基于CRISPR/dCpf1的氧化葡萄糖酸杆菌转录抑制系统的构建与应用

A CRISPR/dCpf1-based transcriptional repression system for Gluconobacter oxydans

氧化葡萄糖酸杆菌(Gluconobacter oxydans)因具有快速不完全氧化糖醇化合物的能力而被广泛应用于工业中。然而,适用于氧化葡萄糖酸杆菌的基因编辑工具较为缺乏,科研人员对其进行代谢改造受到很大的限制。近年来,规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)系统在基因组编辑和转录调控中的应用极大地提高了微生物基因组的编辑效率。为了实现氧化葡萄糖酸杆菌的转录调控,本研究构建了由CRISPR/dCpf1介导的基因转录抑制系统。通过在氧化葡萄糖酸杆菌表达失活的Cpf1蛋白(dCpf1)和含有19 nt重复序列的crRNA,使得该系统在基因转录中表现出有效的抑制效果,其对于单基因的抑制水平最高可达97.9%,且可同时应用于多基因的抑制并表现出很强的抑制能力。为了验证dCpf1介导的CRISPR干扰(CRISPR interference,CRISPRi)系统在代谢工程中的应用效果,将该系统应用于L-山梨糖的代谢途径和呼吸链的调控中,对山梨糖代谢途径关键脱氢酶进行了鉴定,确定了细胞色素bo_(3)氧化酶对细胞生长的作用。该CRISPRi系统的开发有效填补了目前氧化葡萄糖酸杆菌中基因调控方法的短缺,为代谢工程改造氧化葡萄糖酸杆菌提供了有效的基因调控工具。

Gluconobacter oxydans are widely used in industrial due to its ability of oxidizing carbohydrate rapidly.However,the limited gene manipulation methods and less of efficient gene editing tools impose restrictions on its application in industrial production.In recent years,the clustered regularly interspaced short palindromic repeats(CRISPR)system has been widely used in genome editing and transcriptional regulation which improves the efficiency of genome editing greatly.Here we constructed a CRISPR/dCpf1-mediated gene transcriptional repression system,the expression of a nuclease inactivation Cpf1 protein(dCpf1)in Gluconobacter oxydans together with a 19 nt direct repeats showed effective repression in gene transcription.This system in single gene repression had strong effect and the relative repression level had been increased to 97.9%.While it could be applied in multiplex gene repression which showed strong repression ability at the same time.Furthermore,this system was used in the metabolic pathway of L-sorbose and the regulatory of respiratory chain.The development of CRISPR transcriptional repression system effectively covered the shortage of current gene regulation methods in G.oxydans and provided an efficient gene manipulation tool for metabolic engineering modification in G.oxydans.