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

本项目将以基础原料为起始原料，在试管中研究鞘脂合成合成膜泡的机理。囊泡合成技术是一种新颖的技术，囊泡在合成生物学、新的保健技术和工业生物技术方面具有潜在的用途，因此有可能为生物经济做出贡献。该项目还将有助于培训高中生、本科生和研究生，并将用于公共宣传工作。该项目由罗格斯大学（美国）、杜克大学（美国）和爱丁堡大学（英国）的研究人员合作完成。合成生物学的一个首要目标是使合成细胞以一种更可预测和可靠的方式构建。天然细胞产生复杂的分子和高阶结构，如作为半透性外脂质屏障的细胞膜。细胞还显示出根据环境变化（如营养物质）改变其膜组成并保护细胞免受外部威胁（如毒素、病毒）的能力。以前的工作主要集中在由简单磷脂形成的膜上，但SynBioSphinx项目将研究鞘脂，因为它们存在于真核细胞膜和越来越多的重要微生物中。真核鞘脂酶是膜结合的，这阻碍了鞘脂囊泡的体外合成。相反，组装核心鞘脂的细菌酶是可溶的，鞘脂产生生物新月形茎杆菌是一个理想的研究系统。此外，细菌膜中的鞘脂导致对噬菌体的敏感性增加，以及对抗生素多粘菌素b的耐药性增加。基于适应度，适应性恢复，选择筛选将用于鉴定细菌鞘脂产生途径中的基因/酶。质谱法将跟踪标记底物（如重l -丝氨酸）与细菌膜和鞘脂的结合。高通量筛选策略将确定新的糖基转移酶，这将改变含鞘脂细菌和合成细胞膜的生物物理特性。合成生物学/质谱联用方法将确定四种目标生物催化剂的最佳遗传回路，从而从小分子代谢物中构建短而有效的鞘脂途径。最后，在所选构建体的体外转录/翻译将实现无细胞的新生囊泡合成，这将通过显微镜技术进行监测。这个英美合作项目由美国国家科学基金会和英国生物技术和生物科学研究委员会支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project will investigate the mechanism of sphingolipid synthesis to produce synthetic membrane vesicles in a test-tube starting from basic starting materials. The vesicle synthesis technology is novel, and the vesicles have potential uses in synthetic biology, new healthcare technologies and industrial biotechnology and hence the potential to contribute to the bioeconomy. This project will also contribute to the training of high school, undergraduate and graduate students and will be used in public outreach efforts. This project is a collaboration between researchers at Rutgers University (US), Duke University (US), and the University of Edinburgh (UK). 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 the SynBioSphinx project will study sphingolipids since they are found in eukaryotic cell membranes and an increasing number of important microbes. Eukaryotic sphingolipid enzymes are membrane bound and this has hampered the in vitro synthesis of sphingolipid-containing vesicles. In contrast, bacterial enzymes that assemble the core sphingolipids are soluble and the sphingolipid-producing organism Caulobacter crescentus is an ideal system to study. Moreover, sphingolipids in the bacterial membrane lead to increased sensitivity to bacteriophage, as well as resistance to the antibiotic polymyxin B. A fitness-based, adaptive resilience, selection screen will be used to identify the genes/enzymes in the bacterial sphingolipid-producing pathway. Mass spectrometry will track the incorporation of labelled substrates (e.g. heavy L-serine) into bacterial membranes and sphingolipids. A high-throughput screening strategy will identify novel glycosyltransferases that will alter the biophysical properties of the sphingolipid-containing bacterial and synthetic cell membranes. Combined synthetic biology/mass spectrometry approaches will identify the optimal genetic circuits of four target biocatalysts to build a short, efficient sphingolipid pathway from small molecule metabolites. Lastly, in vitro transcription/translation of the selected constructs will deliver cell-free synthesis of de novo vesicles which will be monitored via microscopy techniques.This collaborative US/UK project is supported by the US National Science Foundation and the UK Biotechnology and Biological Sciences Research Council.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.