Cell-free synthesis system-assisted pathway bottleneck diagnosis and engineering in Bacillus subtilis

• DOI: 10.1016/j.synbio.2020.06.006
• Publication Date: 2020-06-23T18:17:42Z
• Authors: Rongzhen Tian; Minghu Wang; Jintian Shi; Xiaolong Qin; Haoyu Guo; Xuanjie Jia; Jianghua Li; Long Liu; Guocheng Du; Jian Chen; Yanfeng Liu
• Keywords: Metabolic Engineering; Synthetic Biology; Metabolic pathway; Cell-free protein synthesis

Metabolic engineering is a key technology for cell factories construction by rewiring cellular resources to achieve efficient production of target chemicals. However, the existence bottlenecks in synthetic pathway can seriously affect efficiency, which also one core issues metabolic engineers solve. Therefore, developing an approach diagnosing potential faster and simpler manner great significance accelerate construction. The cell-free reaction system based on lysates transfer reactions from vivo vitro, providing flexible access directly change protein metabolite variables, thus provides solution rapid identification bottlenecks. Here, bottleneck diagnosis N-acetylneuraminic acid (NeuAc) biosynthesis industrially important chassis microorganism Bacillus subtilis was performed using synthesis system. Specifically, highly B. NeuAc de novo firstly constructed, had 305-fold rate than that enabled fast dynamics analysis. Next, through addition all intermediates combination with substrate glucose respectively, it found insufficient phosphoenolpyruvate supply Rational NeuAc-producing further eliminate bottleneck. By down-regulating expression level pyruvate kinase throughout growth phase or only stationary inhibitory N-terminal coding sequences (NCSs) growth-dependent regulatory NCSs maximal titer increased 2.0-fold. Our study method diagnosis, may help cycle design, build, test learn engineering.