i.e., filtration or membrane-based separation [32,33]. There are of course also more complex examples where a combination of several differences in physicochemical properties is exploited to separate compounds from complex reaction mixtures [34]. These differences are case dependent, which makes the choice and options of suitable ISPR strategies case specific.…”

Section: Ispr In Biocatalysismentioning

confidence: 99%

“…It is pointed out in the table how the implementation of various ISPR strategies in relation to the different examples benefits the overall processes. Even though the examples solely focus on enzyme-catalysis-based applications in relation to the pharmaceutical industry, the ISPR strategies are also highly relevant to other biocatalytic applications [30], e.g., in many fermentation-based processes [25, 34,36]. Hence, the examples demonstrate the current and future value of ISPR technologies, in an era with increasing focus on implementing biocatalysis in the fine chemical and pharmaceutical industries.…”

Section: Ispr In Biocatalysismentioning

confidence: 99%

A Microfluidic Toolbox for the Development of In-Situ Product Removal Strategies in Biocatalysis

Heintz¹,

Mitić²,

Ringborg³

et al. 2016

J Flow Chem

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A microfluidic toolbox for accelerated development of biocatalytic processes has great potential. This is especially the case for the development of advanced biocatalytic process concepts, where reactors and product separation methods are closely linked together to intensify the process performance, e.g., by the use of in-situ product removal (ISPR). This review provides a general overview of currently available tools in a microfluidic toolbox and how this toolbox can be applied to the development of advanced biocatalytic process concepts. Emphasis is placed on describing the possibilities and advantages of the microfluidic toolbox that are difficult to achieve with conventional batch-processbased technologies. Application of this microfluidic toolbox will potentially make it possible to intensify biocatalytic reactions and thereby facilitate the development towards novel and advanced biocatalytic processes, which in many cases have proven too difficult in conventional batch equipment.

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“…Thus, it avoids the loss of biomass because of organic solvent toxicity. [5] Secondly, the rising bubbles also contribute to homogenize fermentation broth. The last but not the least, for most of aerobic fermentation, the bubbles provide oxygen for cells to metabolize.…”

Section: Introductionmentioning

confidence: 99%

Process improvement for fermentation coupling with foam separation: a convenient strategy for cell recycle

Zhang

Dong

et al. 2015

Asia-Pacific J Chem Eng

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Applications of fermentation coupling with foam separation technology show great advantages in many natural biosurfactants fermentation such as nisin, surfactin, and polymyxin E. However, only a few reports focus on process improvement, i.e. cells overflowing with foam. Because the cells also have hydrophobicity, it will overflow during foam separation, which decrease the productivity and contaminate the environment. Therefore, it is important to prevent cell from overflowing. In this study, a new approach was proposed to increase yield, using fermentation coupling with foam separation technology. Meanwhile, we tried to prevent cell from overflowing. This strategy was characterized by enrichment, recovery percentage, and inactivity percentage for product and cells. The results indicated pH was the most important factor in foam separation of polymyxin E. The optimal condition for fermentation coupling with foam separation was determined. The maximum total titer of polymyxin E reached 917 932.31 U (1147.4 U/mL) using coupling strategy that was 1.36 times more than total titer in shaken-flask culture. Additionally, the adsorption mechanism for polymyxin E and cells was studied. The results indicated polymyxin E were mainly adsorbed on bubble, while the cells were partly in liquid form and partly adsorbed on undesired protein. Undesired protein can be considered as a stabilizer in foam separation of polymyxin E.

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In Situ Product Removal (ISPR) in Whole Cell Biotechnology During the Last Twenty Years

Cited by 114 publications

References 244 publications

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

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

A Microfluidic Toolbox for the Development of In-Situ Product Removal Strategies in Biocatalysis

Process improvement for fermentation coupling with foam separation: a convenient strategy for cell recycle

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