Global transcriptional rewiring and dual-compartment engineering for efficient 3-hydroxypropionic acid production in Saccharomyces cerevisiae.
3-Hydroxypropionic acid is an important malonyl-CoA-derived platform chemical whose efficient biosynthesis is constrained by limited utilization of malonyl-CoA across subcellular compartments. Using a biosensor-guided transcription factor mutagenesis screen combined with transcriptomic and functional validation, we identify four positive mutants NRG1_R224H, STB3_L52Y, PDR1_T820C and PGD1_V243D that increase cytosolic malonyl-CoA accumulation by globally reprogramming transcription to reinforce central carbon flux and acetyl-CoA precursor supply, and remodel amino acid, redox, and lipid metabolism to favor malonyl-CoA accumulation. We further utilize mitochondrial malonyl-CoA for 3-HP production through dynamic control of HFA1 and optimized POS5 expression, and further develop a dual-compartment coordination strategy to efficiently exploit cytosolic and mitochondrial malonyl-CoA pools. Integration of optimized pathways in diploid strains enables coordinated precursor utilization, achieving 81.8 g/L 3-HP in 5-L fed-batch fermentation, the highest titer reported to date in Saccharomyces cerevisiae. This work establishes a generalizable framework for multi-compartment malonyl-CoA utilization in eukaryotic cell factories.