Project description:We investigated the changes of gene expression in PHA-producing Pseudomonas putida KT2440 cultivated under elevated pressure (7 bar) and under combined elevated pressure (7 bar) and elevated dissolved oxygen tension by means of DNA microarrays. RNA samples were isolated from cells cultivated in chemostat under very well defined growth conditions (growth rate, medium, temperature, pH,...)
Project description:Free Fatty Acid receptor 2 (FFA2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using a Designer Receptor Exclusively Activated by Designer Drugs (DREADD) variant of human FFA2 we explored the activation and phosphorylation profile of the receptor, both in a heterologous cell line and in tissues from a transgenic knock-in mouse line expressing this DREADD. FFA2 phospho-site specific antisera targeting either pSer296/pSer297 or pThr306/pThr310 provided sensitive biomarkers of both constitutive and agonist-mediated phosphorylation as well as an effective means to visualise agonist-activated receptors in situ. In white adipose tissue phosphorylation of residues Ser296/Ser297 was enhanced upon agonist activation whilst Thr306/Thr310 did not become phosphorylated. By contrast, in immune cells from Peyer’s patches Thr306/Thr310 become phosphorylated in a strictly agonist-dependent fashion. This was also true in enteroendocrine cells of the colon. The concept of phosphorylation bar-coding has centred to date on the potential for different agonists to promote distinct receptor phosphorylation patterns. Here we demonstrate that this occurs for the same agonist-receptor pairing in different patho-physiologically relevant target tissues. This may underpin why a single G protein-coupled receptor can generate different functional outcomes in a tissue-specific manner.
Project description:Bialaphos resistance (BAR) and phosphinothricin acetyltransferase (PAT) genes, which convey resistance to the broad-spectrum herbicide phosphinothricin (also known as glufosinate) via N-acetylation, have been globally used in basic plant research and genetically engineered crops. Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids, indirect effects of BAR-containing transgenes in planta, including modified amino acid levels, have been seen but without the identification of their direct causes. Combining metabolomics, plant genetics, and biochemical approaches, we show that transgenic BAR indeed converts two plant endogenous amino acids, aminoadipate and tryptophan, to their respective N-acetylated products in several plant species examined. We report the crystal structures of BAR, and further delineate structural basis for its substrate selectivity and catalytic mechanism. Through structure-guided protein engineering, we generated several BAR variants that display significantly reduced nonspecific activities compared to its wild-type counterpart in vivo. Our results demonstrate that transgenic expression of enzymes can result in unintended off-target metabolism arising from enzyme promiscuity. Understanding of such phenomena at the mechanistic level can facilitate the design of maximally insulated systems featuring heterologously expressed enzymes.
