A Synthetic Malonyl-CoA Metabolic Oscillator in <i>Komagataella phaffii</i>

Abstract

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 (<i>fapO</i>) of FapR and the core promoter of P_<i>AOX1</i> (cP_<i>AOX1</i>), 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)<i>fapO</i>-P_<i>AOX1</i> carrying an activation/deactivation regulation module was built. Meanwhile, 24 promoter variants of P_<i>GAP</i> with <i>fapO</i> inserted were designed and tested using the fusion regulator, giving a sensor for Prm1-FapR/P_<i>GAP</i>-(+22) <i>fapO</i> 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)<i>fapO</i>-P_<i>AOX1</i> and the Prm1-FapR/P_<i>GAP</i>-(+22)<i>fapO</i> 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.