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

BBSRC-NSF/BIO。

• 批准号: 2031948
• 负责人: Eric Klein
• 金额: $ 51.6万
• 依托单位: Rutgers University Camden
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2031948&HistoricalAwards=false
• 关键词: BBSRC; NSF; BIO.; SynBioSphinx; building

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.

本课题将研究鞘脂合成的机理，从基本的起始原料开始，在试管中生产合成膜囊泡。 囊泡合成技术是新颖的，并且囊泡在合成生物学、新的医疗保健技术和工业生物技术中具有潜在的用途，因此具有对生物经济做出贡献的潜力。 该项目还将有助于培训高中生、本科生和研究生，并将用于公共外联工作。该项目是罗格斯大学（美国）、杜克大学（美国）和爱丁堡大学（英国）的研究人员之间的合作。合成生物学的一个首要目标是以更可预测和可靠的方式构建合成细胞。 天然细胞产生复杂的分子和更高级的结构，例如作为半渗透性外部脂质屏障的细胞膜。 细胞还显示出改变其膜组成以响应环境变化（例如营养物）和保护细胞免受外部威胁（例如毒素、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，这阻碍了含鞘脂囊泡的体外合成。 相比之下，组装核心鞘脂的细菌酶是可溶的，并且鞘脂生产生物体新月柄杆菌（Caulobacter crescentus）是理想的研究系统。 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的抗性。将使用基于适应性的适应性弹性选择筛选来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（例如重L-丝氨酸）掺入细菌膜和鞘脂中。 高通量筛选策略将确定新的糖基转移酶，将改变生物物理特性的鞘脂含细菌和合成细胞膜。 结合合成生物学/质谱方法将确定四种目标生物催化剂的最佳遗传电路，以从小分子代谢物中建立短而有效的鞘脂途径。 最后，所选构建体的体外转录/翻译将提供从头囊泡的无细胞合成，这将通过显微镜技术进行监测。英国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理事会支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准。

项目成果

Caulobacter requires anionic sphingolipids and deactivation of fur to lose lipid A

柄杆菌需要阴离子鞘脂和毛皮失活才能失去脂质 A

• DOI: 10.1101/2022.01.20.477143
• 发表时间: 2022
• 期刊: bioRxiv
• 作者: Zik, J;Yoon, S;Guan, Z;Skidmore, G;Gudoor, R;Davies, K;Deutschbauer, A;Goodlett, D;Klein, E;Ryan, K.
• 通讯作者: Ryan, K.