2019BBSRC-NSF/BIO. SynBioSphinx: building designer lipid membranes for adaptive resilience to environmental challenges.

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Animal and bacterial cells have membranes. These are protective, water-resistant shells that are composed of molecules with a water-loving (hydrophilic) head group and a long, water-hating (hydrophobic) tail. This large family of molecules are called lipids and include fats and cholesterol. One particular sub-family of lipids are sphingolipids (SLs) and ceramides which have long fatty tails. SLs sometimes have sugars attached and are known glycosphingolipids, GSLs. The SLs not only allow membranes to resist water and let nutrients in and waste out, they have also been found to stimulate the human immune system. SL levels fluctuate but are also tightly controlled. Large changes in cellular SL levels are a sign that something has gone wrong and are strongly linked with diseases such as Alzheimer's, asthma, cancer and nerve-wasting.An exciting area of research is the discovery that humans are hosts for many different types of bacteria that also make SLs, ceramides and GSLs. Collectively these bugs are known as the microbiota/microbiome and they live in our gut, on our skin and in our mouths. They are "good" bacteria - beneficial to our health. My USA collaborator recently discovered that bacteria (Caulobacter) growing in fresh water also make SLs and we are only now discovering why bacteria have such SLs. In our project we want to take advantage of SLs and use them to make membrane vesicles (like tiny soap bubbles) in a test-tube starting from basic starting materials. These vesicles are currently made chemically but a goal is to mimic nature and design cell-like, SL-containing vesicles ourselves. It is hoped that these man-made vesicles will have uses in new healthcare technologies e.g. drug delivery and detector molecules. To make the SLs we need to work in a multi-step pathway using simple building blocks. The production steps are catalysed (sped up) by molecular machines called enzymes. Research has focused on the enzymes involved in human and plant SL biosynthesis but very little is known about SL biosynthesis in bacteria. We will use these bugs as a source of the enzymes that will make SLs. If they make enough of them they will naturally come together to form synthetic vesicles. Unlike the human enzymes which need membranes to be active, the bacterial enzymes are active in water - this makes everything a lot easier, quicker and more efficient and we will make vesicles in a more controlled way. We will begin with the enzyme SPT that uses two main building blocks - an amino acid, L-serine and a long chain fatty acid, to make the first SL. We will then add one enzyme at a time to the test tube and monitor the SL formation using a technique called mass spectrometry which measures the exact weight of the molecule. As we progress the enzyme and chemistry work, my collaborators will also put the SL-producing bacteria under attack from two outside agents - an antibiotic and a bacteriophage (like a virus). The SLs in the membrane can protect them or make them more sensitive to these threats so we will use this powerful screening technique to identify the complete bacterial SL and GSL biosynthetic pathway. Then we will combine both parts of the project to pull all the enzymes together in a test tube.One scientific goal is to be able to build up designer natural and non-natural molecules in self-sufficient metabolic networks using a concept known as synthetic biology. This involves engineering concepts to design, build and test collections of biologically- and chemically-catalysed reactions. We measure the output (e.g. SLs/vesicles), learn from that process, then go around the cycle repeatedly until we find the most efficient route. It is hoped that we can use these methods to design and control life-like systems from the bottom up. The results of SynBioSphinx will be of use to academic and industrial scientists from many disciplines who are building new molecules in new ways.