Membrane stress caused by octanoic acid in Saccharomyces cerevisiae.

In order to compete with petroleum-based fuel and chemicals, engineering a robust biocatalyst that can convert renewable feedstocks into biorenewable chemicals, such as carboxylic acids, is increasingly important. However, product toxicity is often problematic. In this study, the toxicity of the carboxylic acids hexanoic, octanoic, and decanoic acid on Saccharomyces cerevisiae was investigated, with a focus on octanoic acid. These compounds are completely inhibitory at concentrations of magnitude 1 mM, and the toxicity increases as chain length increases and as media pH decreases. Transciptome analysis, reconstruction of gene regulatory network, and network component analysis suggested decreased membrane integrity during challenge with octanoic acid. This was confirmed by quantification of dose-dependent and chain length-dependent induction of membrane leakage, though membrane fluidity was not affected. This induction of membrane leakage could be significantly decreased by a period of pre-adaptation, and this pre-adaptation was accompanied by increased oleic acid content in the membrane, significantly increased production of saturated lipids relative to unsaturated lipids, and a significant increase in the average lipid chain length in the membrane. However, during adaptation cell surface hydrophobicity was not altered. The supplementation of oleic acid to the medium not only elevated the tolerance of yeast cells to octanoic acid but also attenuated the membrane leakiness. However, while attempts to mimic the oleic acid supplementation effects through expression of the Trichoplusia ni acyl-CoA Δ9 desaturase OLE1(TniNPVE desaturase) were able to increase the oleic acid content, the magnitude of the increase was not sufficient to reproduce the supplementation effect and increase octanoic acid tolerance. Similarly, introduction of cyclopropanated fatty acids through expression of the Escherichia coli cfa gene was not helpful for tolerance. Thus, we have provided quantitative evidence that carboxylic acids damage the yeast membrane and that manipulation of the lipid content of the membrane can increase tolerance, and possibly production, of these valuable products.

Application of a generalized MWC model for the mathematical simulation of metabolic pathways regulated by allosteric enzymes.

In our effort to elucidate the systems biology of the model organism, Escherichia coli, we have developed a mathematical model that simulates the allosteric regulation for threonine biosynthesis pathway starting from aspartate. To achieve this goal, we used kMech, a Cellerator language extension that describes enzyme mechanisms for the mathematical modeling of metabolic pathways. These mechanisms are converted by Cellerator into ordinary differential equations (ODEs) solvable by Mathematica. In this paper, we describe a more flexible model in Cellerator, which generalizes the Monod, Wyman, Changeux (MWC) model for enzyme allosteric regulation to allow for multiple substrate, activator and inhibitor binding sites. Furthermore, we have developed a model that describes the behavior of the bifunctional allosteric enzyme aspartate kinase I-homoserine dehydrogenase I (AKI-HDHI). This model predicts the partition of enzyme activities in the steady state which paves the way for a more generalized prediction of the behavior of bifunctional enzymes.

Human T-cell lymphotropic virus type I-transformed T-cells have a partial defect in ceramide synthesis in response to N-(4-hydroxyphenyl)retinamide.

Treatment with the synthetic retinoid HPR [N-(4-hydroxyphenyl)-retinamide] causes growth arrest and apoptosis in HTLV-I (human T-cell lymphotropic virus type-I)-positive and HTLV-I-negative malignant T-cells. It was observed that HPR-mediated growth inhibition was associated with ceramide accumulation only in HTLV-I-negative cells. The aim of the present study was to investigate the mechanism by which HPR differentially regulates ceramide metabolism in HTLV-I-negative and HTLV-I-positive malignant T-cells. Clinically achievable concentrations of HPR caused early dose-dependent increases in ceramide levels only in HTLV-I-negative cells and preceded HPR-induced growth suppression. HPR induced de novo synthesis of ceramide in HTLV-I-negative, but not in HTLV-I-positive, cells. Blocking ceramide glucosylation in HTLV-I-positive cells, which leads to accumulation of endogenous ceramide, rendered these cells more sensitive to HPR. Exogenous cell-permeant ceramides that function partially by generating endogenous ceramide induced growth suppression in all tested malignant lymphocytes, were consistently found to be less effective in HTLV-I-positive cells confirming their defect in de novo ceramide synthesis. Owing to its multipotent activities, the HTLV-I-encoded Tax protein was suspected to inhibit ceramide synthesis. Tax-transfected Molt-4 and HELA cells were less sensitive to HPR and C6-ceramide mediated growth inhibition respectively and produced lower levels of endogenous ceramide. Together, these results indicate that HTLV-I-positive cells are defective in de novo synthesis of ceramide and that therapeutic modalities that bypass this defect are more likely to be successful.

Application of a generalized MWC model for the mathematical simulation of metabolic pathways regulated by allosteric enzymes.

In our effort to elucidate the systems biology of the model organism, Escherichia coli, we have developed a mathematical model that simulates the allosteric regulation for threonine biosynthesis pathway starting from aspartate. To achieve this goal, we used kMech, a Cellerator language extension that describes enzyme mechanisms for the mathematical modeling of metabolic pathways. These mechanisms are converted by Cellerator into ordinary differential equations (ODEs) solvable by Mathematica. In this paper, we describe a more flexible model in Cellerator, which generalizes the Monod, Wyman, Changeux (MWC) model for enzyme allosteric regulation to allow for multiple substrate, activator and inhibitor binding sites. Furthermore, we have developed a model that describes the behavior of the bifunctional allosteric enzyme aspartate Kinase I-Homoserine Dehydrogenase I (AKI-HDHI). This model predicts the partition of enzyme activities in the steady state which paves a way for a more generalized prediction of the behavior of bifunctional enzymes.