Redesign of an Escherichia coli Nissle treatment for phenylketonuria using insulated genomic landing pads and genetic circuits to reduce burden

To build therapeutic strains, Escherichia coli Nissle (EcN) have been engineered to express antibiotics, toxin-degrading enzymes, immunoregulators, and anti-cancer chemotherapies. For efficacy, the recombinant genes need to be highly expressed, but this imposes a burden on the cell, and plasmids are difficult to maintain in the body. To address these problems, we have developed landing pads in the EcN genome and genetic circuits to control therapeutic gene expression. These tools were applied to EcN SYNB1618, undergoing clinical trials as a phenylketonuria treatment. The pathway for converting phenylalanine to trans-cinnamic acid was moved to a landing pad under the control of a circuit that keeps the pathway off during storage. The resulting strain (EcN SYN8784) achieved higher activity than EcN SYNB1618, reaching levels near when the pathway is carried on a plasmid. This work demonstrates a simple system for engineering EcN that aids quantitative strain design for therapeutics. Comparative gene expression profiling analysis of RNA-seq data for WT E. coli Nissle 1917 cells and engineered derivatives (EcN_SensorArrayOnly EcN_NOTgate_PsrA, EcN_NOTgate_HlyIIR, EcN_NOTgate_BM3R1, EcN_NOTgate_AmtR, EcN_NOTgate_QacR, EcN_NOTgate_LitR, EcN_NOTgate_AmeR, EcN_NOTgate_IcaR, EcN_NOTgate_BetI, EcN_NOTgate_PhlF)

为构建治疗用工程菌株，研究者已对大肠杆菌Nissle（Escherichia coli Nissle, EcN）进行遗传改造，使其能够表达抗生素、毒素降解酶、免疫调节因子及抗癌化疗药物。为实现高效疗效，重组基因需实现高水平表达，但这会给宿主细胞带来代谢负荷，且质粒在体内难以稳定维持。为解决上述问题，本研究开发了EcN基因组中的整合着陆位点（landing pad）以及用于调控治疗基因表达的遗传回路。上述工具已应用于EcN SYNB1618菌株，该菌株作为苯丙酮尿症治疗药物正处于临床试验阶段。研究中将苯丙氨酸转化为反式肉桂酸的代谢通路整合至该整合着陆位点，并由可在储存状态下关闭该通路的遗传回路进行调控。所得工程菌株（EcN SYN8784）的活性高于EcN SYNB1618，其酶活水平接近该通路以质粒形式携带时的表达水平。本研究构建了一套简便的EcN工程化系统，可为治疗用菌株的定量设计提供有力支撑。本研究对野生型大肠杆菌Nissle 1917（WT E. coli Nissle 1917）细胞及其工程化衍生菌株（EcN_SensorArrayOnly、EcN_NOTgate_PsrA、EcN_NOTgate_HlyIIR、EcN_NOTgate_BM3R1、EcN_NOTgate_AmtR、EcN_NOTgate_QacR、EcN_NOTgate_LitR、EcN_NOTgate_AmeR、EcN_NOTgate_IcaR、EcN_NOTgate_BetI、EcN_NOTgate_PhlF）的RNA-seq数据开展了比较基因表达谱分析。