Biochemistry, Biophysics and Molecular Biology Department, Iowa State University, Ames, Iowa 50011, United States.
Chemical and Biological Engineering Department, Iowa State University, Ames, Iowa 50011, United States.
ACS Synth Biol. 2021 Apr 16;10(4):716-723. doi: 10.1021/acssynbio.0c00501. Epub 2021 Mar 24.
Cell-free protein expression (CFPS) from cell lysate is an established chemical biology technique. Common efforts to improve synthesis capacity, such as strain engineering and process improvements, have overlooked the opportunity to increase productivity by reducing the dependence on limited, dissolved oxygen. Here we demonstrate conditioning cells for anaerobic respiration which increases the initial protein expression rate up to 4-fold and increases titer by 50% as compared to traditional aerobic cell lysate when using sfGFP as a reporter protein in CFPS reactions run at atmospheric conditions. This enhancement is even more significant when run in an oxygen-depleted environment, where anaerobic respiration preconditioned cells increase yield when supplemented with nitrite as a terminal electron acceptor (TEA). Furthermore, we test knockout mutants to determine key proteins responsible for enhancing the anaerobically prepared CFPS lysate. Further improvements could be made in preconditioning cells by increasing expression levels of critical pathway enzymes or by screening other TEA.
无细胞蛋白质表达 (CFPS) 来自细胞裂解物，是一种已确立的化学生物学技术。常见的提高合成能力的努力，如菌株工程和工艺改进，忽略了通过减少对有限溶解氧的依赖来提高生产力的机会。在这里，我们证明了细胞的厌氧呼吸条件，与传统的有氧细胞裂解物相比，当在大气条件下的 CFPS 反应中使用 sfGFP 作为报告蛋白时，该条件可将初始蛋白质表达率提高 4 倍，并将滴度提高 50%。在缺氧环境中运行时，这种增强更为显著，在缺氧环境中，厌氧呼吸预处理的细胞在补充亚硝酸盐作为末端电子受体 (TEA) 时会增加产量。此外，我们测试了敲除突变体，以确定增强厌氧制备 CFPS 裂解物的关键蛋白。通过增加关键途径酶的表达水平或筛选其他 TEA，可以进一步改善细胞的预处理。
