Targeted Growth Medium Dropouts Promote Aromatic Compound Synthesis in Crude E. coli Cell-Free Systems,ACS Synthetic Biology

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Targeted Growth Medium Dropouts Promote Aromatic Compound Synthesis in Crude E. coli Cell-Free Systems
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-10-12 , DOI: 10.1021/acssynbio.9b00524
Benjamin Mohr ₁ , Richard J Giannone ₂ , Robert L Hettich _{1,

2} , Mitchel J Doktycz _{1,

3}

Affiliation

Progress in cell-free protein synthesis (CFPS) has spurred resurgent interest in engineering complex biological metabolism outside of the cell. Unlike purified enzyme systems, crude cell-free systems can be prepared for a fraction of the cost and contain endogenous cellular pathways that can be activated for biosynthesis. Endogenous activity performs essential functions in cell-free systems including substrate biosynthesis and energy regeneration; however, use of crude cell-free systems for bioproduction has been hampered by the under-described complexity of the metabolic networks inherent to a crude lysate. Physical and chemical cultivation parameters influence the endogenous activity of the resulting lysate, but targeted efforts to engineer this activity by manipulation of these nongenetic factors has been limited. Here growth medium composition was manipulated to improve the one-pot in vitro biosynthesis of phenol from glucose via the expression of Pasteurella multocida phenol-tyrosine lyase in crude E. coli lysates. Crude cell lysate metabolic activity was focused toward the limiting precursor tyrosine by targeted growth medium dropouts guided by proteomics. The result is the activation of a 25-step enzymatic reaction cascade involving at least three endogenous E. coli metabolic pathways. Additional modification of this system, through CFPS of feedback intolerant AroG improves yield. This effort demonstrates the ability to activate a long, complex pathway in vitro and provides a framework for harnessing the metabolic potential of diverse organisms for cell-free metabolic engineering. The more than 6-fold increase in phenol yield with limited genetic manipulation demonstrates the benefits of optimizing growth medium for crude cell-free extract production and illustrates the advantages of a systems approach to cell-free metabolic engineering.

中文翻译：

靶向生长培养基去除促进粗大肠杆菌无细胞系统中的芳香族化合物合成

无细胞蛋白质合成 (CFPS) 的进展激发了对细胞外工程复杂生物代谢的兴趣重新燃起。与纯化的酶系统不同，粗制无细胞系统可以以一小部分成本进行制备，并且包含可以激活用于生物合成的内源性细胞途径。内源性活动在无细胞系统中发挥重要作用，包括底物生物合成和能量再生；然而，粗裂解物固有的代谢网络的复杂性被低估了，因此使用粗制无细胞系统进行生物生产受到了阻碍。物理和化学培养参数影响所得裂解物的内源活性，但通过操纵这些非遗传因素来设计这种活性的有针对性的努力受到限制。通过在大肠杆菌粗裂解物中表达多杀巴斯德氏菌苯酚-酪氨酸裂解酶，从葡萄糖体外生物合成苯酚。通过蛋白质组学指导的靶向生长培养基脱落，粗细胞裂解物代谢活动集中在限制性前体酪氨酸上。结果是激活了涉及至少三个内源性大肠杆菌代谢途径的 25 步酶促反应级联。该系统的额外修改，通过反馈不耐受 AroG 的 CFPS 提高了产量。这项工作证明了在体外激活长而复杂的途径的能力并为利用不同生物体的代谢潜力进行无细胞代谢工程提供了一个框架。在有限的遗传操作下苯酚产量增加了 6 倍以上，这证明了优化无细胞粗提物生产的生长培养基的好处，并说明了无细胞代谢工程的系统方法的优势。

更新日期：2020-11-21

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