Genome scrambling and biosensor technologies for production of high value chemicals in synthetic yeast (Chem@Sc2.0)

酵母合成基因组计划是迄今为止最大的合成生物学项目，它为酵母酿酒酵母2.0(Sc2.0)提供了染色体loxP位点，使合成染色体重组和通过loxP介导的进化进行修改成为可能(Scrble，Science 2017,355,1040-1045)。在这个项目中，我们将利用Sc2.0作为一个生产有价值的天然产品(NP)的平台，包括基本的抗生素(如自然催化2018，1,977-984)、抗癌剂、免疫抑制剂和他汀类药物(重磅炸弹药物)。与野生型酿酒酵母不同，Sc2.0拥有人工合成的染色体(重新设计的化学合成的DNA序列)，使基因组能够被打乱，产生基因被重排、缺失或复制的突变。我们已经证明，通过将异源生物合成基因簇引入Sc2.0，有可能产生具有增强的目标化合物产生能力的扰乱突变体(自然普通。2018、9、1936)。然而，筛选大量(数十亿)由基因组扰乱产生的Sc2.0突变是具有挑战性的。为此，我们将开发基于模块化核糖开关组件的生物传感器(例如J.Am化学。SoC。2015、137、9015-9021和J.Am化学。SoC。2014年、第136、10615-10624)，可与靶NP结合并触发荧光反应。通过在Sc2.0中引入生物传感器以及目标NP生物合成所需的基因，我们将能够快速选择产生最高水平目标化合物的杂乱突变细胞；这些细胞将是荧光的，可以使用荧光激活的细胞分选器轻松分离。产生所需化合物产量最高的菌株将受到生物传感器筛选的进一步争抢，并将对它们的基因组进行测序。该项目将提供途径(代谢)工程的范式转变，这可能导致更具成本效益和可持续的抗生素生产，以对抗抗菌素耐药性(AMR)，以及其他基本药物。这个雄心勃勃的项目提供合成生物学方面的尖端培训，涵盖NP生物合成、基于RNA的生物传感器和遗传学。该项目将由Jason Micklefield教授和Patrick Cai教授监督，总部设在曼彻斯特生物技术学院(MIB)和位于密歇根大学的SYNBIOCHEM中心，拥有世界级的设施和培训机会。

The yeast synthetic genome project, the largest Synthetic Biology project to date, provides yeast, Saccharomyces cerevisiae 2.0 (Sc2.0), with chromosomal loxP sites, enabling Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE, Science 2017, 355, 1040-1045). In this project, we will exploit Sc2.0 as a platform for producing valuable natural products (NP), including essential antibiotics (e.g. Nature Catalysis 2018, 1, 977-984), anticancer agents, immunosuppressants and statins (blockbuster drugs). Unlike the wild-type S. cerevisiae, Sc2.0 has synthetic chromosomes (re-designed DNA sequences chemically synthesized), enabling the genome to be scrambled, generating mutants where genes have been rearranged, deleted or duplicated. We have shown that, by introducing heterologous biosynthetic gene clusters into Sc2.0, it is possible to generate scrambled mutants with enhanced capacity for producing target compounds (Nature Commun. 2018, 9, 1936). However, screening large numbers (billions) of Sc2.0 mutants generated by genome scrambling is challenging. To this end, we will develop biosensors, based on modular riboswitch components (e.g. J. Am. Chem. Soc. 2015, 137, 9015-9021 & J. Am. Chem. Soc. 2014, 136, 10615-10624), that can bind to the target NP and trigger a fluorescent response. By introducing biosensors into Sc2.0, along with the genes required for the biosynthesis of the target NP, we will be able to rapidly select the scrambled mutant cells that produce the highest levels of the target compound; these cells will be fluorescent and can be easily separated using a fluorescence activated cell sorter. Strains producing highest yields of desired compounds, will be subjected to further rounds of scrambling with biosensor screening, and their genomes will be sequenced. The project will provide a paradigm shift in pathway (metabolic) engineering, that could lead to more cost-effective and sustainable production of antibiotics required to combat antimicrobial resistance (AMR), as well as other essential medicines. This ambitious project provides cutting-edge training in synthetic biology, spanning NP biosynthesis, RNA-based biosensors and genetics. The project will be supervised by Profs Jason Micklefield and Patrick Cai, and will be based in the Manchester Institute of Biotechnology (MIB) and SYNBIOCHEM centre at the UoM, with world-class facilities and training opportunities.