| Metabolic engineering has become a commonly used tool for improving the titer and yield of various value-added molecules that are naturally produced and heterologously synthesized by microorganisms.Traditional methods have focused on static control,e.g.,over-expression of enzymes in the rate-limiting pathway,knockout or knockdown of the competitive pathways,control of ATP supply,equilibrium of redox reactions,and precursor metabolism rewiring,in order to redistribute the flux of steady-state pathways.However,such engineered strains often do not dynamically control gene expression and are particularly sensitive to environmental disturbances.Dynamic control strategies can rebalance cell growth,metabolism and production responding to environmental conditions in cells or the fermentation media,enabling cells spontaneously sensing and responding to changing conditions.Dynamic regulation better balances cell growth,metabolism and production of target products and can reduce the accumulation of toxic intermediates.Dynamic control of enzymes in heterologous pathways can also reduce protein co-expression burden.Recently,advances in promoter libraries,high-throughput screening and synthetic biology techniques have well supported the development of dynamic control.However,reports on dynamic control mainly focus on prokaryotic cells,which may ascribe to the complicated transcriptional regulation mechanism of yeast or other higher eucaryotic cells.Malonyl-CoA(Mal-CoA)is a key metabolic molecule that participates in a diverse range of physiological responses and can act as the building block for a variety of value-added pharmaceuticals and chemicals.The cytosolic Mal-CoA concentration is usually very low,and neither online nor off-line technique of Mal-CoA measure is available although it is so important for the optimium process control.Therefore,if the cell can be dynamically regulated responding to the change of Mal-CoA concentration,it will be beneficial for the stable formation and efficient transformation of Mal-CoA in cell.Nevertheless,there are few reports on the dynamic control of Mal-CoA in eukaryotic cells.K.phaffii is one of the most widely used eukaryotic expression hosts.It has also attracted extensive attention as a chassis host of synthetic biology in recent years.K.phaffii has been recognized by the FDA as a generally recognized as safe(GRAS)strain and can be used in the pharmaceutial and food industries.In this study,the Mal-CoA biosensor was designed and applicated in dynamic metabolic control in K.phaffii.Two Mal-CoA biosensors with reverse regulation mode were first constructed for application in K.phaffii.A yeast transcription activator Prm1 was fused with a bacterial Mal-CoA sensitive transcription repressor FapR to form a synthetic regulatory protein,Prm1-FapR.Then two oppositely regulated biosensors were engineered.A total of 18 hybrid promoter var iants carrying the operator sequence(fapO)of fapR and core promoter of PAOX1(cPAOX1)naturally regulated by Prm1 were designed,and their promoter activities regulated by the Prm1-FapR were tested.By this,a sensor of Prm1-FapR/(-52)fapO-PAOX1 carrying activation/deactivation regulation module was constructed.Meanwhile,24 promoter variants of PGAP with insertion of fapO inside were designed and tested with the fusion regulator,giving a sensor of Prm1-FapR/PGAP-(+22)fapO harboring repression/derepression regulation module.Both sensors were subsequently integrated into a single cell,which exhibited correct metabolic switching by GFP and mCherry reporters after manipulation of cytosolic Mal-CoA levels.Furtherly,the Prm1-FapR/(-52)fapO-PAOX1 and the Prm1-FapR/PGAP-(+22)fapO were used to control the Mal-CoA source and sink pathway,respectively,for the synthesis of 6-methylsalicylic acid(6-MSA).This finally led to an oscillatory metabolic mode of cytosolic Mal-CoA.Such metabolator is useful in exploring potential industrial and biomedical applications not limited by natural cellular behavior.Afterwards,a dynamically controlled yeast biosynthetic system was engineered for improving biosynthesis of Mal-CoA derived bioactive chemicals.This system allows continuous polyketide biosynthesis but automatically controlled the fatty acid biosynthesis by"on" or "off" according to the high or low malonyl level.As described above,a synthetic regulatory protein fusing a yeast activator Prm1 with a bacterial repressor FapR was proved to intensively combine with a hybrid promoter(-7)fapo-cPPRM1 and activate gene transcription.To ensure a promoted production of 6-MSA in ethanol medium and allow an intensive expression of Prm1-FapR,the initial constitutive prmoter of PPRM1 was replaced by an ethanol inducible promoter of PICL1.Expression mode by this biological device Prm1-FapR/(-7)fapO-cPAOX1 was not affected by intracellular malonyl-CoA levels.Further,9 promoter variants of PGAP with insertion of fapO at various sites were tested with the Prm1-FapR.It gave a biosensor of Prm1-FapR/PGAP-(+2)fapO that showed low expression capacity and regulation behavior of malonyl-CoA low-level repression/high-level derepression.Both devices were subsequently integrated into a single cell of K.phaffii,for which fatty acid synthesis module was driven by the Prm1-FapR/PGAP-(+2)fapO,while polyketide(6-MSA)synthesis module was expressed by the Prm1-FapR/(-7)fapO-cPAOX1.The integrated system allowed continuous polyketide synthesis and conditional fatty acid synthesis on malonyl-CoA level.The 6-MSA production was increased by 110%in this system,proving the improved redirection of the malonyl-CoA flux to the target compound.The improved polyketide production proved the optimal redirection of the malonyl-CoA flux to the target compound.It provides a new strategy for synthesis of malonyl-CoA derived compounds in eukaryotic chassis hosts. |
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