Engineered dynamic distribution of malonyl-CoA flux for improving polyketide biosynthesis in Komagataella phaffii

Malonyl-CoA is a basic but limited precursor for the biosynthesis of various bioactive compounds and life-supporting fatty acids in cells. This study develops a biosynthetic system to dynamically redirect malonyl-CoA flux and improve production of malonyl-CoA derived polyketide (6-MSA) in Komagataella phaffii. A synthetic regulatory protein fusing a yeast activator Prm1 with a bacterial repressor FapR was proved to work with a hybrid promoter (-7)fapO-cP_AOX1 and activate gene expression. Expression mode by the Prm1-FapR/(-7)fapO-cP_AOX1 device was not affected by intracellular malonyl-CoA levels. Further, 9 promoter variants of P_GAP with insertion of fapO at various sites were tested with the Prm1-FapR. It generated a biosensor of Prm1-FapR/P_GAP-(+2)fapO with regulation behavior of malonyl-CoA-low-level repression/high-level derepression. Both devices were subsequently integrated into a single cell, for which fatty acid synthesis module was driven by Prm1-FapR/P_GAP-(+2)fapO but 6-MSA synthesis module was expressed by Prm1-FapR/(-7)fapO-cP_AOX1. The integrated system allowed continuous polyketide synthesis but malonyl-CoA-high-level "on"/low-level "off" fatty acid synthesis. This design finally increased 6-MSA production capacity by 260 %, proving the positive effects of dynamic malonyl-CoA distribution to the target compounds. It provides a new strategy for synthesis of malonyl-CoA derived compounds in eukaryotic chassis hosts.