A Synthetic Malonyl-CoA Metabolic Oscillator in Komagataella phaffii
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 malon...
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Published in | ACS synthetic biology Vol. 9; no. 5; pp. 1059 - 1068 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
15.05.2020
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Subjects | |
Online Access | Get full text |
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Summary: | 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/(−52)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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 2161-5063 2161-5063 |
DOI: | 10.1021/acssynbio.9b00378 |