Tools for the determination of population heterogeneity caused by inhomogeneous cultivation conditions

Lemoine, Anja; Delvigne, Frank; Bockisch, Anika; Neubauer, Peter; Junne, Stefan

doi:10.1016/j.jbiotec.2017.03.020

Cited by 34 publications

(31 citation statements)

References 81 publications

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“…Noise in gene expression was found to be one major cause of population heterogeneity influencing bioprocess efficiency . Upon occurrence, it leads to a broadening of distributions or multimodality . Noise was found to be minimized in the expression of genes that are essential and contribute to cellular growth whereas it was elevated in genes that mediate the response to environmental changes, for example, the stress response, energy metabolism, and carbon utilization .…”

Section: Introductionmentioning

confidence: 99%

The effect of acetate on population heterogeneity in different cellular characteristics of Escherichia coli in aerobic batch cultures

Heins

Lundin

Nunes

et al. 2019

Biotechnology Progress

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Acetate as the major by-product in industrial-scale bioprocesses with Escherichia coli is found to decrease process efficiency as well as to be toxic to cells, which has several effects like a significant induction of cellular stress responses. However, the underlying phenomena are poorly explored. Therefore, we studied time-resolved population heterogeneity of the E. coli growth reporter strain MG1655/pGS20PrrnBGFPAAV expressing destabilized green fluorescent protein during batch growth on acetate and glucose as sole carbon sources. Additionally, we applied five fluorescent stains targeting different cellular properties (viability as well as metabolic and respiratory activity). Quantitative analysis of flow cytometry data verified that bacterial populations in the bioreactor are more heterogeneous in growth as well as stronger metabolically challenged during growth on acetate as sole carbon source, compared to growth on glucose or acetate after diauxic shift. Interestingly, with acetate as sole carbon source, significant subpopulations were found with some cells that seem to be more robust than the rest of the population. In conclusion, following batch cultures population heterogeneity was evident in all measured parameters.Our approach enabled a deeper study of heterogeneity during growth on the favored substrate glucose as well as on the toxic by-product acetate. Using a combination of activity fluorescent dyes proved to be an accurate and fast alternative as well as a supplement to the use of a reporter strain. However, the choice of combination of stains should be well considered depending on which population traits to aim for. K E Y W O R D Sflow cytometry, growth on acetate, metabolic activity, population heterogeneity, reporter strain, viability

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Section: Introductionmentioning

confidence: 99%

The effect of acetate on population heterogeneity in different cellular characteristics of Escherichia coli in aerobic batch cultures

Heins

Lundin

Nunes

et al. 2019

Biotechnology Progress

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“…Thereby, cells diversify their expression profile of certain proteins to have a broader range of response possibilities to environmental changes [7,10,11]. Noise influences essential cellular functions like regulation, survival, differentiation and the stress response by a global regulatory feedback system [7,[12][13][14]. In studies of noise in gene expression an inverse correlation between gene expression and noise could be established [8].…”

mentioning

confidence: 99%

“…Additionally, their fluorescence characteristics (excitation/emission) have to be compatible with the flow cytometers' lasers and filters [1]. However, so far mostly single reporter strains, expressing one fluorescent protein to follow one cellular event of interest, are used when studying population heterogeneity [3,12]. Then, other cellular characteristics remain in the dark, if one does not deploy global omics-methods.…”

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confidence: 99%

Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses

et al. 2020

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Background: Today there is an increasing demand for high yielding robust and cost efficient biotechnological production processes. Although cells in these processes originate from isogenic cultures, heterogeneity induced by intrinsic and extrinsic influences is omnipresent. To increase understanding of this mechanistically poorly understood phenomenon, advanced tools that provide insights into single cell physiology are needed. Results: Two Escherichia coli triple reporter strains have been designed based on the industrially relevant production host E. coli BL21(DE3) and a modified version thereof, E. coli T7E2. The strains carry three different fluorescence proteins chromosomally integrated. Single cell growth is followed with EmeraldGFP (EmGFP)-expression together with the ribosomal promoter rrnB. General stress response of single cells is monitored by expression of sigma factor rpoS with mStrawberry, whereas expression of the nar-operon together with TagRFP657 gives information about oxygen limitation of single cells. First, the strains were characterized in batch operated stirred-tank bioreactors in comparison to wildtype E. coli BL21(DE3). Afterwards, applicability of the triple reporter strains for investigation of population heterogeneity in bioprocesses was demonstrated in continuous processes in stirred-tank bioreactors at different growth rates and in response to glucose and oxygen perturbation simulating gradients on industrial scale. Population and single cell level physiology was monitored evaluating general physiology and flow cytometry analysis of fluorescence distributions of the triple reporter strains. Although both triple reporter strains reflected physiological changes that were expected based on the expression characteristics of the marker proteins, the triple reporter strain based on E. coli T7E2 showed higher sensitivity in response to environmental changes. For both strains, noise in gene expression was observed during transition from phases of non-growth to growth. Apparently, under some process conditions, e.g. the stationary phase in batch cultures, the fluorescence response of EmGFP and mStrawberry is preserved, whereas TagRFP657 showed a distinct response. Conclusions: Single cell growth, general stress response and oxygen limitation of single cells could be followed using the two triple reporter strains developed in this study. They represent valuable tools to study population heterogeneity in bioprocesses significantly increasing the level of information compared to the use of single reporter strains.

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“…Virtually no negative impact on C. glutamicum cultivations regarding growth or product formation was observed by repeated oxygen perturbations in the cultivations with mineral salt medium. Consequently, we assume that growth retardation and aggregation observed in our previous scale‐down studies are caused by the combination of oscillating complex media substrate components and oxygen supply …”

Section: Discussionmentioning

confidence: 99%

Carboxylic acid consumption and production by Corynebacterium glutamicum

Conrady

Lemoine

Limberg

et al. 2019

Biotechnology Progress

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Corynebacterium glutamicum is well-known as an industrial workhorse, most notably for its use in the bulk production of amino acids in the feed and food sector. Previous studies of the effect of gradients in scale-down reactors with complex media disclosed an accumulation of several carboxylic acids and a parallel decrease of growth and product accumulation. This study, therefore, addresses the impact of carboxylic acids, for example, acetate and L-lactate, on the cultivation of the cadaverine producing strain C. glutamicum DM1945Δact3:P tuf -ldcC opt and their potential role in scale up related performance losses. A fluctuating power input in shake flask and stirred tank cultivations with mineral salt was applied to mimic discontinuous oxygen availability.Results demonstrate, whenever sufficient oxygen was available, C. glutamicum recovered from previously occurring stressful conditions like an oxygen limiting phase.Reassimilation of acids was detected simultaneously. In cultures, which were supplemented with either acetate or L-lactate, a rapid cometabolization of both acids in presence of glucose was observed, showing conversion rates of 7.8 and 3.8 mmol g cell dry weight −1 hr −1 , respectively. Uptake of these acids was accompanied by increased oxygen consumption. Proteins related to oxidative stress response, glycogen synthesis, and the main carbon metabolism were found in altered concentrations under oscillatory cultivation conditions. (Proteomics data are available via ProteomeXchange with identifier PXD012760). Virtually no impact on growth or product formation was observed. We conclude that the reduced growth and product formation in scale-down cultivations when complex media was used is not caused by the accumulation of carboxylic acids. K E Y W O R D S carboxylic acid, Corynebacterium glutamicum, heterogeneity, oxygen oscillation, scale down

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Tools for the determination of population heterogeneity caused by inhomogeneous cultivation conditions

Cited by 34 publications

References 81 publications

The effect of acetate on population heterogeneity in different cellular characteristics of Escherichia coli in aerobic batch cultures

The effect of acetate on population heterogeneity in different cellular characteristics of Escherichia coli in aerobic batch cultures

Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses

Carboxylic acid consumption and production by Corynebacterium glutamicum

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