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A Synthetic Malonyl-CoA Metabolic Oscillator in Komagataella phaffii.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-04-10 , DOI: 10.1021/acssynbio.9b00378 Jiao Wen 1 , Lin Tian 1 , Mingqiang Xu 1 , Xiangshan Zhou 1 , Yuanxing Zhang 1, 2 , Menghao Cai 1, 2
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-04-10 , DOI: 10.1021/acssynbio.9b00378 Jiao Wen 1 , Lin Tian 1 , Mingqiang Xu 1 , Xiangshan Zhou 1 , Yuanxing Zhang 1, 2 , Menghao Cai 1, 2
Affiliation
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Malonyl-CoA is a key metabolic molecule that participates in a diverse range of physiological responses and can act as a building block for a variety of value-added pharmaceuticals and chemicals. The cytosolic malonyl-CoA concentration is usually very low, and thus dynamic metabolic control of malonyl-CoA variation will aid its stable formation and efficient consumption. We developed a synthetic malonyl-CoA metabolic oscillator in yeast. A synthetic regulatory protein, Prm1-FapR, was constructed by fusing a yeast transcriptional activator, Prm1, with a bacterial malonyl-CoA-sensitive transcription repressor, FapR. Two oppositely regulated biosensors were then engineered. A total of 18 hybrid promoter variants were designed, each carrying the operator sequence (fapO) of FapR and the core promoter of PAOX1 (cPAOX1), which is naturally regulated by Prm1. The promoter activities of these variants, regulated by Prm1-FapR, were tested. Through this process, a sensor for Prm1-FapR/(-52)fapO-PAOX1 carrying an activation/deactivation regulation module was built. Meanwhile, 24 promoter variants of PGAP with fapO inserted were designed and tested using the fusion regulator, giving a sensor for Prm1-FapR/PGAP-(+22) fapO that contained a repression/derepression regulation module. Both sensors were subsequently integrated into a single cell, which exhibited correct metabolic switching of eGFP and mCherry reporters following manipulation of cytosolic malonyl-CoA levels. The Prm1-FapR/(-52)fapO-PAOX1 and the Prm1-FapR/PGAP-(+22)fapO were also used to control the malonyl-CoA source and sink pathways, respectively, for the synthesis of 6-methylsalicylic acid. This finally led to an oscillatory metabolic mode of cytosolic malonyl-CoA. Such a metabolator is useful in exploring potential industrial and biomedical applications not limited by natural cellular behavior.
中文翻译：
蛇形果蝇中的合成丙二酰辅酶A代谢振荡器。
丙二酰辅酶A是参与各种生理反应的关键代谢分子，可以作为多种增值药物和化学药品的基础。胞质丙二酰-CoA的浓度通常非常低，因此动态控制丙二酰-CoA变异的代谢将有助于其稳定形成和有效消耗。我们在酵母中开发了合成的丙二酰辅酶A代谢振荡器。通过将酵母转录激活因子Prm1与细菌丙二酰辅酶A敏感的转录抑制因子FapR融合来构建合成的调节蛋白Prm1-FapR。然后设计了两个相反调节的生物传感器。共设计了18个杂种启动子变体，每个变体带有FapR的操纵子序列（fapO）和PAOX1的核心启动子（cPAOX1），这是由Prm1自然调节的。测试了由Prm1-FapR调节的这些变体的启动子活性。通过此过程，构建了带有激活/停用调节模块的Prm1-FapR /（-52）fapO-PAOX1传感器。同时，使用融合调节剂设计并测试了插入有fapO的PGAP的24个启动子变体，从而为Prm1-FapR / PGAP-（+ 22）fapO提供了一个传感器，该传感器包含一个阻抑/阻抑调节模块。随后将两个传感器都整合到单个细胞中，该细胞在操纵胞质丙二酰辅酶A水平后表现出eGFP和mCherry报告基因正确的代谢转换。Prm1-FapR /（-52）fapO-PAOX1和Prm1-FapR / PGAP-（+ 22）fapO也分别用于控制丙二酰辅酶A的来源和吸收途径，以合成6-甲基水杨酸。最终导致细胞质丙二酰辅酶A的振荡代谢模式。这种代谢物可用于探索不受天然细胞行为限制的潜在工业和生物医学应用。
更新日期：2020-03-31
中文翻译：
蛇形果蝇中的合成丙二酰辅酶A代谢振荡器。
丙二酰辅酶A是参与各种生理反应的关键代谢分子，可以作为多种增值药物和化学药品的基础。胞质丙二酰-CoA的浓度通常非常低，因此动态控制丙二酰-CoA变异的代谢将有助于其稳定形成和有效消耗。我们在酵母中开发了合成的丙二酰辅酶A代谢振荡器。通过将酵母转录激活因子Prm1与细菌丙二酰辅酶A敏感的转录抑制因子FapR融合来构建合成的调节蛋白Prm1-FapR。然后设计了两个相反调节的生物传感器。共设计了18个杂种启动子变体，每个变体带有FapR的操纵子序列（fapO）和PAOX1的核心启动子（cPAOX1），这是由Prm1自然调节的。测试了由Prm1-FapR调节的这些变体的启动子活性。通过此过程，构建了带有激活/停用调节模块的Prm1-FapR /（-52）fapO-PAOX1传感器。同时，使用融合调节剂设计并测试了插入有fapO的PGAP的24个启动子变体，从而为Prm1-FapR / PGAP-（+ 22）fapO提供了一个传感器，该传感器包含一个阻抑/阻抑调节模块。随后将两个传感器都整合到单个细胞中，该细胞在操纵胞质丙二酰辅酶A水平后表现出eGFP和mCherry报告基因正确的代谢转换。Prm1-FapR /（-52）fapO-PAOX1和Prm1-FapR / PGAP-（+ 22）fapO也分别用于控制丙二酰辅酶A的来源和吸收途径，以合成6-甲基水杨酸。最终导致细胞质丙二酰辅酶A的振荡代谢模式。这种代谢物可用于探索不受天然细胞行为限制的潜在工业和生物医学应用。
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