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

2019BBSRC-NSF/BIO。

基本信息

批准号：
BB/T016841/1
负责人：
Dominic Campopiano
金额：
$ 49.08万
依托单位：
University of Edinburgh
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FT016841%2F1
关键词：
2019BBSRC NSF BIO.SynBioSphinx building

项目摘要

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.

动物和细菌细胞都有膜。这些是保护性防水壳，由具有亲水（亲水）头基和长憎水（疏水）尾部的分子组成。这个大分子家族被称为脂质，包括脂肪和胆固醇。脂质的一个特殊亚家族是鞘脂 (SL) 和具有长脂肪尾的神经酰胺。 SL 有时附有糖，称为鞘糖脂 (GSL)。 SL 不仅可以让膜防水、让营养物质进入和排出，而且还被发现可以刺激人体免疫系统。 SL 水平有所波动，但也受到严格控制。细胞 SL 水平的巨大变化是出现问题的迹象，并且与阿尔茨海默病、哮喘、癌症和神经衰弱等疾病密切相关。一个令人兴奋的研究领域是发现人类是许多不同类型细菌的宿主还生产 SL、神经酰胺和 GSL。这些细菌统称为微生物群/微生物组，它们生活在我们的肠道、皮肤和口腔中。它们是“好”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（柄杆菌）也能产生 SL，而我们现在才发现为什么细菌具有这种 SL。在我们的项目中，我们希望利用 SL 的优势，并用它们从基本起始材料开始在试管中制造膜囊泡（如微小的肥皂泡）。这些囊泡目前是通过化学方法制备的，但我们的目标是模仿自然并自己设计细胞样的、含有 SL 的囊泡。希望这些人造囊泡能够在新的医疗技术中得到应用，例如：药物输送和检测分子。为了制作 SL，我们需要使用简单的构建块以多步骤的方式进行工作。生产步骤由称为酶的分子机器催化（加速）。研究重点是参与人类和植物 SL 生物合成的酶，但对细菌中 SL 生物合成知之甚少。我们将使用这些错误作为制造 SL 的酶的来源。如果它们制造了足够多的它们，它们就会自然地聚集在一起形成合成囊泡。与需要膜才能激活的人类酶不同，细菌酶在水中具有活性 - 这使得一切变得更加容易、更快和更有效，我们将以更受控的方式制造囊泡。我们将从酶 SPT 开始，它使用两个主要组成部分 - 氨基酸、L-丝氨酸和长链脂肪酸，来制造第一个 SL。然后，我们一次将一种酶添加到试管中，并使用一种称为质谱法的技术来监测 SL 的形成，该技术可测量分子的精确重量。随着酶和化学工作的进展，我的合作者还将使产生 SL 的细菌受到两种外部物质的攻击——抗生素和噬菌体（如病毒）。膜中的 SL 可以保护它们或使它们对这些威胁更加敏感，因此我们将使用这种强大的筛选技术来识别完整的细菌 SL 和 GSL 生物合成途径。然后我们将结合该项目的两个部分，将所有酶放在试管中。一个科学目标是能够使用合成生物学的概念在自给自足的代谢网络中构建设计天然和非天然分子。这涉及设计、构建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 SL/囊泡），从该过程中学习，然后重复循环，直到找到最有效的路线。希望我们能够利用这些方法自下而上地设计和控制栩栩如生的系统。 SynBioSphinx 的结果将为来自多个学科的学术和工业科学家所用，他们正在以新的方式构建新分子。