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

2019BBSRC-NSF/BIO。

• 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

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.

动物和细菌的细胞都有膜。这些是保护性的防水外壳，由具有亲水（亲水）头部基团和长的憎水（疏水）尾部的分子组成。这个大家族的分子被称为脂质，包括脂肪和胆固醇。脂质的一个特定亚家族是具有长脂肪尾的鞘脂（SL）和神经酰胺。SL有时会连接糖，称为鞘糖脂（glycosphingolipids，GSL）。SL不仅允许膜抵抗水分，让营养物质进入并排出废物，还被发现可以刺激人体免疫系统。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信号，与阿尔茨海默氏症、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，这些细菌也会产生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-有益于我们的健康。我的美国合作者最近发现，在淡水中生长的细菌（柄杆菌）也会产生SL，我们现在才发现为什么细菌会产生SL。在我们的项目中，我们希望利用SL，并使用它们在试管中从基本的起始材料开始制造膜囊泡（如微小的肥皂泡）。这些囊泡目前是用化学方法制造的，但我们的目标是模仿自然，设计出类似细胞的、含有SL的囊泡。人们希望这些人造囊泡将用于新的医疗保健技术，例如药物输送和检测分子。为了制造SL，我们需要使用简单的构建模块在多步骤途径中工作。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，但对细菌中SL生物合成知之甚少。我们将利用这些细菌作为制造SL的酶的来源。如果它们产生足够多的蛋白质，它们会自然地聚集在一起形成合成囊泡。与人类酶需要膜才能活化不同，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。我们将开始与酶SPT使用两个主要的建筑块-氨基酸，L-丝氨酸和长链脂肪酸，使第一SL。然后，我们将一次添加一种酶到试管中，并使用一种称为质谱法的技术来监测SL的形成，该技术可以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敏感，因此我们将使用这种强大的筛选技术来鉴定完整的细菌SL和GSL生物合成途径。然后，我们将联合收割机结合项目的两个部分，将所有的酶一起放入试管中，其中一个科学目标是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天然和非天然分子。这涉及设计，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SL/囊泡），从该过程中学习，然后重复循环，直到我们找到最有效的路线。希望我们可以使用这些方法自下而上地设计和控制类生命系统。SynBioSphinx的研究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的分子。

项目成果

Abstract 1349: Convergent evolution of bacterial ceramide synthesis

摘要 1349：细菌神经酰胺合成的趋同进化

• DOI: 10.1016/j.jbc.2023.103790
• 发表时间: 2023
• 期刊: Journal of Biological Chemistry
• 影响因子: 4.8
• 作者: Klein E