Novel antimicrobial discovery using high-throughput pathway assembly and robotics

分子生物学的最新进展，特别是我们读取和(最重要的)写入基因组序列的能力的提高，导致了基因工程领域，即合成生物学领域的新一轮兴奋。合成生物学具有很高的生物技术潜力。它最有前途的应用领域之一是产生新的高价值生物活性天然产品，包括抗生素和抗癌药物。克服全球抗菌素耐药性危机的一种方法是通过加速发现新的抗生素来保持抗药性游戏的领先地位。大多数商业抗生素是在放线菌物种中发现的，但这些生物很难操纵，需要很长时间才能生长，因此不适合用于高通量途径组装和机器人。我们的目标是利用合成生物学发现新的抗菌剂。我们已经确定了编码I型聚酮合成酶(一类负责各种抗菌剂和抗癌剂的酶)的生物合成途径，该酶首次可以在大肠杆菌中表达。这一突破将使我们能够利用大肠杆菌作为生产和发现新型抗生素的宿主，利用合成生物学方法，如高通量途径组装和机器人技术。目的：1.从基因组序列中鉴定并表达产生核心I型聚酮结构所需的几个最小单位，并对所获得的化学结构进行分析。通过a)使用不同的具有分支和长度的起始单元来创建不同的分子；b)引入修饰酶，例如水解酶、还原酶、P450、糖基酶，以获得新的末端化合物，从而使所获得的化学结构多样化和修饰。将通过计算分析来选择合适的修饰酶。利用机器人技术和微流控微滴系统开发高通量途径组装和分析。该分析将基于与高通量自动化相结合的目标代谢组学策略(质谱学)。使用CRISPR/CAS9基因组编辑系统等，进一步重新设计和重构已创建的生物合成途径。通过蛋白质进化提高酶活性。这个项目非常适合生物分析、生物技术和生物化学专业的学生，对现代微生物学、合成生物学和高通量机器人/自动化技术有浓厚的兴趣，并愿意学习后基因组数据生成和分析所需的跨学科技能。

Recent advances in molecular biology, in particular our improved ability to read and (most importantly) write genomic sequences, have led to renewed excitement in the area of genetic engineering, i.e. Synthetic Biology. Synthetic Biology has high biotechnological potential. One of its most promising application areas is the generation of new high-value bioactive natural products, including antibiotics and anticancer drugs. One approach to overcome the worldwide antimicrobial resistance crisis is to stay ahead of the resistance game by accelerating the discovery of novel antibiotics. Most commercial antibiotics were discovered in actinomycete species, but these organisms are difficult to manipulate and take long to grow and are thus not suitable for use in high-throughput pathway assembly and robotics. We aim to harness synthetic biology for the discovery of novel antimicrobials. We have identified biosynthesis pathways which encode for a type I polyketide synthase (a class of enzymes responsible for a variety of antimicrobial and anticancer agents) which can be expressed in E. coli for the first time. This breakthrough will allow us to use E. coli as a host for the production and discovery of novel antibiotics, exploiting synthetic biology approaches such as high-throughput pathway assembly and robotics. Aim: 1. Identify and express several minimal units required to produce the core type I polyketide structures in E. coli which are identified computationally from genome sequences and analyse the obtained chemical structure.2. Diversify and modify the obtained chemical structures by a) using different starter units with branching and length to create diverse molecules; b) introducing modifying enzymes, e.g. hydrolase, reductase, P450, glycosylases, to obtain novel end compounds. Suitable modifying enzymes will be selected using computational analysis.3. Develop high-throughput pathway assembly and analysis using robotics as well as a microfluidic picodroplet system. The analysis will be based on a targeted metabolomics strategy (mass spectroscopy) coupled to high-throughput automation.4. Further redesign and refactor the created biosynthesis pathways using, for example, the CRISPR/Cas9 genome editing system. Improve enzyme activity using protein evolution. Determine the chemical structures of newly produced compounds using several analytical methods, including mass spectroscopy and NMR.This project is ideal for bioanalytical, biotechnology and biochemistry students, with a strong interest in modern microbiology, synthetic biology and high-throughput robotic/automation techniques, and a willingness to learn the interdisciplinary skills required for postgenomic data generation and analysis.