Flow-cytometric detection of changes in the physiological state ofE. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation

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...

Section: Discussionsupporting

confidence: 60%

mentioning

confidence: 99%

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

Bühler

Park

Blank

et al. 2008

“…The power of the technique lies in the use of specific, fluorescent dyes to discriminate between vital (i.e., active and viable) and compromised (or dead) cells. For instance, propidium iodide (PI), bis-(1,3-dibutylbarbituric acid)trimethine oxonol (BOX) (30), and ethidium bromide (EB) (34,46) can be used to stain defective cells with permeabilized membranes and damaged membrane integrity, while 5(6)-carboxyfluorescein diacetate (cFDA) (26) is used to stain intact cells with nonspecific esterase activity.…”

mentioning

confidence: 99%

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Hyka

Züllig

Ruth

et al. 2010

Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut ؉ (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.Changes to the product quantity and quality as well as the robustness of bioprocesses can be triggered by a number of factors which affect the physiological state of the microorganisms. The expression of a foreign gene, the processing of the recombinant protein, and the exposure of the cell to metabolites, inductors, substrates, unfavorable environmental conditions, high cell density (HCD), and/or "aging" can all result in markedly different physiological responses (17).The interpretation of bioprocess data often fails to reflect the actual state of individual cells within a population. It is based typically on performance characterization using concentration measurements and the simplifying assumption of uniform ("averaged") performance of each cell. This practice (39) provides an example of the failure to distinguish between a homogenous population of equally compromised microorganisms and a heterogeneous population of cells each performing differently. An understanding of the state of the individual cells is critical in achieving high efficiency in recombinant protein production. Only by knowing the number of (vital) cells that express the target molecule at the highest possible rate can the number of such cells within the population be maximized and the propo...

“…Flow cytometry (FC) has evolved as an outstanding tool in bioprocesses due to its usefulness in cell physiology monitoring (5, 12). The persistence of nonculturable cells during microbial fermentation has been attributed to changes in water activity, acidity, redox potential, nutrient availability, and starvation (14,17,18,24,25) or to the use of preserved starter cultures (20). Additionally, the quantification of catalytic activity is critical to bioprocess optimization, as it measures the individual contributions of different cell subpopulations to the global process (2, 13).…”

mentioning

confidence: 99%

Quantitative Approach to Determining the Contribution of Viable-but-Nonculturable Subpopulations to Malolactic Fermentation Processes

Quirós

Herrero

Garcı́a

et al. 2009

Different sizes of viable-but-nonculturable cell subpopulations of a lactic acid bacterium strain were induced by adding increasing amounts of SO 2 . The experimental data obtained here were fitted to a segregated kinetic model developed previously. This procedure allowed us to determine in quantitative terms the contribution of this physiological state to malolactic fermentation.The persistence of stressed, damaged, or viable-but-nonculturable (VBNC) cells during microbial fermentation underlines the requirement of alternative methods for detecting and characterizing these emergent states not otherwise detectable by traditional culture-based methods (13). Flow cytometry (FC) has evolved as an outstanding tool in bioprocesses due to its usefulness in cell physiology monitoring (5, 12). The persistence of nonculturable cells during microbial fermentation has been attributed to changes in water activity, acidity, redox potential, nutrient availability, and starvation (14,17,18,24,25) or to the use of preserved starter cultures (20). Additionally, the quantification of catalytic activity is critical to bioprocess optimization, as it measures the individual contributions of different cell subpopulations to the global process (2, 13). Despite the loss of culturability under standard conditions, it is strongly suspected that VBNC cells remain alive, maintain the transport system and biosynthesis, and are able to metabolize substrates (16,26). Gene expression has also been demonstrated previously (9, 23). However, although the physiology, biochemistry, and genetics of the VBNC state have been studied over the years, its functionality and biological implication are still issues under intense debate (1,21,22).In this work, cider malolactic fermentation (MLF) was selected as a model system to clarify the role played by VBNC cells in bioprocesses. MLF was carried out under different SO 2 concentrations (0, 30, and 60 ppm total) for inducing VBNC states. The fermentation medium was sterile apple must or "green" cider (obtained just after alcoholic fermentation and containing 5.6% [vol/vol] ethanol), obtained as previously described (11). Sodium bisulfite was used for SO 2 treatments. MLF was carried out in duplicate at 22°C statically in 250-ml bottles. An indigenous strain of Lactobacillus hilgardii was inoculated at an optical density at 600 nm of ϳ0.1 to start MLF. Flasks were shaken just before sampling in order to homogenize the biomass content. Samples were taken aseptically at time intervals until malic acid was consumed (Յ0.5 g liter Ϫ1 ), and cells were collected and processed for further analysis as described previously (20). Supernatants were filtered (0.45 m pore size) and frozen (Ϫ20°C) until chemical analysis. The amount of malic acid was determined by highpressure liquid chromatography (Alliance 2690; Waters) with a photodiode array detector (Waters 996), as reported previously (19).Evolution of bacterial subpopulations during MLF. Viable cells (measured as CFU ml Ϫ1 ) were monitored by a plate counting metho...