Overexpression of a 123-kDa anion transport inhibitor binding protein and two cytoskeleton proteins in Drosophila Kc cell variants resistant to disulfonic stilbenes

Drugs of the disulfonic stilbene class, which inhibit anion transport in the cell membrane in many cell types, have been found to inhibit anion transport and cell growth in Drosophila Kc cells. Cell variants selected by a stepwise selection protocol for the ability to grow in the presence of the disulfonic stilbenes are severalfold resistant to growth inhibition by the drugs. Both the resistant populations and a cloned cell line show dramatic overexpression of three polypeptides. The most highly overproduced protein is a 123-kDa plasma membrane protein which binds the reversible anion transport inhibitor, flufenamic acid, in a protection biotinylation experiment. The 123-kDa putative anion transport protein copurifies with, and immunologically cross-reacts with, two detergent-insoluble cytoskeleton proteins of 46- and 62-kDa molecular weight, which are each overexpressed more than 8-fold in the variants. Resistance to growth inhibition by the disulfonic stilbenes and amplified expression of the 123-, 62-, and 46-kDa proteins are simultaneously lost over a period of 30 weeks in the absence of selective conditions, suggesting that the function of the overproduced polypeptides is related to growth control in Drosophila cells.     

PspA adopts an ESCRT-III-like fold and remodels bacterial membranes

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A dynamic metabolite valve for the control of central carbon metabolism

Successful redirection of endogenous resources into heterologous pathways is a central tenet in the creation of efficient microbial cell factories. This redirection, however, may come at a price of poor biomass accumulation, reduced cofactor regeneration and low recombinant enzyme expression. In this study, we propose a metabolite valve to mitigate these issues by dynamically tuning endogenous processes to balance the demands of cell health and pathway efficiency. A control node of glucose utilization, glucokinase (Glk), was exogenously manipulated through either engineered antisense RNA or an inverting gene circuit. Using these techniques, we were able to directly control glycolytic flux, reducing the specific growth rate of engineered Escherichia coli by up to 50% without altering final biomass accumulation. This modulation was accompanied by successful redirection of glucose into a model pathway leading to an increase in the pathway yield and reduced carbon waste to acetate. This work represents one of the first examples of the dynamic redirection of glucose away from central carbon metabolism and enables the creation of novel, efficient intracellular pathways with glucose used directly as a substrate.