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

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.

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.

The research carried out in this project will have impacts across a wide group of beneficiaries.
Academia. Understanding the pathway for the de novo production of sphingolipid (SL)-containing lipid membranes will benefit researchers who have studied SL biosynthesis. The technical challenges of using membrane-bound, eukaryotic SL enzymes has prevented in vitro SL production. The SynBioSphinx pathway will deliver a combined chemo- and biocatalytic route that uses soluble enzymes and readily available building blocks. Recombinant enzymes can be used to optimise the yield of SL vesicles but we will also apply in vitro/translation expression methods to generate a cell-free system. The SL products will be measured using mass spectrometry (MS) using our upgraded high resolution Bruker FT-ICR Solarix MS. The methodology and analytical data will benefit the metabolomics/lipidomics fields. The speed with which genomic sequence data is generated has provided a glut of new genes/enzymes where low sequence homology fails to assign a function. The antibiotic/phage HTP phenotypic screen of Caulobacter will be of use to the broader fields of gene annotation, antibiotic resistance and phage research. Additionally, the SynBioSphinx project will link chemistry with the Edinburgh Genome Foundry to deliver designed, built and tested expressible constructs suitable for SL synthesis and membrane formation. This proof-of-concept will be of use to the synthetic biology community who wish to assemble optimised complex biochemical pathways to make natural and unnatural molecules. In the future our system could be automated through droplet technology, to allow screening of many parameters and increase reproducibility. Finally, there is a long term goal to functionalise lipid vesicles with biological and synthetic components in order to engineer artificial cells and our work will add SLs and GSLs for application in this field.
Economic Impact. This project will benefit the industrial biotechnology and healthcare sectors. These industries wish to adopt new technologies/concepts such as synthetic biology. The design/build/test cycle can be applied to the HTP screening, identification and characterisation of a new method to produce complex molecules. There is also a desire for greener, less wasteful technologies where biocatalysts can be used to make drugs, industrial chemicals (e.g. biosurfactants) and biofuels. Drug companies have explored new technologies such as synthetic membranes (e.g. Strat-M) to replace animal derived products for testing. A long term goal is to produce synthetic model systems and robust artificial cells that can be used to screen functions such as biomarker sensing and drug delivery. The incorporation of SLs into these new materials will expand the chemical breadth of such systems. In recent years SL-containing extracellular vesicles (EVs), particularly exosomes, have emerged as new players in the progression of certain diseases such as Alzheimer's. These EVs show promise as possible diagnostic markers but natural sources of EVs contain a complex mixture of lipids enriched in SLs. Our model SL-containing membranes will have potential use in this field in being able to generate synthetic vesicles with defined components.
Societal Impact. The UK government's Bioeconomy Strategy (2018-30) for clean growth supports the drawing together of industrial biotechnology and synthetic biology platforms. This aligns with the public demand to replace environmentally damaging and unsustainable chemical manufacturing methods. Moreover, we are ethically obliged to replace animal-derived products especially those used in the healthcare and cosmetic industries. We will make a contribution to both areas by developing bio-inspired routes to complex molecules (ceramides) that are already used in the skin care industry. This will be an excellent proof-of-concept study to articulate the jargon-heavy language of synthetic biology to a wider audience.