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

动物和细菌细胞都有膜。这些是保护性的防水外壳，由具有亲水(亲水)头部基团和长长的憎水(疏水)尾巴的分子组成。这个大家族的分子被称为脂类，包括脂肪和胆固醇。神经鞘脂(SLS)和神经酰胺是一种特殊的脂肪亚家族，它们有长长的脂肪尾巴。SLS有时带有糖，称为神经鞘糖脂，简称GSLS。SLS不仅可以使膜抵抗水分，让营养物质进入和排出，而且还被发现可以刺激人类的免疫系统。SL水平波动，但也受到严格控制。细胞SL水平的大幅变化是出了问题的迹象，与阿尔茨海默氏症、哮喘、癌症和神经损伤等疾病密切相关。一个令人兴奋的研究领域是，人类是许多不同类型细菌的宿主，这些细菌也会产生SLS、神经酰胺和GSLS。这些细菌统称为微生物区系/微生物组，它们生活在我们的肠道、皮肤和口腔中。它们是有益的细菌--对我们的健康有益。我的美国合作者最近发现，在淡水中生长的细菌(Caulbacter)也会产生SLS，我们现在才发现细菌为什么会有这种SLS。在我们的项目中，我们想要利用SLS，并使用它们在试管中从基本的起始材料开始制造膜泡(像微小的肥皂泡)。这些囊泡目前是通过化学方法制造的，但一个目标是模仿自然，自己设计像细胞一样的、含有SL的囊泡。人们希望这些人造囊泡将在新的医疗技术中使用，例如药物输送和检测器分子。为了制作SLS，我们需要使用简单的积木在一个多步骤的路径上工作。生产步骤由称为酶的分子机器催化(加速)。研究主要集中在参与人类和植物SL生物合成的酶上，但对细菌中SL的生物合成知之甚少。我们将使用这些细菌作为产生SLS的酶的来源。如果它们制造了足够的量，它们就会自然地聚集在一起，形成合成的囊泡。与需要膜才能激活的人类酶不同，细菌酶在水中是活跃的--这使得一切都变得更容易、更快、更有效，我们将以更可控的方式制造囊泡。我们将从SPT酶开始，它使用两个主要的构建块--L-丝氨酸和长链脂肪酸--来制造第一个SL。然后，我们将一次向试管中添加一种酶，并使用一种名为质谱学的技术来监测SL的形成，该技术测量分子的确切重量。随着酶和化学工作的进展，我的合作者们还将使产生SL的细菌受到两种外部因素的攻击--一种抗生素和一种噬菌体(如病毒)。膜上的SLS可以保护它们或使它们对这些威胁更加敏感，因此我们将利用这一强大的筛选技术来鉴定细菌SL和GSL的完整生物合成途径。然后，我们将结合该项目的两个部分，将所有酶放在一个试管中。一个科学目标是能够利用合成生物学的概念，在自给自足的代谢网络中建立设计的自然和非自然分子。这涉及到设计、建造和测试生物和化学催化反应集合的工程概念。我们测量输出(例如SLS/囊泡)，从这个过程中学习，然后反复循环，直到我们找到最有效的路线。希望我们能用这些方法自下而上地设计和控制逼真的系统。SynBioSphinx的结果将对来自许多学科的学术和工业科学家有用，他们正在以新的方式构建新分子。

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.