Enhancing control of cell-free metabolism through pH modulation
Enhancing control of cell-free metabolism through pH modulation
复制标题
DOI：
10.1093/synbio/ysz027
复制
发表时间：
2020-01-01
影响因子：
3.2
通讯作者：
Jewett, Michael C.
中科院分区：
文献类型：
--
作者：
Karim, Ashty S.;Rasor, Blake J.;Jewett, Michael C.
在非模式生物中合成生物基产品的工程代谢可能具有挑战性。一个具体的挑战是，生物合成途径通常是由来源于不同生物体的候选酶构建的，由于其细胞环境（例如pH、辅因子平衡）的差异，这可能证明难以在重组宿主中实施。为了解决这个问题，我们报告了一种无细胞合成生物学方法，用于了解在一系列环境条件下的代谢，特别是在不同的pH值下。杆菌作为模型，我们应用该方法研究了pH对梭菌正丁醇生物合成途径的影响。大肠杆菌裂解物。具体来说，我们利用开放的，无细胞的反应环境，探索pH值以外的可居住的范围E。大肠杆菌，揭示了化学环境如何影响天然金属和异源酶之间的相互作用。我们发现，从乙酰辅酶A生产丁醇的最适pH值明显低于在E.基于大肠杆菌的粗裂解物。此外，pH值是在无细胞环境中激活代谢途径时需要考虑的一个重要因素，因为它对反应产率或酶活性有影响，后者在本研究中对一系列极端微生物的醇脱氢酶进行了证明。最终，通过pH控制改变代谢将允许无细胞系统用于研究生物体的代谢状态，并确定合适的酶用于途径工程。
Engineering metabolism for the synthesis of bio-based products in non-model organisms can be challenging. One specific challenge is that biosynthetic pathways are often built from enzyme candidates sourced from diverse organisms, which can prove difficult to implement in recombinant hosts due to differences in their cellular environments (e.g. pH, cofactor balance). To address this problem, we report a cell-free synthetic biology approach for understandingmetabolism in a range of environmental conditions, specifically under varied pH. The key idea is to control the pH of Escherichia coli-based cell-free systems for assessing pathway performance using enzymes sourced fromorganisms other than E. coli. As amodel, we apply this approach to study the impact of pH on the n-butanol biosynthesis pathway derived from clostridia in E. coli lysates. Specifically, we exploit the open, cell-free reaction environment to explore pH outside the habitable range of E. coli, revealing insights into how chemical context impacts the interaction between nativemetabolism and heterologous enzymes. We find that the pH optimumfor butanol production from acetyl-CoA is substantially lower than the optimal pH of glycolysis in E. coli-based crude lysates. In addition, pH is an essential factor to consider when activatingmetabolic pathways in the cell-free environment due to its effect on reaction yield or enzyme activity, the latter of which is demonstrated in this work for alcohol dehydrogenases from a range of extremophiles. Ultimately, altering metabolism through pH control will allow cell-free systems to be used in studying the metabolic state of organisms and identify suitable enzymes for pathway engineering.