Abstract

One overarching goal of synthetic biology is to enable the building of synthetic cells in a more predictable and reliable manner. Natural cells generate complex molecules and higher order structures such as the cell membrane that acts as a semi-permeable, external lipid barrier. Cells also display an ability to alter their membrane composition in response to environmental changes (e.g. nutrients) and protect the cell from external threats (e.g. toxins, viruses). Previous work has focused on membranes formed from simple phospholipids but our SynBioSphinx project will study sphingolipids (SLs) since they are found in eukaryotic cell membranes and an increasing number of important microbes. Eukaryotic SL enzymes are membrane bound and this has hampered the in vitro synthesis of SL-containing vesicles. In contrast, bacterial enzymes that assemble the core SLs are soluble and the glycosphingolipid (GSLs)-producing Caulobacter crescentus is an ideal system to study. Moreover, GSLs in the bacterial membrane lead to increased sensitivity to bacteriophage, as well as resistance to the antibiotic polymyxin B. We will use this as a fitness-based, adaptive resilience, selection screen to identify the genes/enzymes in the bacterial SL-producing pathway. We will also use mass spectrometry to track the incorporation of labelled substrates (e.g. heavy L-serine) into bacterial membranes and SLs. A HTP screening strategy will identify novel glycosyltransferases (GT) that will alter the biophysical properties of the GSL-containing bacterial and synthetic cell membranes. We will apply a combined synthetic biology/MS analysis approach to identify the optimal genetic circuits of four target biocatalysts to build a short, efficient SL pathway from known metabolites (serine, fatty acids, ATP, NADH, CoASH). We will use in vitro transcription/translation of the selected constructs to deliver cell-free synthesis of de novo vesicles and monitor these by microscopy techniques.