Targeted Growth Medium Dropouts Promote Aromatic Compound Synthesis in Crude E. coil Cell-Free Systems
Targeted Growth Medium Dropouts Promote Aromatic Compound Synthesis in Crude E. coil Cell-Free Systems
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DOI：
10.1021/acssynbio.9b00524
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发表时间：
2020-11-20
影响因子：
4.7
通讯作者：
Doktycz, Mitchel J.
中科院分区：
文献类型：
--
作者：
Mohr, Benjamin;Giannone, Richard J.;Doktycz, Mitchel J.
无细胞蛋白质合成（CFPS）的进展激发了人们对细胞外复杂生物代谢工程的兴趣。与纯化的酶系统不同，粗无细胞系统可以以一小部分成本制备，并且含有可以被激活用于生物合成的内源性细胞途径。内源性活性在无细胞系统中执行基本功能，包括底物生物合成和能量再生;然而，使用粗无细胞系统进行生物生产受到粗裂解物固有的代谢网络的未充分描述的复杂性的阻碍。物理和化学培养参数影响所得裂解物的内源性活性，但通过操纵这些非遗传因子来工程化该活性的有针对性的努力受到限制。在此，通过在粗大肠杆菌中表达多杀性巴氏杆菌酚-酪氨酸裂解酶，控制生长培养基组成以改善从葡萄糖一锅法体外生物合成苯酚。大肠杆菌裂解物。通过蛋白质组学指导的靶向生长培养基脱落，将粗细胞裂解物代谢活性集中于限制性前体酪氨酸。其结果是激活了一个25步的酶促反应级联，涉及至少三个内源性E。大肠杆菌代谢途径。该系统的额外修改，通过反馈不耐受AroG的CFPS提高产率。这项工作证明了在体外激活一个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超过6倍，证明了优化生长培养基用于粗无细胞提取物生产的益处，并说明了无细胞代谢工程的系统方法的优点。
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.