Evolution-inspired engineering of non-ribosomal peptide synthetase assembly lines to create novel bioactive scaffolds

微生物和植物为世界提供了相当数量的用于医药和农业的生物活性化合物。这些化合物结构的微小变化可能会影响它们作为抗生素和抗癌剂(用于医院治疗严重疾病)和杀虫剂(用于农业保护农作物)的有效性。这些生物活性化合物中的许多是由称为非核糖体多肽合成酶(NRPS)的大型多酶系统在细菌中产生的，这些系统作为流水线工作，形成最终产品。这些装配线由一组被称为生物合成基因簇(BGC)的基因在细菌基因组中编码。通过移除、复制或替换编码这些装配线的BGC的不同部分，细菌可以修改最终产品。我的项目旨在利用公开可用的细菌基因组序列数据，使用生物信息学技术识别假单胞菌中这些BGC变异的自然例子，并对这些变异进行分类。我们可以使用这些数据来查看自然用来修改BGC的确切位置，并应用这些规则进行新的更改，同时不会扰乱装配线的整体功能。负责制造称为奥法米德A的天然产物的BGC将被捕获，并随后通过移除、复制或替换不同的区段进行修改。最初，我将重新创建已知BGC的示例来试验修饰策略，但稍后我将应用此技术来创建新的BGC变体和新的天然产品。这种创造BGC变体的合成生物学方法将绕过从难以获得的来源获得细菌菌株的需要，并导致新支架的创建。通过这种方法产生的天然产物变体将进行抗菌、抗癌和生物杀虫特性测试。该项目的成果将加速NRPS系统的合理设计和生物工程，从而创造出具有临床、工业和农业有益特性的新化合物。

Microorganisms and plants supply the world with a considerable number of bioactive compounds used in medicine and agriculture. Small changes in the structure of these compounds can influence their efficacy as antibiotics and anti-cancer agents (used in hospitals to treat serious diseases), and pesticides (used in agriculture to protect crops).Many of these biologically active compounds are produced in bacteria by large multi-enzyme systems known as non-ribosomal peptide synthetases (NRPSs), that work as an assembly line to create the final product. These assembly lines are encoded in bacterial genomes by sets of genes known as biosynthetic gene clusters (BGCs). By removing, duplicating, or substituting different sections of the BGCs encoding these assembly lines, bacteria can modify the final product.My project aims to exploit publicly available bacterial genome sequence data to identify natural examples of these BGC variations in Pseudomonas bacteria using bioinformatics techniques and catalogue these variants. We can use this data to see the exact positions employed by Nature to modify the BGCs and apply these rules to make novel alterations while not disrupting the overall function of the assembly line. The BGC responsible for making the natural product called orfamide A will be captured and subsequently modified through the removal, duplication, or substitution of different sections. Initially, I will re-create examples of known BGCs to trial the modification strategy, but I will later apply this technique to create novel BGC variants and new natural products. This synthetic biology approach to creating BGC variants will bypass the need to acquire bacterial strains from difficult to access sources and lead to the creation of novel scaffolds. The natural product variants resulting from this approach will be tested for antimicrobial, anti-cancer, and biopesticidal properties.The outcomes of this project will accelerate the rational design and bioengineering of NRPS systems enabling novel compounds with beneficial properties for clinical, industrial, and agricultural to be created.