BBSRC-NSF/BIO. SynBioSphinx: building designer lipid membranes for adaptive resilience to environmental challenges

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This project will investigate the mechanism of sphingolipid synthesis to produce synthetic membrane vesicles in a test-tube starting from basic starting materials. The vesicle synthesis technology is novel, and the vesicles have potential uses in synthetic biology, new healthcare technologies and industrial biotechnology and hence the potential to contribute to the bioeconomy. This project will also contribute to the training of high school, undergraduate and graduate students and will be used in public outreach efforts. This project is a collaboration between researchers at Rutgers University (US), Duke University (US), and the University of Edinburgh (UK). 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 the SynBioSphinx project will study sphingolipids since they are found in eukaryotic cell membranes and an increasing number of important microbes. Eukaryotic sphingolipid enzymes are membrane bound and this has hampered the in vitro synthesis of sphingolipid-containing vesicles. In contrast, bacterial enzymes that assemble the core sphingolipids are soluble and the sphingolipid-producing organism Caulobacter crescentus is an ideal system to study. Moreover, sphingolipids in the bacterial membrane lead to increased sensitivity to bacteriophage, as well as resistance to the antibiotic polymyxin B. A fitness-based, adaptive resilience, selection screen will be used to identify the genes/enzymes in the bacterial sphingolipid-producing pathway. Mass spectrometry will track the incorporation of labelled substrates (e.g. heavy L-serine) into bacterial membranes and sphingolipids. A high-throughput screening strategy will identify novel glycosyltransferases that will alter the biophysical properties of the sphingolipid-containing bacterial and synthetic cell membranes. Combined synthetic biology/mass spectrometry approaches will identify the optimal genetic circuits of four target biocatalysts to build a short, efficient sphingolipid pathway from small molecule metabolites. Lastly, in vitro transcription/translation of the selected constructs will deliver cell-free synthesis of de novo vesicles which will be monitored via microscopy techniques.This collaborative US/UK project is supported by the US National Science Foundation and the UK Biotechnology and Biological Sciences Research Council.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.