A synthetic genomics platform for antibiotic discovery

抗生素耐药性是一种“慢性大流行”。联合国最近的一份报告估计，到2050年，全球每年将有1000万人死于艾滋病，其经济影响可与2008年金融危机相提并论。开发工具来解决抗菌素化合物的耐药性具有迫切的重要性，并已被确定为BBSRC展望英国生物科学的关键目标。非核糖体肽(NRP)代表着一种不同类别的次级代谢物，包括许多抗生素和抗生素前体。它们通常由氨基酸组成，这些氨基酸通过酶作用组装，然后被一系列剪裁酶修饰。通过这种方式，核心抗生素分子可以通过许多方式进行修饰，潜在地绕过抗生素耐药性机制。我们已经证明，使用合成生物学方法可以在酵母中实现NRP抗生素的生产。酵母拥有无与伦比的遗传工具箱，为发现新型NRP衍生抗生素开辟了新的可能性。在这个项目中，学生将建立一个合成酵母平台，用于生产和筛选具有抗生素特性的新型NRP分子。在旋转项目中建立的细菌共培养系统将用于通过扰乱基因组进化来选择一株能够增强青霉素生产的菌株。然后，该菌株将作为基础NRP生产菌株，用于裁剪酶组合的组合原型。生物信息学工具将被用于从基因组数据集中识别候选裁剪酶基因，以便在我们的系统中进行测试。基因将被克隆到金门文库中，并采用与加扰兼容的格式。不同基因组合的文库将被引入到NRP产生菌中，并将使用SCRIBE来进一步增加宿主基因组和文库的多样性。这些高度多样化的文库将与具有不同抗药性特征的细菌菌株进行共培养。酵母细胞在这些条件下的生存将依赖于细菌生长的抑制，这种抑制方式避开了相应的抗生素耐药机制。在化验中幸存下来的菌株将被分离并完全鉴定，以识别任何用作抗生素或抑制抗生素耐药性机制的新化合物。这项工作将建立合成酵母方法发现抗生素的可行性，并可能产生新的抗菌化合物。随后，该系统可以扩展到包括更广泛的候选剪裁酶和额外的NRP分子。监督小组-本杰明·布朗特是合成基因组学专家，也是国际合成酵母基因组计划的成员。他是第一个成功将NRP抗生素生产到酿酒酵母中的团队的成员，也是第一个证明可以使用SCRIBLE来提高包括青霉素在内的工程途径的产量的团队。John Heap是代谢工程的组合方法和应用合成生物学技术改良生物技术菌株方面的专家。

Antibiotic resistance is the "slow pandemic". A recent UN report estimates that by 2050 it will be responsible for 10 million deaths a year globally, with an economic impact comparable to the 2008 financial crisis. Development of tools to tackle resistance to antimicrobial compounds is of pressing importance and has been identified as a key objective in the BBSRC's Forward Look for UK Bioscience.Nonribosomal peptides (NRPs) represent a diverse class of secondary metabolites and include many antibiotics and antibiotic precursors. They typically consist of amino acids that are assembled enzymatically and then modified by an array of tailoring enzymes. In this way, a core antibiotic molecule can be modified in many ways, potentially circumventing antibiotic resistance mechanisms. We have shown that NRP antibiotic production can be achieved in yeast using a synthetic biology approach. Yeast has an unparalleled genetic toolbox available, opening up new possibilities for discovering novel NRP-derived antibiotics In this project, the student will establish a synthetic yeast platform for production and screening of novel NRP molecules for antibiotic properties. The bacterial co-culture system established in the rotation project will be used to select a strain that is enhanced for penicillin production through SCRaMbLE genome evolution. This strain will then act as the base NRP-production strain for combinatorial prototyping of tailoring enzyme combinations.Bioinformatics tools will be used to identify candidate tailoring enzyme genes from genomic datasets to be tested in our system. Genes will be cloned into a Golden Gate library with SCRaMbLE-compatible formatting. Libraries of different gene combinations will be introduced to the NRP-production strain and SCRaMbLE will be used to further increase host genome and library diversity. These massively diversified libraries will be co-cultured with bacterial strains with different antibiotic resistance profiles. Survival of the yeast cells in these conditions will be reliant on the inhibition of bacterial growth in a way that evades the respective antibiotic resistance mechanism. Strains surviving the assays will be isolated and fully characterised to identify any novel compounds that act as antibiotics or that inhibit antibiotic resistance mechanisms. This work will establish the feasibility of a synthetic yeast approach to antibiotic discovery and may yield novel antimicrobial compounds. The system could subsequently be expanded to include a wider range of candidate tailoring enzymes and additional NRP molecules. Supervision Team - Benjamin Blount is an expert in synthetic genomics and a member of the International Synthetic Yeast Genome Project. He was part of the first team to successfully engineer NRP antibiotic production into S. cerevisiae yeast and was the first to demonstrate that SCRaMbLE can be used to improve production from engineered pathways, including penicillin. John Heap is an expert in combinatorial approaches to metabolic engineering and the application of synthetic biology techniques to the improvement of strains for biotechnology.