Flux Control at the Malonyl-CoA Node through Hierarchical Dynamic Pathway Regulation in Saccharomyces Cerevisiae

The establishment of a heterologous pathway in a microbial host for the production of industrially relevant chemicals at high titers and yields requires efficient adjustment of the central carbon metabolism to ensure that flux is directed towards the product of interest. To achieve this, we established a ligand responsive transcription factor based sensor module in Saccharomyces cerevisiae and applied the concept for enzyme engineering and controlling flux at the malonyl-CoA node. We present a novel approach for dynamic modulation of pathway flux and enzyme expression levels. It is based on a hierarchical dynamic control system around the key pathway intermediate malonyl-CoA. The upper level of the control system ensures down-regulation of endogenous use of malonyl-CoA for fatty acid biosynthesis, which results in accumulation of this pathway intermediate. The lower level of the control system is based on use of a novel biosensor for malonyl-CoA to activate expression of a heterologous pathway using this metabolite for production of 3-hydroxypropionic acid (3-HP). The malonyl-CoA sensor was developed based on the FapR transcription factor of Bacillus subtilis, and it demonstrates one of the first applications of a bacterial metabolite sensor in yeast. Introduction of the dual pathway control increased the production of 3-HP by 10-fold. The approach can be seen as a prototype of a general strategy to engineer and dynamically control metabolic pathways for balancing precursor supply and can also be applied for production of other malonyl-CoA derived products.