Precise control of lycopene production to enable a fast-responding, minimal-equipment biosensor.
Precise control of lycopene production to enable a fast-responding, minimal-equipment biosensor.
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DOI：
10.1016/j.ymben.2017.07.004
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发表时间：
2017-09
影响因子：
8.4
通讯作者：
Styczynski MP
中科院分区：
文献类型：
--
作者：
McNerney MP;Styczynski MP
色素代谢物在针对低资源地区的生物传感器中具有巨大的潜力，因为传感器的输出可以在没有任何设备的情况下得到解释。然而，在不受欢迎的情况下完全抑制色素的产生是具有挑战性的，因为即使是少量的酶也可以催化产生大量可见的色素。红色素番茄红素可能特别有用，因为它在多色素类胡萝卜素途径中的位置，但常用的诱导启动子系统不能抑制番茄红素的生产。在本文中，我们设计了一种在没有诱导剂的情况下完全抑制番茄红素生产的系统，并在诱导后两小时内产生可见的番茄红素。我们设计了Lac、Ara和T7系统，使其具有高达10倍的抗性，但这些改进的系统仍然不能完全抑制番茄红素。事实证明，翻译修饰在控制番茄红素方面要有效得多。通过降低crtEBI基因上核糖体结合位点的强度，我们能够在诱导的3-4小时内完全抑制番茄红素并产生可见的番茄红素。最后，我们加入甲氧戊酸途径酶来提高诱导后番茄红素的产量，并证明了补充代谢前体可以将着色时间减少到1.5小时左右。总体而言，与之前报告的战略相比，这意味着响应时间减少了一个数量级以上。这里使用的方法证明了荧光报告和代谢物报告之间的脱节，有助于将番茄红素用作报告，并可能推广到其他需要精确控制代谢物产生的系统。
Pigmented metabolites have great potential for use in biosensors that target low-resource areas, since sensor output can be interpreted without any equipment. However, full repression of pigment production when undesired is challenging, as even small amounts of enzyme can catalyze the production of large, visible amounts of pigment. The red pigment lycopene could be particularly useful because of its position in the multi-pigment carotenoid pathway, but commonly used inducible promoter systems cannot repress lycopene production. In this paper, we designed a system that could fully repress lycopene production in the absence of an inducer and produce visible lycopene within two hours of induction. We engineered Lac, Ara, and T7 systems to be up to 10 times more repressible, but these improved systems could still not fully repress lycopene. Translational modifications proved much more effective in controlling lycopene. By decreasing the strength of the ribosomal binding sites on the crtEBI genes, we enabled full repression of lycopene and production of visible lycopene in 3–4 hours of induction. Finally, we added the mevalonate pathway enzymes to increase the rate of lycopene production upon induction and demonstrated that supplementation of metabolic precursors could decrease the time to coloration to about 1.5 hours. In total, this represents over an order of magnitude reduction in response time compared to the previously reported strategy. The approaches used here demonstrate the disconnect between fluorescent and metabolite reporters, help enable the use of lycopene as a reporter, and are likely generalizable to other systems that require precise control of metabolite production.
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影响因子：
4.7
作者：
Brockman IM;Prather KL
通讯作者：
Prather KL
影响因子：
64.5
作者：
Pardee K;Green AA;Ferrante T;Cameron DE;DaleyKeyser A;Yin P;Collins JJ
通讯作者：
Collins JJ
DOI：
10.1126/science.1191652
发表时间：
2010-10-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Ajikumar PK;Xiao WH;Tyo KE;Wang Y;Simeon F;Leonard E;Mucha O;Phon TH;Pfeifer B;Stephanopoulos G
通讯作者：
Stephanopoulos G
影响因子：
8.4
作者：
Jin, Yong-Su;Stephanopoulos, Gregory
通讯作者：
Stephanopoulos, Gregory
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.