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大量用于医学和农业的生物活性化合物。这些化合物结构的微小变化会影响其作为抗生素和抗癌剂（用于医院治疗严重疾病）以及杀虫剂（用于农业保护作物）的功效。许多这些生物活性化合物是由称为非核糖体肽合成酶（NRPS）的大型多酶系统在细菌中产生的，该系统作为装配线生产最终产品。这些装配线在细菌基因组中由称为生物合成基因簇（BGC）的基因组编码。通过去除、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变异的自然实例，并对这些变异进行编目。我们可以使用这些数据来查看Nature修改BGC的确切位置，并应用这些规则进行新的修改，同时不破坏装配线的整体功能。负责制造称为orfamide A的天然产物的BGC将被捕获，随后通过去除，复制或替换不同部分进行修改。最初，我将重新创建已知BGC的例子来尝试修改策略，但我稍后将应用这种技术来创建新的BGC变体和新的天然产物。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，并导致创造新的支架。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型化合物。

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.