文献类型：期刊文献

英文题名：A Synthetic Malonyl-CoA Metabolic Oscillator in Komagataella phaffii

作者：Wen, Jiao[1];Tian, Lin[1];Xu, Mingqiang[1];Zhou, Xiangshan[1];Zhang, Yuanxing[1,2];Cai, Menghao[1,2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Shanghai Collaborat Innovat Ctr Biomfg, Shanghai 200237, Peoples R China

年份：2020

卷号：9

期号：5

起止页码：1059

外文期刊名：ACS SYNTHETIC BIOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000535294200011)】；

基金：This work was supported by the Shanghai Rising-Star Program, China (19QA1402700), Fundamental Research Funds for the Shanghai Science and Technology Innovation Action Plan (17JC1402400), National Natural Science Foundation of China (31870073), the 111 Project of China (B18022), Fundamental Research Funds for the Central Universities, China (22221818014), Research Program of State Key Laboratory of Bioreactor Engineering. We thank Dr. Jianye Xia for the help of malonyl-CoA extraction and analysis.

语种：英文

摘要：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 P-AOX1 (cP(AOX1)), 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-P-AOX1 carrying an activation/deactivation regulation module was built. Meanwhile, 24 promoter variants of P(GAP )with fapO inserted were designed and tested using the fusion regulator, giving a sensor for Prm1-FapR/P-GAP-(+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/(-S2)fapO-P-AOX1 and the Prm1-FapR/P-GAP-(+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.