Medium optimization for the production of lipstatin by Streptomyces toxytricini using full factorial design of experiment

Abstract: Full factorial design of experiment for medium optimization was employed for lipstatin production by Streptomyces toxytricini in shake flask study. The full factorial DOE was very much effective in screening of nutritional parameters within the stipulated time frame in a limited number of experiments. A maximum lipstatin production was achieved 3.290 g/l with the following optimized factors: soya flour 35g/l and soya oil 25g/l. Validation experiments were also carried out to verify the adequacy and the accuracy of the model. The results also give a scope for large scale fermentation of lipstatin production. [Luthra, U., Kumar, H., Kulshreshtha, N., Tripathi, A., Trivedi, A., Khadpekar, S., Chaturvedi, A. and Dubey, R.C. Medium optimization for the production of lipstatin by Streptomyces toxytricini using full factorial design of experiment. Nat Sci 2013;1

Operating regimes in a single enzymatic cascade at ensemble-level.

Single enzymatic cascade, ubiquitously found in cellular signaling networks, is a phosphorylation-dephosphorylation reaction cycle causing a transition between inactive and active states of a protein catalysed by kinase and phosphatase, respectively. Steady-state information processing ability of such a cycle (e.g., MAPK cascade) has been classified into four qualitatively different operating regimes, viz., hyperbolic (H), signal-transducing (ST), threshold-hyperbolic (TH) and ultrasensitive (U). These four regimes represent qualitatively different dose-response curves, that is, relationship between concentrations of input kinase (e.g., pMEK) and response activated protein (e.g., pERK). Regimes were identified using a deterministic model accounting for population-averaged behavior only. Operating regimes can be strongly influenced by the inherently present cell-to-cell variability in an ensemble of cells which is captured in the form of pMEK and pERK distributions using reporter-based single-cell experimentation. In this study, we show that such experimentally acquired snapshot pMEK and pERK distribution data of a single MAPK cascade can be directly used to infer the underlying operating regime even in the absence of a dose-response curve. This deduction is possible primarily due to the presence of a monotonic relationship between experimental observables RIQR, ratio of the inter-quartile range of the pERK and pMEK distribution pairs and RM, ratio of the medians of the distribution pair. We demonstrate this relationship by systematic analysis of a quasi-steady state approximated model superimposed with an input gamma distribution constrained by the stimulus strength specific pMEK distribution measured on Jurkat-T cells stimulated with PMA. As a first, we show that introduction of cell-to-cell variability only in the upstream kinase achieved by superimposition of an appropriate input pMEK distribution on the dose-response curve can predict bimodal response pERK distribution in ST regime. Implementation of the proposed method on the input-response distribution pair obtained in stimulated Jurkat-T cells revealed that while low-dosage PMA stimulation preserves the H regime observed in resting cells, high-dosage causes H to ST regime transition